DEPARTMENT OF ENTOMOLOGY, FACULTY OF AGRICULTURE, …prr.hec.gov.pk/jspui/bitstream/123456789/7973/1/Muhammad_Yasin... · ENTOMOPATHOGENS AS POTENTIAL BIOCONTROL AGENTS AGAINST RED

ENTOMOPATHOGENS AS POTENTIAL BIOCONTROL AGENTS AGAINST

RED PALM WEEVIL, RHYNCHOPHORUS FERRUGINEUS (OLIVIER)

By MUHAMMAD YASIN

M.Sc. (Hons.) Entomology

A thesis submitted in partial fulfillment of the

requirements for the degree of

DOCTOR OF PHILOSPHY (Ph.D.)

In

ENTOMOLOGY

DEPARTMENT OF ENTOMOLOGY, FACULTY OF AGRICULTURE, UNIVERSITY OF AGRICULTURE,

FAISALABAD, PAKISTAN 2016

DECLARATION

I hereby declare that that the contents of the thesis “Entomopathogens as Potential Biocontrol

Agents Against Red Palm Weevil, Rhynchophorus ferrugineus (Olivier)” are product of my own

research and no part has been copied from published sources (except references, standard

mathematical or genetic models/equations/formulas/protocols etc.). I further declare that this

work has not been submitted for the award of any other degree/diploma. The University may

take action if the information provided found inaccurate at any stage (In case of any default, the

scholar will be proceeded against as per HEC plagiarism policy).

Muhammad Yasin

The Controller of Examinations,

University of Agriculture,

Faisalabad.

We, the Supervisory Committee , certify that the contents and

form of thesis submitted by Muhammad Yasin, Registration No. 2005-

ag-1886 have been found satisfactory and recommend that it be

processed for evaluation, by the External Examiner (s) for the award of

degree.

Supervisory Committee

Chairman ____________________________________

Dr. Waqas Wakil

Member ____________________________________

Prof. Dr. Muhammad Jalal Arifl

Member ____________________________________

Prof. Dr. Shahbaz Talib Sahil

This thesis is dedicated with Love and Respect to My Parents

ACKNOWLEDGEMENTS

First of all I would like to bow my head before “ALMIGHTY ALLAH” the Omnipotent, the Omnipresent, the Merciful, the Beneficial who presented me in a Muslim community and also bestowed and blessed me with such a lucid intelligence as I could endeavor my services toward this manuscript. Countless salutations are upon the HOLY PROPHET MUHAMMAD (May Peace Be Upon Him), the fountains of knowledge, who has guided his “Ummah” to seek knowledge for cradle to grave.

The work presented in this manuscript was accomplished under the sympathetic attitude, animate directions, observant pursuit, scholarly criticism, cheering perspective and enlightened supervision of Dr. Waqas Wakil, Assistant Professor, Department of Entomology, University of Agriculture, Faisalabad. His thoughtful guidance helped me in all the time of research, writing of dissertation/publications etc. and his rigorous critique improved my overall understanding of the subject. I am grateful to his ever inspiring guidance, keen interest, scholarly comments and constructive suggestions throughout my PhD studies.

I wish to acknowledge my deep sense of profound gratitude to the worthy member of my research

supervisory panel Dr. Muhammad Jalal Arif and Dr. Shahbaz Talib Sahi, for their constructive criticism, illuminating and inspiring guidance and continuous encouragement throughout course of my study. I really have no words to express my sincere thankful feelings and emotions for all my Teachers, seniors and friends especially Dr. M. Usman Ghazanfar, Dr. M. Khalid Bashir, Dr. M. Ejaz Ashraf, Dr. Mirza Abdul Qayyum, Dr. Kashif Ali, Dr. M. Tahir and Dr. M. Irfan Akram for their cooperation, well wishes and moral support from time to time during the course of study. I am also thankful to my junior fellows specially Muhammmad Farooq, Muhammad Asif, Muhammad Ismail, Muhammad Usman, Muhammad Shoaib Qazi, Muhammad Ahmad Rana, Shumaila Rasoool, Sehrish Gulzar, Zeshna Khaliq, Muhammad Tufail, Mehwish Ayaz, Kanza Syed and Ayesha Faraz for their support towards my PhD studies. Words are lacking to express my humble obligation to my affectionate grandparents, Father, Mother, Brothers, Sisters, uncles, aunts, cousins and specially my lovely wife and my children Rehmin Fatima and Muhammad Abdullah for patience in absence of their Dad, and all family members for their love, good wishes, inspirations and unceasing prayers for me, without which the present destination would have been mere a dream.

I would like to express my deepest gratitude to the Higher Education Commission, Islamabad

(Pakistan) for the scholarship under PhD Indigenous Fellowship Program is greatly acknowledged. I would like to say special thanks to Dr. Richard Stouthamer and Paul Rugman-Jones UCR, USA for accepting me towards IRSIP as an international student. Last but least I will pay my special thanks to Ms. Aima Bashir who always prayed for my success, may God bless her.

Muhammad Yasin

i

CONTENTS

Chapter 1. Introduction……………………………………………………………………. 01

Chapter 2. Literature Reviewed

2.1 Invasive Red Palm Weevil (RPW)……………………………… 07

2.2 Taxonomic position ……………………………………………... 07

2.3 Classification…………………………………………………….. 07

2.4 Spatial distribution………………………………………………. 07

2.5 Control measures………………………………………………… 08

2.5.1 Microbial control………………………………………………… 08

2.5.2 History of microbial control……………………………………... 08

2.5.3 Entomopathogenic Fungi (EPFs)………………………………... 10

2.5.3.1 History…………………………………………………………… 10

2.5.3.2 Geographical distribution and occurrence……………………….. 11

2.5.3.3 Classification…………………………………………………….. 11

2.5.3.4 Host range……………………………………………………….. 11

2.5.3.5 Mode of infection………………………………………………... 12

2.5.3.6 Enzymes and toxins of EPFs…………………………………….. 12

2.5.3.7 Chitinases………………………………………………………... 12

2.5.3.8 Proteases and peptidases………………………………………… 13

2.5.3.9 Lipases…………………………………………………………… 13

2.5.3.10 Toxins……………………………………………………………. 13

2.5.3.11 Destruxins………………………………………………………... 13

2.5.3.12 Oosporein………………………………………………………... 14

2.5.3.13 Beauvericin and beauveriolide…………………………………... 14

2.5.3.14 Bassianolide……………………………………………………... 14

2.5.3.15 Beauveriolide……………………………………………………. 14

2.5.3.16 Host range……………………………………………………….. 14

2.5.3.17 Effect of abiotic factors………………………………………….. 15

2.5.3.17.1 Temperature……………………………………………………... 15

2.5.3.17.2 Relative humidity………………………………………………... 15

2.5.3.18 Effect of EPFs on non-target organisms………………………… 15

2.5.3.19 Integration of EPFs with other control measures……………….. 16

2.5.3.20 EPF against RPW………………………………………………... 17

2.5.3.21 Natural incidence of EPFs on RPW……………………………... 17

2.5.3.22 Susceptibility of RPW to EPFs infections under lab condition…. 18

2.5.3.23 Field and Semi-field assessment of fungi for RPW management.. 19

2.5.4 Endophytic fungi………………………………………………… 20

2.5.5 Future prospects of entomopathogenic fungi.................................. 20

2.5.6 Entomopathogenic Nematodes (EPNs)........................................... 21

2.5.6.1 Natural incidence………………………………………………… 21

2.5.6.2 Susceptibility of RPW to EPNs infections under lab conditions... 22

2.5.6.3 Field and semi-field assessment of EPNs for RPW management.. 22

2.5.6.4 Interactions between EPNs and pesticides...................................... 23

2.5.7 Entomopathogenic Bacteria............................................................. 24

ii

2.5.7.1 History............................................................................................... 24

2.5.7.2 Classification…………………………………………………….. 24

2.5.7.3 Life cycle………………………………………………………… 25

2.5.7.4 Ecology…………………………………………………………... 25

2.5.7.5 Mechanism of action…………………………………………….. 26

2.5.7.6 Commercial formulations………………………………………... 26

2.5.7.7 Methods of applications of Bt products…………………………. 27

2.5.7.8 Superiority of Bt products over synthetic insecticides…………... 27

2.5.7.9 Concerns to use of Bt……………………………………………. 27

2.5.7.10 Interaction of Bt products and other toxins……………………… 28

2.5.7.11 Effect of Bacillus thuringiensis on non-target invertebrates…….. 28

2.5.7.12 Mode of infection………………………………………………... 29

2.5.7.13 Important entomopathogenic bacteria…………………………… 29

2.5.7.13.1 Bacillus thuringiensis……………………………………………. 29

2.5.7.13.1 Paenibacillus popilliae…………………………………………... 30

2.5.7.13.2 Brevibacillus laterosporus………………………………………. 30

2.5.7.13.3 Bacillus subtilis………………………………………………….. 30

2.5.7.13.4 Bacillus sphaericus……………………………………………… 30

2.5.7.13.5 Wolbachia………………………………………………………... 31

2.5.7.14 Host range of B. thuringiensis…………………………………… 31

2.5.7.15 Natural incidence………………………………………………… 32

2.5.7.16 Susceptibility of RPW to EPB under lab conditions….………… 32

2.5.7.17 Field and Semi-field assessment of EPB for RPW management.. 32

2.5.8 Microbial control agents as a component of RPW IPM…………. 33

2.5.9 Ecological engineering and agricultural practices to conserve

microbial control agents………………………………………….

33

2.5.10 Biotechnological approaches to enhance virulence of microbial

control agents…………………………………………………….

34

2.6 References……………………………………………………….. 35

Chapter 3

Genetic variation among populations of Red Palm Weevil

Rhynchophorus ferrugineus (Olivier) (Coleoptera:

Curculionidae) from the Punjab and Khyber Pakhtunkhwa

provinces of Pakistan

Abstract………………………………………………………….. 61

3.1 Introduction ……………………………………………………... 62

3.2 Materials and methods…………………………………………... 63

3.2.1 Specimen collections…………………………………………….. 63

3.2.2 DNA extraction and amplification………………………………. 63

3.2.3 Cleaning and sequencing………………………………………… 64

3.2.4 Genetic analysis………………………………………………….. 64

3.3 Results…………………………………………………………… 64

3.4 Discussion……………………………………………………….. 65

3.5 References……………………………………………………….. 68

iii

Chapter 4 Resistance to commonly used insecticides and phosphine

(PH3) against Rhynchophorus ferrugineus (Olivier)

(Coleoptera: Curculionidae) in Punjab and Khyber

Pakhtunkhwa, Pakistan

Abstract………………………………………………………….. 74

4.1 Introduction……………………………………………………… 75


4.2.1 RPW collection and rearing……………………………………... 76

4.2.2 Test chemicals…………………………………………………… 76

4.2.3 Generation of phosphine gas…………………………………...... 76

4.2.4 Bioassay…………………………………………………………. 77

4.3 Statistical analysis……………………………………………….. 77

4.4 Results…………………………………………………………… 78

4.4.1 Imidacloprid……………………………………………………... 78

4.4.2 Spinosad…………………………………………………………. 78

4.4.3 Lambda cyhalothrin……………………………………………… 78

4.4.4 Chlorpyrifos…………………………………………………....... 78

4.4.5 Profenophos……………………………………………………… 78

4.4.6 Deltamethrin……………………………………………………... 78

4.4.7 Cypermethrin…………………………………………………….. 79

4.4.8 Phosphine………………………………………………………... 79

4.5 Discussion……………………………………………………….. 79

4.6 References……………………………………………………….. 81

Chapter 5

Insecticidal potential of Beauveria bassiana and Metarhizium

anisopliae isolates against Rhynchophorus ferrugineus

(Olivier) (Coleoptera: Curculionidae)

Abstract………………………………………………………….. 89

5.1 Introduction……………………………………………………… 90



5.2.2 Culture collection………………………………………………... 91

5.2.3 Isolation from RPW cadavers…………………………………… 92

5.2.4 Screening assay …………………………………………………. 92

5.2.5 Virulence assay………………………………………………….. 92

5.2.6 Statistical analysis……………………………………………….. 93

5.3 Results…………………………………………………………… 93

5.3.1 Screening assay …..……………………………………………… 93

5.3.2 Virulence assay………………………………………………….. 93

5.4 Discussion……………………………………………………….. 94

5.5 References……………………………………………………….. 97

iv

Chapter 6 Combined effectiveness of endophytically colonized

Beauveria bassiana and Bacillus thuringiensis against

Rhynchophorus ferrugineus (Olivier) (Coleoptera:

Curculionidae)

Abstract………………………………………………………….. 111

6.1 Introduction ……………………………………………………... 112


6.2.1 RPW collection and rearing…………………………………….. 113

6.2.2 Preparation of fungi……………………………………………… 113

6.2.3 Preparation of Bacillus thuringiensis spore-crystal mixtures…… 114

6.2.4 Screening of fugal isolates……………………………………… 114

6.2.5 Bioassay procedure……………………………………………… 114

6.2.6 Bioassay on development of R. ferrugineus……………………... 115

6.2.7 Bioassay on larval development ………………………………… 115


6.3 Results …………………………………………………………... 115

6.3.1 Fungal colonization of date palm petioles……………………….. 115

6.3.2 Toxicity of microbial agents…………………………………….. 116

6.3.3. Development of R. ferrugineus………………………………….. 116

6.3.4 Effect on larval development……………………………………. 117

6.4 Discussion ……………………………………………………….. 117

6.5 References……………………………………………………….. 120

Chapter 7 Integrated effect of entomopathogenic fungi and

entomopathogenic nematodes against Rhynchophorus

ferrugineus (Olivier) (Coleoptera: Curculionidae)

Abstract………………………………………………………….. 134

7.1 Introduction……………………………………………………… 135



7.2.2 Nematode………………………………………………………... 136


7.2.4 Treatment with entomopathogenic fungi………………………... 136

7.2.5 Treatment with H. bacteriophora ………………………………. 137

7.2.6 Treatment with entomopathogenic fungi and nematode ………... 137

7.2.7 Effects of entomopathogens on R. ferrugineus development…… 137

7.2.8 Effects of entomopathogens on larval development…………….. 138


7.3 Results………………………………………………………….... 138

7.3.1 Entomopathogenic fungi and nematode interaction……………... 138

7.3.2 Development of R. ferrugineus …………………………………. 139

7.3.3 Effect on larval development ………………………………….... 139

7.4 Discussion……………………………………………………….. 139

7.5 References……………………………………………………….. 142

v

Chapter 8 Combined toxicity of Beauveria bassiana, Bacillus

thuringiensis and entomopathogenic nematodes against red

palm weevil Rhynchophorus ferrugineus (Olivier)

(Coleoptera: Curculionidae)

Abstract………………………………………………………….. 155

8.1 Introduction……………………………………………………… 156


8.2.1 RPW collection………………………………………………….. 157

8.2.2 Preparation of B. thuringiensis spore-crystal mixtures………….. 157

8.2.3 Entomopathogenic nematode……………………………………. 157


8.2.5 Treatment with B. bassiana……………………………………… 158

8.2.6 Treatment with B. thuringiensis…………………………………. 158

8.2.7 Treatment with H. bacteriophora……………………………….. 158

8.2.8 Treatment with B. bassiana, Bt-k and H. bacteriophora………... 159

8.2.9 Sporulation and Nematode production………………………….. 159


8.3 Results…………………………………………………………… 159

8.3.1 Mortality of larvae and adult…………………………………….. 159

8.3.2 Mycosis and sporulation…………………………………………. 160

8.3.3 Insects affected by EPNs and EPNs production…………………. 160

8.4 Discussion……………………………………………………….. 160

8.5 References……………………………………………………….. 162

Summary………………………………………………………… 172

vi

List of Tables

No Title Page

No.

Table 3.1 Sampling information for RPW populations collected from date palm

Phoenix dactylifera in Punjab and KPK provinces of Pakistan……………...

70

Table 3.2 Genetic characterization of five RPW populations from the Punjab and

KPK provinces of Pakistan based on a 528 bp section of the mitochondrial

COI gene. For population abbreviations, see Table 1………………………..

70

Table 3.3 Variation in a 528 bp segment of the cytochrome oxidase subunit I (COI)

region of mitochondrial DNA (mtDNA) of Rhynchophorus ferrugineus.

Average number of pairwise nucleotide differences (k) within (diagonal

element) and between (below diagonal) populations in the Punjab and KPK

provinces of Pakistan. For population abbreviations………………………...

71

Table 4.1 Geographical characteristics of the localities from where R. ferrugineus

populations were collected in Punjab and Khyber Pakhtunkhwa, Pakistan….

84

Table 4.2 Resistance to commonly used insecticides and phosphine against

susceptible strains and field-collected populations of R. ferrugineus………..

85

Table 5.1 Characterization of B. bassiana and M. anisopliae isolates obtained from

soils and insect cadavers……………………………………………………..

103

Table 5.2 Factorial analysis of screening and mycosis of R. ferrugineus exposed to B.

bassiana and M. anisopliae isolates………………………………………….

104

Table 5.3 Percentage pathogenicity (%±SE) of 19 isolates of B. bassiana and M.

anisopliae isolates against R. ferrugineus larvae after 12 days of incubation.

104

Table 5.4 Percentage pathogenicity (%±SE) of 19 isolates of B. bassiana and M.

anisopliae isolates against R. ferrugineus adult after 12 days of incubation...

105

Table 5.5 Factorial analysis for virulence of B. bassiana and M. anisopliae isolates

against larvae and adult of R. ferrugineus……………………………………

106

Table 5.6 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 7 days of

exposure treated with B. bassiana and M. anisopliae isolates…………….…

106

Table 5.7 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 14 days

of exposure treated with B. bassiana and M. anisopliae isolates…………….

107

Table 5.8 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 21 days

of exposure treated with B. bassiana and M. anisopliae…………………….

107

Table 5.9 LC50 and LC90 values of B. bassiana and M. anisopliae isolates tested

against larvae and adult of R. ferrugineus …………………………………..

108

Table 5.10 LT50 and LT90 values of B. bassiana and M. anisopliae isolates tested

against larvae of R. ferrugineus………………………………………………

109

Table 5.11 LT50 and LT90 values of B. bassiana and M. anisopliae isolates tested

against adult of R. ferrugineus……………………………………………….

110

Table 6.1 Percentage of petiole fragments colonized by entomopathogenic (E) and

other (O) fungi in live palm petioles experiments……………………………

124

Table 6.2 Factorial analysis of mortality, pupation, adult emergence and egg eclosion

of R. ferrugineus exposed to endophytically colonized B. bassiana and B.

thuringiensis………………………………………………………………….

125

Table 6.3 Mean mortality (%±SE) of 2nd, 4th and 6th instar larvae of R. ferrugineus

vii

treated with endophytic B. bassiana (Bb: 2 cm away from inoculation site)

and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination

(means followed by the same letter within each treatment are not

significantly different; HSD test P≤0.05) …………………………………...

126

Table 6.4 Pupation, adult emergence and egg eclosion (%±SE) of 2nd, 4th and 6th instar

larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 2 cm

away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg

ml-1) alone and in combination (means followed by the same letter within

each treatment are not significantly different; HSD test P≤0.05)……………

127

Table 6.5 Growth parameters e.g. larval duration (days), larval weight (grams) pre-

pupal duration (days), pre-pupal weight (grams), pupal duration (days),

pupal weight (grams), adult longevity (days) and adult weight (grams)

(%±SE) of 2nd instar larvae of R. ferrugineus treated with endophytic B.

bassiana (Bb: 4 cm away from inoculation site) and Bt-k (Bt1: 10 µg; Bt2:

15 µg; Bt3: 20 µg ml-1) alone and in combination (means followed by the

same letter within each treatment are not significantly different; HSD test

P≤0.05) ……………………………………………………………………..

128

Table 6.6 Analysis of Co-variance for 10th instar larvae of R. ferrugineus regarding

weight gain, frass production and diet consumption when treated with

endophytic B. bassiana (Bb: 4 cm away from inoculation site) and Bt-k (Bt:

10 µg ml-1). Initial weight of larvae and diet consumption were taken as

covariate……………………………………………………………………...

129

Table 7.1 Mean mortality (%±SE) of 2nd instar larvae of R. ferrugineus treated with B.

bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M.

anisopliae were used each @ 1×106 spore ml-1 and H. Bacteriophora was

applied @ 100 IJs ml-1………………………………………………………..

146

Table 7. 2 Mean mortality (%±SE) of 4th instar larvae of R. ferrugineus treated with B.



applied @ 100 IJs ml-1………………………………………………………..

147

Table 7.3 Mean mortality (%±SE) of 6th instar larvae of R. ferrugineus treated with B.



applied @ 100 IJs ml-1………………………………………………………..

148

Table 7.4 Factorial analysis of pupation, adult emergence and egg eclosion of R.

ferrugineus exposed to B. bassiana, M. anisopliae and H. Bacteriophora…..

148

Table 7.5 Pupation, adult emergence and egg eclosion (%±SE) of 2nd, 4th and 6th instar

R. ferrugineus larvae treated with B. bassiana, M. anisopliae and H.

Bacteriophora. B. bassiana and M. anisopliae were used each @ 1×106

spore ml-1 and H. Bacteriophora was applied @ 100 IJs ml-1. Mean sharing

the same letters are not significantly different. Means sharing the same

letters within columns are not significantly different……………………….

149

Table 7.6 Effect of B. bassiana, M. anisopliae and H. Bacteriophora on the

development of R. ferrugineus. B. bassiana and M. anisopliae were used

each @ 1×104 spore ml-1 and H. Bacteriophora was applied @ 50 IJs ml-1.

Mean sharing the same letters are not significantly different………………..

150

viii

Table 7.7 Analysis of co-variance for 2nd, 4th and 6th instar larvae of R. ferrugineus

regarding weight gain and frass production at a given level of diet

consumption when treated with B. bassiana and H. Bacteriophora alone

and in combination. Initial weight of larvae and diet consumption were

taken as covariate…………………………………………………………….

151

Table 8.1 ANOVA parameters for the main effects and associated interactions for

mortality levels of R. ferrugineus larvae and adults………………………….

166

Table 8.2 Mean mortality (%±SE) of R. ferrugineus populations collected from

Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg

g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs)

applied alone or in combination after 7 days of exposure (means followed

by the same letter within each treatment and insect populations not


166







167






significantly different; HSD test P≤0.05)……………………………………

167

ix

List of Figures

No. Title Page

No.

Figure 3.1 Map of collection sites in Punjab and KPK provinces of Pakistan………….. 71

Figure 3.2 Distribution of mitochondrial haplotypes across five populations of RPW

from the Punjab and KPK provinces of Pakistan……………………………

72

Figure 3.3 Relationships between four Pakistani COI haplotypes and 48 others are

occurring around the world. Haplotype network constructed from 539 COI

sequences (each 528 bp long) generated by the present study and three

earlier studies (see text). Each haplotype is represented by an oval or for

that with the highest outgroup probability, a rectangle. Size of each

haplotype is indicative of the number of specimens sharing that haplotype;

also given inside each haplotype. H1-43 is numbered according to El

Mergawy et al. (2011) and Rugman-Jones et al. (2013); H44-50

corresponds to additional haplotypes from Wang et al. (2015); and H51-52

are new to this study…………………………………………………………

73

Figure 4.1 Map of collection sites in Punjab and Khyber Pakhtunkhwa provinces of

Pakistan (1. Bahawalpur 2. Rahim Yar Khan 3. Vehari 4. Dera Ghazi Kahn

5. Muzaffargarh 6. Layyah 7: Dera Ismail Khan)………………………….

87

Figure 4.2 Resistance ratio (RR) of chemical insecticides and phosphine against

susceptible strains and field-collected populations of R. ferrugineus

populations of R. ferrugineus from various localities in Punjab and Khyber

Pakhtunkhwa, Pakistan………………………………………………………

88

Figure 6.1 Mean mycosis (%±SE) in cadavers of R. ferrugineus treated with

endophytic B. bassiana (Bb: 2 cm away from inoculation site) and Bt-k

(Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination (means

followed by the same letter within each treatment are not significantly

different; HSD test P≤0.05)………………………………………………….

130

Figure 6.2 Sporulation (conidia ml-1) on R. ferrugineus cadavers treated with

endophytic B. bassiana (Bb: 2 cm away from inoculation site) and Bt-k

(Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination (means

followed by the same letter within each treatment are not significantly

different; HSD test P≤0.05)………………………………………………….

130

Figure 6.3 Diet consumption (grams) in 10th instar larvae of R. ferrugineus treated

with endophytic B. bassiana (Bb: 6 cm away from inoculation site) and Bt-

k (Bt: 10 µg ml-1) ……………………………………………………………

131

Figure 6.4 Frass production (grams) in 10th instar larvae of R. ferrugineus treated with


10 µg ml-1) …………………………………………………………………..

132

Figure 6.5 Weight gain (grams) in 10th instar larvae of R. ferrugineus treated with


10 µg ml-1) ………………………………………………………………….

133

Figure 7.1 Diet consumption in last instar larvae of R. ferrugineus when treated with

B. bassiana and H. bacteriophora…………………………………………...

152

Figure 7.2 Frass production in last instar larvae of R. ferrugineus when treated with B.

x

bassiana and H. bacteriophora……………………………………………… 153

Figure 7.3 Weight gain in last instar larvae of R. ferrugineus when treated with B.

bassiana and H. bacteriophora………………………………………………

154

Figure 8.1a Mean mycosis (%±SE) in larvae of R. ferrugineus populations collected

from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k

(70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs)

applied alone or in combination (means followed by the same letter within

each treatment are not significantly different; HSD test

P≤0.05)………………………………………………………………………

168

Figure 8.1b Mean mycosis (%±SE) in adults of R. ferrugineus populations collected





P≤0.05)…………………………..…………………………………………

168

Figure 8.2a Sporulation (conidia ml-1) in larvae of R. ferrugineus populations collected





P≤0.05)………………………………………………………………………

169

Figure 8.2b Sporulation (conidia ml-1) in adult of R. ferrugineus populations collected





P≤0.05)……………………………………………………………………….

169

Figure 8.3a R. ferrugineus larvae affected by H. bacteriophora (%±SE) from different

populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y.

Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H.

bacteriophora (300 IJs) applied alone or in combination (means followed

by the same letter within each treatment are not significantly different; HSD

test P≤0.05) ………………………………………………………………….

170

Figure 8.3b R. ferrugineus adult affected by H. bacteriophora (%±SE) from different

populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y.

Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H.

bacteriophora (300 IJs) applied alone or in combination (means followed

by the same letter within each treatment are not significantly different; HSD

test P≤0.05) …………………............................................................... ..........

170

Figure 8.4a Nematode production (IJs ml-1) in larvae of R. ferrugineus affected by H.

bacteriophora from different populations collected from Layyah, D.G.

Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B.

bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone

or in combination (means followed by the same letter within each treatment

are not significantly different; HSD test P≤0.05)……………………………

171

Figure 8.4b Nematode production (IJs ml-1) in adult of R. ferrugineus affected by H.

xi

bacteriophora from different populations collected from Layyah, D.G.

Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B.

bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone

or in combination (means followed by the same letter within each treatment

are not significantly different; HSD test P≤0.05)……………………………

171

Abstract

The red palm weevil red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera:

Curculionidae) is one of the voracious pest among invasive insect pests. Pakistani populations of

R. ferrugineus distributed among different areas have been found genetically diverse. Four

different haplotypes were recorded across Punjab and Khyber Pakhtunkhwa (KPK) provinces.

Haplotype H1, H5, H51 were found from all collection sites while H52 was rare haplotype that

was only present in populations of Layyah and Dera Ghazi Khan only. Study indicated the native

range of R. ferrugineus instead of invaded from other parts of the world, since the weevil was

recorded from Pakistan in 1913 and may have been present before that. The populations of R.

ferrugineus have gained resistance to commonly used chemical insecticides and phosphine due

to the excessive and unwise use of these chemical insecticides. Resistance against seven different

populations of R. ferrugineus was determined from very low to low and moderate to high level

of resistance against commonly used insecticides. Phosphine, cypermethrin and deltamethrin

exhibited high resistance against almost all the populations. To overcome the insecticide

resistance entomopathogens were evaluated against R. ferrugineus. Nineteen different isolates of

Beauveria bassiana s.l. and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales) were

screened which exhibited varying level of susceptibility towards larvae and adult. Three isolates

of B. bassiana (WG-41, WG-42 and WG-43) and two isolates of M. anisopliae (WG-44 and

WG-45) exhibited highest larval and adult mortality after 12 days of application and the same

isolates were tested for their virulence at different exposure intervals against larvae and adult

which caused almost 100% mortality for certain isolates. B. bassiana are capable of colonizing

endophytically in live date palm petioles even after 30 days of inoculation and can significantly

reduce the weevil population when exposed to the endophytically colonized date palm petiole

pieces. Moreover Bacillus thuringiensis var. kurstaki (Bt-k) is also an effective agent that can

cause detrimental effects on R. ferrugineus survival alone and in combination with

endophytically colonized date palm. Both agents exerted influence on developmental parameters

such as larval duration, larval weight, pre-pula duration, pre-pupal weight, pupal duration, pupal

weight, adult longevity (male and female) and adult weight (male and female) etc. Moreover,

diet consumption, frass production and weight gain was affected by the treatments applied.

Entomopathogenic fungi in integration with Heterorhabditis bacteriophora Poinar applied either

simultaneously or in sequential manners exert detrimental effects on growth and development of

R. ferrugineus larvae. Combined application of three agents i.e. B. bassiana, Bt-k and H.

bacteriophora also suppress the larvae and adult population collected from 4 different areas of

Punjab and KPK, Pakistan under laboratory conditions. Hence we can use microbial

entomopathogens against this voracious pest which are safer to human beings and compatible to

environment.

1

CHAPTER 1

Introduction

Date palm is a tropical fruit crop which belongs to the family Arecaceae. It is a main

source of dietary fiber and provides livelihood to a number of peoples in the old and new world.

The tree is named as “tree of life” in the Bible due to its long life (100 year), productivity and

elevated nutritional value (UN, 2003). It is one of the oldest cultivated plants on the earth (Lee,

1963; Riad, 2006). Date fruits have key importance in the Muslim culture and among the few

fruits repeatedly mentioned in the Holy Quran, the tree has been mentioned as a humble tree as it

does not affect the growth and development of any other plant (Goes straight and does not pour

the shade on other plants or inhibit their growth). The date palm cultivation may have been

practiced 7000 year ago (Popenoe, 1924), but the domestication of date palm is assumed to be

started in Mesopotamia by 3000 B.C. (Nixon, 1951). Excavation of godowns from Mohenjo

Daro indicated the presence of date seeds which depict the date palm cultivation in Sindh

province of Pakistan since 5000 year back (Marshal, 1931).

Some school of thought believes that Alexander the Great brought the date palm to the

Indian subcontinent (Nixon, 1951; Pasha et al., 1972). But some scientists believe that the date

palm prevailed in subcontinent before the time of Alexander as Greek army use to eat harvested

date from the gardens of Kech valley (Balochistan) when they travel through the Makran coasts

during 4th century BC (Qasim and Naqvi, 2012). Latter on the mass spread of date seeds came to

existence by the arrival of Mohammed Bin Qasim in Sindh as early as 712 AD, when he came

for the preach of Islam. During camping the Arab soldiers (threw) discarded date seeds at that

site which caused spread of date palm cultivation in Sindh valley (Ahmad and Tahir, 2005;

Dhillon et al., 2005; Jatoi et al., 2010). In Indian subcontinent off-shoots of highly potent verities

of date palm (Dayri, Halawy, Khadrawy, Zahidi and Sayer) were imported from Basra (Iraq)

during the colonial period (1910-1912) by the British Indian Government and were planted in

Muzaffargarh and Multan (Punjab, Pakistan) (Milne, 1918). Pakistan is ranked 6 th in date

production in the world which produced 0.6 million tones dates in 2013 cultivated over 93,088

ha (Al-Khayri et al., 2015), largest presence of the dates in the world. The major importer

countries include India, UK, USA, Canada, Malaysia, Germany, Indonesia and Denmark

(Faostat, 2013). In Pakistan the major cultivars are Begum Jangi (Baluchistan), Aseel (Sindh)

and Dhakki of (Dera Ismail Khan). The major date growing areas in Pakistan are Kech (the

administrative center is Turbat), Panjgur, Sukkur and Khairpur, Jhang, Dera Ismail Khan, Dera

Ghazi Khan, Multan, Muzaffargarh and Bahawalpur.

Date palm is attacked by a numerous insect pests and diseases (Al-Doghairi, 2004).

Among these insects Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier)

(Coleoptera: Curculionidae) caused 10-20% loss in production to different varieties of dates in

Pakistan (Baloach et al., 1992). The pest is cryptic in nature and has been found devastating 29

different palm species belonging to 18 genera and 3 families (Malumphy and Moran, 2009;

Hussain et al., 2013). It is a voracious feeder and most prolific. A single female may give birth to

about five million weevils in four generations, within 14 months (Nirula, 1956) which is a very

high reproduction rate (Rahalkar et al., 1972; Avand-Faghih, 1996; Esteban-Duran et al., 1998;

Cabello, 2006). Male RPW produces aggregation pheromones which attract weevils to damaged

plants (Gunawardena and Bandarage, 1995). Larvae are damaging stages that remain confined

within tree trunk, exploiting the stem vascular system and bore into the trunk (heart of the host)

causing death of the palm (Ju et al., 2011; Hussain et al., 2013). Usually 3-4 generations

comprised of different stages of the insect may be seen inside an infested palm (Rahalkar et al.,

2

1972) but in Egypt 21 generations have been reported in a single year by Salama et al. (2002).

This high rate of multiplication may be attributed to continuous egg laying throughout the year,

with some periods being more intense than others. During her life span a single RPW female can

lay 58-760 eggs (Avand-Faghih 1996; Abraham et al. 2002; Kaakeh, 2005; Faleiro, 2006,

Prabhu and Patil, 2009) which incubate for 1-6 days, before hatching into whitish yellow larvae

(grubs), which live for 25-105 days depending on the weather conditions (Wattanapongsiri,

1966; Avand-Faghih, 1996; Abraham et al., 2002). The neonate larvae chew plant fibers and

penetrate the interior leaving behind the chewed-up frass that has a typical fermented odor. The

completely developed grubs pupate in a cocoon fabricated from chewed fibers and the pupal

period lasts for 11-45 days. The life cycle of the pest may vary from just 45-139 days reported

from Philippines and Spain respectively (Esteban-Duran et al., 1998; Murphy and Briscoe,

1999). Adult weevils can interbreed and live within the same host until they are required to

colonize a new palm. If the plant remains untreated the palm can die within 6-8 months (Kurian

and Mathen, 1971; Avand-Faghih, 1996; Rajamanickam et al., 1995).

By now the beetle is distributed over more than 50% of the date palm cultivated areas

worldwide causing wide-spread damage. The ancient records of the beetle date back to the 1750s

when Rhumph first found this species on sago palm Sagu Campas, in Ceylon in 1750-1755. He

also first described the larvae, cocoon and dorsal and ventral views of the adult as Cossus

sagurios. This name was not valid according to the ICZN article 3, 11 (a), and 86. In 1776,

Sulzer identified and described the weevil from India as Curculios hemipterus Linnaeus, 1758.

The weevil was later described as Curculios ferrugineus by Olivier in 1790 and this name is

currently used. When Herbst erected the genus Rhynchophorus, he pointed out that R.

ferrugineus (Olivier) was commonly mistaken for C. hemipterus Linn. He also pointed out the

difference between two species. In 1797 Thunberg described a male specimen from India as

Cordyle sexmaculatus. It was placed in synonymy with R. ferrugineus by Csiki in 1936.

Chevrolate 1882 described the male and female specimens from Assam as “R. indostanus” and a

male from Ceylon as “R. signaticillis” based on the shapes and number of spots on the pronotum

(Wattanapongsiri, 1966).

As described earlier the beetle is native to the Indian sub-continent that was identified by

Olivier in 1790. It was observed first time from India in 1891, but devastation to coconut palms

was not reported until 1906 (Lefroy, 1906). Until 1917, RPW was considered the only pest of

coconut palm but latter on it was found attacking date palms in Punjab, India (Mohan, 1917;

Buxton, 1920). During the same period (1918) RPW also inflicted harmful effects to date palm

in Mesopotamia, but damage authentication was not confirmed by collecting any insect

specimen. Furthermore, RPW is generally considered to be invasive in Pakistan, although it was

first formerly reported in what are now the Multan, Muzaffargarh and Dera Ghazi Khan Districts

of the Pakistani province of Punjab, and the neighboring Indian state of Punjab, almost a century

ago (Mohan, 1917; Milne, 1918). The native range of RPW is thought to be restricted to

Southeast Asia and Melanesia, stretching: through the countries bordering the Bay of Bengal

from Sri-Lanka to the Malayan peninsula and Singapore; through Thailand, Cambodia and

Vietnam; across the South China Sea to Taiwan and the Philippines; and down through the

Sunda Islands (Java, Sumatra and Borneo) (Wattanapongsiri, 1966). Latter on worldwide

distribution of the pest was recorded from Japan in 1975 (Matsuura, 1993).

Since the mid-1980s, weevils advanced westward rapidly from Southern Asia and

Melanesia (Gomez and Ferry, 1999) and the Kingdom of United Arab Emirates (UAE),

Kingdom of Saudi Arabia (KSA) and Oman in 1985 (El-Ezaby, 1997), south of Spain in 1994

3

(Barranco et al., 1996), Savaran region of Iran in 1996 (Avand-Faghih, 1996), Palestine, Israel

and Jordan 1999 (Kehat, 1999) Sharquiya region of Egypt in 1992 (Cox, 1993) in China the

weevil was detected in 2007 (Li et al., 2009) and recently been reported in Cyprus, Morocco,

Italy, France, Turkey, Greece, Portugal, Aruba and Syria (Zhang et al., 2008). The weevil has

invaded every country of Southern, South Eastern and Western Asia (EPPO 2005, 2008) and

lastly in Australia (Li et al., 2009) and California USA (NAPPO, 2010). It was added to the

EPPO Alert list in 1999, since 2006 has been included on the A2 list of pests recommended for

regulation (no. 339) (Melifronidou-Pantelidou, 2009; Nardi et al., 2011).

This pest has spread to different climatic regions including Mediterranean, Monsoon,

coastal, Arid and Semi-Arid (Avand-Faghih, 1996; Faleiro, 2006; El-Mergawy et al., 2011;

Hussain et al., 2014). According to Rahalkar et al. (1972), the environment does not have a

marked influence on the growth and development of the weevil. However, Ramachandran (1991,

1998) revealed variations in morphology and habit of RPW samples collected from different

parts of India and suggested that fecundity and sex ratio may influence F1 and F2 progeny. DNA

finger prints of three morphologically different forms of RPW collected from Egyptian date

plantations indicated major genetic variations in the three forms (Salama and Saker, 2002).

Agro-climatic conditions of the region, morphology of the date palm and modern farming

systems have provided an environment conducive to the rapid establishment of RPW in the

Middle East (Abraham et al., 1998).

Different control tactics have been employed against RPW within an IPM strategy. The

main component used against RPW is phyto-sanitation, mechanical control and deployment of

chemical insecticides (Nirula, 1956; Abraham, 1971; Butani, 1975; Faleiro, 2006; Ajlan et al.,

2000), use of plant extracts (Nassar and Abdullah, 2001), and pheromone trapping (Hagley,

1965; Hallet et al., 1993; Oehlschlager et al., 1993). Chemical insecticides are efficient in RPW

control but they are short-lived and need to be applied periodically with possible negative

consequences for human health and the induction of resistance in the insect (Abraham et al.,

1998; Ferry and Gomez, 2002; Faleiro, 2006; Llácer et al., 2012a). Moreover, the unwise use of

chemical insecticides has led the resistance against this pest (Abraham et al., 1998);

To combat this problem some control measures should be initiated that are

environmentally friendly and compatible with human health. Bio-control agents are an alternate

method to potentially replace many chemical pesticides now used against RPW (Gauglar and

Kaya, 1990). Entomopathogenic fungi (EPFs), entomopathogenic bacteria and

entomopathogenic nematodes (EPNs) have been found very effective against a vast array of

insect orders. Microorganisms have been successfully used to control a number of insect pests of

economic importance (Francesca et al., 2015). Among them EPFs are an important microbial

control agent and their effectiveness has been studied by a number of scientists (Murphy and

Briscoe, 1999; Faleiro, 2006), particularly Beauveria bassiana (Balsamo) Vuillemin

(Ascomycota: Clavicipitaceae) and, to a lesser extent, Metarhizium anisopliae (Metschnikoff)

Sokorin (Ascomycota: Clavicipitaceae) (Deadman et al., 2001; Ghazavi and Avand-Faghih,

2002). Microbiological treatments with B. bassiana and M. anisopliae offer an alternative and

bio-rational pest management strategy (Inglis et al., 2001) and this is the alternate method where

potential replacement of many chemical pesticides may occur against RPW (Gauglar and Kaya,

1990; Merghem, 2011; Deadman et al., 2001;Gindin et al., 2006; El-Sufty et al., 2007; 2009;

2011;Sewify et al., 2009; Torta et al., 2009; Vitale et al., 2009; Dembilio et al., 2010; Francardi

et al., 2012).

4

EPFs cause natural epizootics in insect pests through contact to the host body following

penetrating, germination and proliferation into the host body and ultimately killing the host

(Zimmermann, 2007). Infection is caused by direct application or by horizontal transmission

from one developmental stage to the other (subsequent developmental stages) (Lacey et al.,

1999; Quesada-Moraga et al., 2004). Similarly, mechanical transmission within populations has

also been recorded for M. anisopliae, B. bassiana and Isaria fumosorosea (Lacey et al., 1999;

Quesada-Moraga et al., 2004, 2008). These peculiar characteristics enable EPFs to combat

concealed insect pests. Same is the case with RPW, whose most stages live into the tree trunk,

enabling the pest to direct contact with the treatments applied except the adult stages which can

be infected on emergence.

Being the main pathogens of Lepidopteran pests EPFs can actively participate in the

control of coleopteran pests as well due to its active mode of infestation on the outer surface of

host cuticle (Hajek and St Leger, 1994; Lacey et al., 1999). Some researchers believe that EPFs

can be effective as bio-control agents against RPW (Lacey et al., 1999; Dembilio et al., 2010;

Francardi et al., 2013; Ricaño et al., 2013). The recent identification of strains of M. anisopliae

and B. bassiana with high virulence against the RPW has increased the possibility of a more

efficient microbiological control of the Curculionid. Entomopathogenic bacteria also play a

significant role in managing insect pest populations which include the members of genus

Bacillus (Salama et al., 2004). A number of species from this genus are successfully deployed

against a variety of insect pests including the member of order Coleoptera; the species are stage

specific. Bacillus thuringiensis (Bt), B. lentimorbus, B. sphaericus and B. popilliae synthesize

insecticidal proteins (Bulla et al., 1975).

B. thuringiensis is a spore-forming gram positive bacterium is the selectively toxic

products of Bt; products that are harmless to mammals and acceptable to environment (Entwistle

et al., 1993). B. thuringiensis produces protein crystal when larvae ingested this crystal protein

through his food, crystal protein dissolved in the alkaline environment of larval midgut. Actual

toxic fragment (protein) is produced when the dissolved crystal protein is proteolytically

processed. This proteoltically processed protein adheres to the intima membrane of midgut

columnar cells. The spores are produced on the epithelial cell membranes by membrane bound

proteins. Finally, as a result of spore formation the cells of the larvae die (Bauer, 1995; Aronson

et al., 1986; Gill et al., 1992). Manachini et al. (2009) evaluated a Bt based commercial product

registered against coleopteran pests for the control of RPW and found the pathogenicity with

small increase in concentrations than the recommended dose for Coleoptera. Experiments

showed that the total number of circulating hemocytes (mainly the plasmatocytes) gradually

decreased after 19 hours when RPW larvae fed with Bt spores. In this experiment for the first

time a very high number of Bt vegetative forms were recorded in the hemolymph of RPW larvae

after exposure to the Bt commercial product.

As far as the mode of action is concerned, the Cry toxins produced after the ingestion of

Bt spores in alkaline media that lyse larval midgut epithelial cells (Bravo et al., 2007). Cell

contents together with other components promote spore germination which leads to the severe

septicemia and ultimately cause the insect death. It has been proposed that during vegetative

growth, Bt release some kind of new insecticidal proteins (Soberón, 2005; Salamitou et al., 2000;

De Maagd et al., 2001; Bravo et al., 2007). The findings that bacteria, as a vegetative form, are

in RPW hemolymph suggests that Bt is able to bypass the various above described steps to reach

the hemolymph and affect the defense system. THC were dramatically reduced, especially the

plasmatocytes. Many parasites must avoid hemocyte-mediated immune responses to growth in

5

host larvae, and many species achieve this by suppressing one or more components of the host

immune defense system, e.g. alteration of THC, inhibition of hemocyte spreading, apoptosis in

circulating hemocytes (Adamo, 2005; Eleftherianos et al., 2008; Ericsson et al., 2009). The

interaction between entomopathogenic bacteria and hemocytes is little studied in insects and the

available literature is mainly on Lepidoptera. However similar results to our findings have been

found in insect response to other entomopathogenic bacteria, for example Bt-k in Trichoplusia ni

(Ericsson et al., 2009) and Photorhabdus in fifth-stage larvae of Manduca sexta (Eleftherianos et

al., 2008). Differences in antibacterial responses have been attributed to bacterial species and

virulence levels (Dettloff et al., 2001; Giannoulis et al., 2007), however some quite important

gaps in understanding general mode of action of Bt still exist (Then, 2009). Indeed, there are

several contradictions among the different models (Then, 2009). Thus, question of how bacteria

act in RPW larvae is still open.

EPNs are obligate parasites in the families Steinernematidae and Heterorhabditidae. They

kill insects with the aid of mutualistic bacteria, which are carried in their intestine (Xenorhabdus

spp. and Photorhabdus spp. are associated with Steinernema spp. and Heterorhabditis spp.,

respectively) (Poinar, 1990). The nematodes complete 2-3 generations within the host, after

which free-living Infective Juveniles (IJs) emerge to seek new hosts (Poinar, 1990). The

pathogenicity of EPNs to Helicoverpa sp. has been demonstrated previously (Bong, 1986).

Furthermore, they have been found effective against a variety of insect pests including foliage

feeders and they have been effective mainly against soil-inhabiting pests (Kaya, 1990). Several

formulations have been developed to improve the activity of nematodes on plant and in stored

products. In coleopteran pests larvae of several weevil species (Coleoptera: Curculionidae) such

as the black vine weevil, Otiorhynchus sulcatus (F.), and the Diaprepes root weevil, Diaprepes

abbreviatus (L.) (Shapiro-Ilan et al., 2002) are susceptible to EPNs. One approach to controlling

H. armigera with EPNs would be to target the larvae when they drop to the ground or after

burrowing into the soil for pupate.

Currently efforts are focused on developing integrated control strategies against RPW by

combining more than one control agent e.g. integrated use of EPFs, EPNs and entomopathogenic

bacteria. Several researchers have demonstrated successful control of multivoltine coleopterans

by combining microbial agents. In view of the extent of damage to the date palms attributed to

RPW, it was considered worthwhile to exploit bio-rational approaches. The present study was

carried out for evaluating the efficacy of different microbial control agents with the aim to meet

the following objectives;

To check the genetic variation among populations of Red Palm Weevil Rhynchophorus

ferrugineus (Olivier) (Coleoptera: Curculionidae) from the Punjab and Khyber

Pakhtunkhwa provinces of Pakistan

To check the resistance to commonly used insecticides and phosphine (PH3) against

Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) in Punjab and Khyber

Pakhtunkhwa, Pakistan

To check the Insecticidal potential of Beauveria bassiana and Metarhizium anisopliae

isolates against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

6

To check the combined effectiveness of endophytically colonized Beauveria bassiana

and Bacillus thuringiensis against Rhynchophorus ferrugineus (Olivier) (Coleoptera:

Curculionidae)

To check the integrated effect of entomopathogenic fungi and entomopathogenic

nematode against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

To evaluate the combined toxicity of Beauveria bassiana, Bacillus thuringiensis and

Heterorhabditis bacteriophora against red palm weevil Rhynchophorus ferrugineus

(Olivier) (Coleoptera: Curculionidae)

7

CHAPTER 2

2.1 Invasive Red Palm Weevil (RPW)

Some invasive insect pests are of great importance due to their habit of severely

damaging the agricultural products, and imposing severe threats to ecology and causing serious

economic losses (Kenis et al., 2009; Simberloff et al., 2013). Resultantly, these annoying pests

cause direct losses of worth thousand million dollars annually and money involved in

management efforts to reduce populations below economic thresh hold level (Pimentel et al.,

2005; Kovacs et al., 2010; Van Driesche et al., 2010; Simberloff et al., 2013). RPW is an

important invasive pest that has invaded more than 50% of the date palm growing areas of the

world. This is attributed to a higher fecundity than most species (Faleiro, 2006), capability to live

and interbreed in the same tree even for several years (Avand-Faghih, 1996; Rajamanickam et

al., 1995), adult flight capacity (Wattanapongsiri, 1966) and pest tolerance to a wide range of

climatic conditions due to its protected habit within palm trees.

2.2 Taxonomic position

A key to revision of this species and related genera was previously provided by

Wattanapongsiri (1966). RPW was classified under order Coleoptera, the family Curculionidae

and the subfamily Rhynchophorinae (Wattanapongsiri, 1966; EPPO, 2007). Synonymously it is

also called Asian Palm Weevil, Indian Palm Weevil or Pakistani Weevil. This genus has 10 other

species, three of them identified from New World, two African and five tropical Asian countries.

Among these R. bilineatus, R. quadrangulus, R. palmarum, R. bilineatus, R. lobatus, R.

distinctus R. ritcheri, R. vulneratus are severe pest of palms (Booth et al., 1990; Hallet et al.,

2004).

2.3 Classification

Kingdom: Animalia (Animals)

Phylum: Arthropoda (Arthropods)

Subphylum: Hexapoda (Hexapods)

Class: Insecta (Insects)

Order: Coleoptera (Beetles)

Suborder: Polyphaga (Water, Rove, Scarab, Leaf and Snout Beetles)

Superfamily: Curculionoidea (Snout and Bark Beetles)

Family: Curculionoidea (Snout and Bark Beetles)

Subfamily: Dryophthorinae

Tribe: Rhynchophorini

Genus: Rhynchophorus

Species: ferrugineus (Red Palm Weevil)

2.4 Spatial Distribution

The aboriginal home of RPW is considered to be the Southern Asia and Melanesia, it is

cryptic in nature and has been reported to attack more than 29 different palm species belonging

to 18 genera and 3 families (Malumphy and Moran, 2009; Hussain et al., 2013), including date

palms and coconut palms, as well as the Mediterranean fan palms, the native Cretan date palms

(Kontodimas et al., 2006; Dembilio et al., 2011) and Canary Islands date palms (Dembilio et al.,

2009). Currently, this pest has spread to many areas of the world; its range now includes much of

8

Asia, regions of Oceania, Southern Europe, Middle-East, North Africa, the Caribbean, and in

October 2010, five specimens belonging to the genus Rhynchophorus sp., were found in southern

California (EPPO, 2008; 2009; 2011).

2.5 Control Measures

Different control practices have been deployed to combat RPW among date palm

growing areas of the world. Treatments revolve around the use of conventional chemical

insecticides, sterile insect techniques and use of semio-chemicals (Paoli et al., 2014) and bio-

control agents (Wattanapongsiri, 1966; Murphy and Briscoe, 1999; Faleiro, 2006a, b).

Integration of RPW associated microbial control agents with other control practices such as bio-

control agents with chemical insecticides and attract-and-kill techniques.

2.5.1 Microbial control

Microbial pest control relies on use of microbes such as EPFs, EPNs, entomopathogenic

bacteria and viruses. Very few researchers have systematically studied the effect of

entomopathogens on RPW (Murphy and Briscoe, 1999; Faleiro, 2006). Conversely,

entomopathogenic microorganisms, in particular mitosporic ascomycetes, have been reported to

naturally regulate RPW populations (Dembilio et al., 2010b). The deployment of microbial

control agents in pest control is an important step towards mitigating reliance on conventional

chemical insecticides. Microorganism exhibit high degrees of host specificity that accounts for

their distinguish ability to search their host. The use of entomopathogenic microbes has a number

of advantages such as safer to environment and non-target organisms, cheaper and self-

perpetuation. Environment friendly control practices against RPW are getting serious attention in

many parts of the world. Concentrations are focused on use of entomopathogens such as EPFs,

EPNs, entomopathogenic bacteria and their integration with the chemical insecticides and plant

extracts.

2.5.2 History of microbial control

Insects and microorganisms have ancient relationships well described by insects

conserved in amber 15 to 20 million years ago, as the collection of several insect cadavers

dressed with entomopathogens like neucleopolyhedrovirus (NPV), EPNs, trypanosomes is

reported (Poinar and Poinar, 2005). It has been an old profession; however, its roots can be

traced back to the time of Aristotle (2700 BC), who observed the diseased silk worm with

whitish growth on the dead larvae of silk worm during 335 BC. It was not until the work of

Agostino Bassi (1773-1856) an Italian lawyer and scientist reported the fungus, B. bassiana on

the larvae as a whitish sooty growth. This led to the germ theory of disease and named

“Calcinaccio” disease because the dead larvae exhibit whitish calcium powder like coverings

(Steinhaus, 1956, 1975).

Agostino Bassi observed that the causal agent of the disease as “vegetable parasite” a

fungus now called B. bassiana that may be transferred through inoculation, contact or by the

ingestion of the leaves by the caterpillar. This was the first research of Bassi which confirmed

that microorganisms could cause disease and also it was the important contribution towards

disproving the idea of spontaneous generation. Calcinaccio disease was found plaguing the silk

industry first in Italy (1805) and then in France (1841). Bassi conducted scientific studies on

Calcinaccio disease in 1807. After long and comprehensive observations, in 1835 Bassi

9

confirmed that this disease causing entity is a living organism which produced whitish growth on

the dead larvae.

He was honored for rescuing the precious and economically important silk industry by

suggesting separation of the rows of caterpillars feeding on the mulberry leaves, disinfection

process, destroying dead cadavers and keeping the rearing room clean and infection free. His

findings were translated and distributed throughout the Europe and greatly helped Louis Pasteur

(1822-1895) to study the cause and potential cure of the disease in Europe (Porter, 1973). In the

same year, a famous Italian naturalist Giuseppe Gabriel Balsamo-Crivelli studied and named the

fungus, Botrytis bassiana in the honor of Bassi (Steinhaus, 1949; Müller-Kögler, 1965; Rehner,

2005). The species B. bassiana came into existence when in 1911 Beauverie studied the fungus

again and Vuillemin created the new genus Beauveria in honor of Beauverie in 1912, since then

the species B. bassiana became the type.

In 1865, French silk industry was badly devastated and Louis Pasteur was asked to

identify the disease. He was not reluctant to accept the offer, although he was not fully aware of

silk worms, he was persuaded by his teacher and friend Senator Jean-Baptist Dumas to move and

consult the famous entomologist Jean Henri Fabre (1823-1915) in Alés Village in the south of

France. After several years he came to the conclusion that two silk worm diseases "pébrine" and

"flacherie" (thought to be caused by bacterium) are responsible for the decline of silk industry.

He proposed that pébrine is characterized by tiny black spots on the surface of dead larvae of silk

worm caused by the microorganism Nosema bombycis, previously described by Nägeli (1857).

For the potential elimination of the disease, he proposed that careful handling, segregating of

healthy and diseased larvae, and well maintained sanitation conditions may be helpful in disease

prevention (Debré, 1998).

During the study it is found that disease can be transmitted by contaminated food, contact

with the infected caterpillar and even from mother to the offspring. This is the first study

demonstrating the vertical transmission of the disease (Pasteur, 1874). He published his findings

in two series to make people aware of silk worm disease and its prevention (Pasteur, 1874). This

work laid the foundation for advances in sericulture in Japan dealing with the molecular and

biochemical biology of the silk worm. The scientists like, Agostino Bassi, Louis Pasteur and Elie

Metchnikoff, the19th century pioneers also proposed that these micro-organisms can be a good

solution for controlling economically important insect pests (Steinhaus, 1956, 1975). The first

half and end of the 19th century was the period of most development in invertebrate pathology, it

was not until the discovery of B. thuringiensis (Bt) Berliner, that practical and massive use of

entomopathogens started (Lacey and Goettel, 1995).

In 1879, Metchnikoff discovered the diseased larvae of wheat cockchafer and later on

Cleonus punctiventris near Odessa (Ukraine). He named this fungus the green muscardine

fungus. The genus Metarhizium was first established by Sorokin (1883). For this fungus, he first

proposed the name Entomophthora anisopliae, and later renamed as Isaria destructor. The

history of the description, discovery the scientific research and on the use of fungus in biological

control is described in detail by Steinhaus (1949) and Müller-Kögler (1965). In the start of 20th

century B. thuringiensis was recovered first time from infected silk worm larvae by a Japanese

bacteriologist (Ishiwata, 1901) and subsequently in 1911 German biologist Berliner re-

discovered the disease. He isolated the bacterium from infected larvae of Mediterranean flour

moth (Berliner, 1915) so named it B. thuringiensis. Because of high and knock down mortality

effects with small amount of B. thuringiensis preparations, the agronomist get aware about the

insecticidal properties of this bacterium. The first B. thuringiensis based commercial formulation

10

“Sporéine” was developed in France in 1938, but the 1st well documented record of commercial

procedure for producing Bt-based product dates from 1959 by the “Bactospéine” under the 1st

French patent as a bio-pesticide formulation. Since after, a vast array of microorganisms like

fungi, bacteria, viruses and protozoans has been identified as potential biocontrol agent against

insect pests (Riba and Silvy, 1989). So far, even though more than 100 species of

entomopathogenic bacteria have been identified, only a few Bacillus species have met with

commercial success, B. thuringiensis in particular (Starnes et al., 1993).

Today a vast array of entomopathogens are deployed against insect pests of agriculture

importance such as fruits, cereals, ornamentals, stored commodities, insect pests of households

and insect vector of medical and veterinary importance (Tanada and Kaya,1993; Lacey and

Kaya, 2007). Microbial control agents used against insect pests includes EPFs, EPNs, viruses,

protozoa and bacteria. Keeping in mind the harmful effects of chemical insecticides and their

impact on the environment and human health, insecticides based on entomopathogens exert only

a small fraction of hazards on environment and human health as compared to the conventional

insecticides. The share of bio-pesticide in crop protection market is about 600 million US$ which

accounts for only 2% of the total pesticides, with about 90% of all bio-pesticide sales involving

products based on B. thuringiensis.

The comparison of microbial pesticides with chemical pesticides is usually exclusively

cost effectiveness. These microbial insecticides particularly offer unique advantages when there

are environmental and human safety concerns along with the increasing need of enriched

biodiversity in an ecosystem and increased activity of natural enemies (Shahid et al., 2012).

Furthermore, ease of application, production on artificial medium and long term storage are

further distinct features of these bio-insecticides over other insect control tactics.

2.5.3 Entomopathogenic Fungi (EPFs)

2.5.3.1 History

The history and research on mycopathogens invading insect pests is ancient. Before the

invention of microscopes fungi could be seen with naked eye and this observation helped to

establish invertebrate pathology as a modern study. Fungi are categorized in a number of taxa

that exhibit greater diversity in properties, requirements and found in all arthropod habitats. As a

result, great attention was diverted to possible use of fungi as microbial control of insect pests.

The fungi are heterotrophic, eukaryotic, absorptive individuals which may develop in different

patterns like diffuse, branched, or tubular body that can reproduce sexually as well as asexually

(Kendrick, 2000). The primitive studies regarding entomopathogenic fungi were conducted

during start of 18th century with an aim to develop control strategies for managing muscardine

diseases of silk worm (Steinhaus, 1975). Bassi (1835 as cited by Steinhaus, 1975) proposed the

germ theory using silkworm and invading fungus, later this was named Beauveria bassiana in

the honor of Bassi. His studies on silkworm disease assisted him to introduce the fungal

biocontrol agents for the control of insect vector that elicit disease in human beings.

The silkworm diseases provided gross root foundations for the control of insect pests by

employing entomopathogens. Nevertheless, the major efforts were attempted in deploying EPFs

for the control of insect pests carried out during 1950’s when chemical insecticides were

invented. There are many fungal based products commercially available worldwide now-a-days

(Shah and Goettel, 1999; Copping, 2001).

EPFs have a long primordial historic recognition; their illustrated descriptions can be

seen centuries back, infection of B. bassiana and Cordyceps sp. to silk worm described in ancient

11

Japanese paintings infections of insects date from the 19th century (Samson et al., 1988). As a

vocation, invertebrate pathology is an organized discipline. Historic stories can be drawn from

the solution of silk worm and honey bee diseases prevention from entomopathogens (Steinhaus,

1956, 1975). Very first reports of managing insect pests in insect pathology with

entomopathogenic fungi were proposed by the legend pioneers like Louis Pasteur, Elie

Metchnikoff and Agostino Bassi (Steinhaus, 1975). Currently numerous entomopathogens are

deploying for managing insect pests in lawn and turf, orchards, glasshouse, ornamentals, row

crops, forestry, range lands, stored products, pest and insect vectors of medical and veterinary

importance (Tanada and Kaya, 1993; Lacey and Kaya, 2007).

2.5.3.2 Geographical distribution and occurrence

Soil is vital source for a number of EPFs especially species belonging to Ascomycota

thus they serve to regulate the insect populations in soil (Keller and Zimmermann, 1989; Hajek,

1997) as most arthropods spent some of their life stages into the soil. The knowledge about the

indigenous isolates of EPFs, their diversity, distribution and composition is key factor to

conserve these indigenous fungal species for the natural control of the insect pest populations

within the agro-ecosystem. The detailed studies have been conducted on the 14 occurrences? of

soil dwelling entomopathogenic fungi in different countries, the data from around the world

suggest them to be ubiquitous inhabitants of the soil (Chandler et al., 1997). The effect of

different factors like climatic conditions, geographical distribution, habitat type, and soil

properties, soil pH (Foth, 1984; Ali-Shtayeh et al., 2002; Padmavathi et al., 2003), soil organic

matter (Milner, 1989; Mietkiewski et al., 1997), soil type (Storey and Gardner, 1988; Rath et al.,

1992; Inglis et al., 2001; Derakhshan, 2008), soil moisture contents (Ali-Shtayeh et al., 2002) on

fungal incidence and dispersal has been studied several times (Vänninen et al., 1989; Chandler et

al., 1997; Meyling and Eilenberg, 2006; Zimmerman, 2007).

The worldwide distribution of EPFs in insects from different habitats is also reported by

many authors (MacLeod, 1954; Evans, 1982; Wraight et al., 1993; Aung et al., 2008; Thakur and

Sandhu, 2010). The naturally occurring EPFs can be obtained by collecting insects from the field

then incubating them under laboratory conditions and checking for outgrowth of fungi (Meyling

and Eilenberg, 2007). EPFs from eight genera (Entomphthora, Batkoa, Conidiobolus, Pandora,

Erynia, Neozygites, Zoospora and Tarichium) have been isolated from aphids. The most

common entomopathogenic fungi, B. bassiana, P. fumosoroseus, and P. farinosus were recently

isolated from some new insect hosts such as beetles of Agrilus species and hairy caterpillar of

Lymantria species from Central India (Thakur and Sandhu, 2010).

2.5.3.3 Classification

Among different fungal divisions, EPFs belongs to Ascomycota, Zygomycota and

Deuteromycota (Samson et al., 1988), Oomycota and Chytridiomycota (classifies within fungi

previously). Many of the genera of EPFs currently under research belong either to the class

Hyphomycetes in the Deuteromycota or to the class Entomophthorales in the Zygomycota.

2.5.3.4 Host range

Fungal infections to the most insect orders with all life stages have been observed, while

infection to the immatures of holometabolous insects have been reported more commonly

(Tanada and Kaya, 1993). The host range may differ significantly among different species of

EPFs and even among different strains of the same single species. For obligate pathogens,

12

specifically restricted to a narrow host range and complicated life cycles associated to their insect

host like Strongwellsea castrans (Phycomycetes: Entomophthoraceae), restricted to flies like

anthomyiid (Eilenberg and Michelsen, 1999) and Entomophthorales, Massospora sp. are

restricted to a single genus belonging to cicadas (Soper, 1974). In contrast, Deuteromycetes,

particularly B. bassiana, have wide host range including numerous genera of insects (McCoy et

al., 1988). It must be kept under consideration that description of host range to some extent

mainly relies on laboratory studies which do not reflect the true picture in nature. Some factors

like insect host, fungal biology and ecology may be responsible for reducing infection in insect

host. It is important to mention that fungi are capable of infecting several other arthropods,

insects and the species which are not pests of cultivated crops (Gibellula spp. predator of spiders

and, Erynia and Cordyceps sp. infects ants).

2.5.3.5 Mode of infection

The fungal infection of insect hosts is a complex process, involving chemical and

physical procedures starting from spore attachment to host death. Following steps are undertaken

during infection process: (1) spore attachment to the host cuticle, (2) germination of fungal

spore, (3) diffusion into the host cuticle, (4) overcoming the immune defense mechanism, (5)

formation and proliferation of hyphal bodies into the hemocoel, (6) saprophytic outgrowth from

the dead host, production and dissemination of new conidia. For the successful attachment,

mainly hydrophobicity of the spore and cuticular surface play significant role. Furthermore, the

germination and infection is influenced by a number of factors e.g., humidity, optimal

temperature, susceptible host stage and cuticular lipids, such as aldehydes, ketones, wax, short-

chain fatty acids, alcohols and esters which may exhibit antimicrobial activity. Generally, fungal

spores breach through the non-sclerotised parts of the cuticle such as joints, between segments or

the mouthparts. The conidial germination starts after 10 h of attachment and may complete by 20

h at 20-25 oC. Before infection process, germ tube produce appressorium or penetration pegs

which is accompanied by mechanical and chemical processes by the production of several

enzymes (Ortiz-Urquiza and Keyhani, 2013).

2.5.3.6 Enzymes and toxins of EPFs

Along with different degrading enzymes (such as lipase, protease, chitinase) which

account for the virulence of different entomopathogenic fungi (Joshi et al., 1995; Fan et al.,

2007), certain secondary metabolites of these fungi also possess insecticidal activities and

contribute to the pathogenesis of the fungal strains (Mollier et al., 1994). Some metabolites may

also act as the defensive tool by protecting the fungi from certain hostile factors such as

competitive micro-organism (Dowd, 1992; Bandani et al., 2000). The type of toxins produced

may also be helpful in defining the mode of action of the entomopathogenic fungi (Vey et al.,

1993). The toxins produced by different entomopathogenic fungi, their role in fungal efficacy

and safety concerns about the utilization of these compounds have been discussed by several

researchers (Roberts, 1981; Strasser et al., 2000; Vey et al., 2001).

2.5.3.7 Chitinases

The chitin is a major constituent of the insect cuticle, therefore, endo and exo chitanses

are important enzymes for the breakdown of N-acetylglucosamine polymer of insect cuticle into

monomers and a key factor determining the fungal virulence (Khachatourians, 1991).

13

Endochitinases, N-acetyl-β-D-glucosaminidases and chitinolytic enzymes from M. anisopliae

and M. flavovirid and B. bassiana were presented in broth culture nourished with insect cuticles.

2.5.3.8 Proteases and peptidases

Chitin and protein are the main constituents of insect cuticle; hence proteases and

peptidases of EPFs is considered key component in degradation of insect cuticle, saprophytic

growth, initiation of prophenol oxidase in insect hemolymph, furthermore they are also

responsible for virulence in EPFs. Chymotrypsin (CHY1) of 374 amino acids, with pI of 5.07

and MW38279 were investigated from M. anisopliae by Screen and St Leger (2000). Some

genes of overlapping response with a unique expiration pattern were observed when encountered

with the cuticle of Blaberus giganteus, Popilla japonica and Lymantria dispar and using cDNA

counted gene expression responses to the cuticles of number of host insects and constructed

microarrays from expressed sequence tags, clone of 837 genes (Freimoser et al., 2005).

2.5.3.9 Lipases

The epicuticle of the insects is chiefly composed of non-polar lipids which play an

important role in chemical signaling between insect host and EPFs (Blomquist and Vogt, 2003),

and keeps cuticular outer surface dry which aids to avoid the penetration of chemicals and

insecticides (Blomquist et al., 1987; Juárez, 1994). They are chemically stable with high

molecular mass, mainly due to the presence of specific physicochemical characteristics, like

number of carbons, length of the chain and the kind and position of double bond and the

functional groups. The long chain HC, free fatty acids, fatty alcohols and wax esters are ample

components of the insect epicuticle. It also contains fats, waxy layers and lipoproteins which act

as a barrier to the action of lipoxygenases and lipases of eontomopathogenic fungus. Among

these compounds some have anti-fungal activities (Khachatourians, 1996) while some other

possess saturated fatty acids which can inhibit the fungal growth.

2.5.3.10 Toxins

The biochemical properties and structure of some major fungal metabolites have been

investigated in detail (Vey et al., 2001), but very few studies have been conducted regarding the

metabolite production under field conditions (Bandani et al., 2000; Strasser et al., 2000). One

major problem to fungal toxins is that one type of fungi can produce variety of bioactive

metabolites and risk assessment to these entire compounds would be enormous. Furthermore,

fate of their toxins is little known in the environment, which would be the key question for their

registration.

2.5.3.11 Destruxins

Destruxins are moderately dissimilar compounds which exist in the form of isomers.

Basically destruxins contain 5 amino acids and α-hydroxy acid which may be found in many

different forms. Till now 28 different but structurally similar destruxins have been isolated from

different EPFs and most of these are discovered from M. anisopliae isolates (Vey et al., 2001).

Insects exhibit varying susceptibility levels to destruxins and Lepidopterans have been reported

as the most susceptible amongst the all studied insect orders (Samuels et al., 1988; Kershaw et

al., 1999). The toxicosis symptoms also vary among insect pests the most peculiar symptom are

an immediate tetanus; which at low concentrations develops for up to three minutes period,

while, brief or no paralysis is depicted at high dose rates (Abalis, 1981; Samuels et al., 1988).

14

2.5.3.12 Oosporein

Oosporein is produced mainly from the soil inhibiting fungi like Beauveria spp. which

contain red colored di-benzoquinone (Eyal et al., 1994). It reacts with amino acids and proteins

through redox reaction by altering the SH-groups and results malfunctioning in enzymes

(Wilson, 1971). Like bassianin and tenellin, oosporein also inhibit the activity of erythrocyte

membrane ATPase which is directly proportional to the dose rate of oosporein. Up to 50%

activity can be ceased at 200g/ml. All these pigments greatly influenced Ca2+-ATPases compared

to the activity of Na+/K+-ATPase. Antibiotic effect of oosporein against gram-positive bacteria

has also been observed with no or little effect on gram negative bacteria (Taniguchi et al., 1984;

Wainwright et al., 1986).

2.5.3.13 Beauvericin and beauveriolide

Beauvericin is also an important toxin isolated from Beauveria, Paecilomyces sp., the

plant pathogenic fungi Polyporus fumosoroseus and Fusarium sp. (Gupta et al., 1991; Plattner

and Nelson, 1994). Gupta et al. (1995) described two different forms of these toxicants

Beauvericin A and B forms K+ and Na+ complexes, which increase the membranes permeability

(Ovchinnikov et al., 1971). It also exhibits antibiotic activity against a number of bacteria like,

Mycobacterium phlei, Escherichia coli, Sarcinea lutea, Bacillus subtilis, Staphylococcus aureus

and Streptococcus faecalis (Ovchinnikov et al., 1971).

2.5.3.14 Bassianolide

Another toxin cyclo-octadepsipeptide also called bassianolide is secreted by B. bassiana

(Suzuki et al., 1977). Bassianolide is also an ionophore which exhibits different reactions with

different hosts (Kanaoka et al., 1978). Very little knowledge about the toxic nature of

bassianolide against plants and animals, the synergistic interaction with the structurally

associated myco-toxin moniliformin may be possible.

2.5.3.15 Beauveriolide

Beauveriolide are isolated from Beauveria spp. which is structurally similar to

bassianolide and beauvericin (Namatame et al., 1999). The toxic effect of beauveriolid towards

plants and animals is still unknown except beauveriolide I (Mochizuki et al., 1993). Overall

these cyclodepsipeptides may still have an unreported health hazard effects is common. Except

the above mentioned metabolites these B. bassiana also produce bassianin, tenellin and two non-

peptide toxins isolated from Beauveria spp. which aid in inhibiting the erythrocyte membrane

ATPases (Jeffs and Khachatourians, 1997).

2.5.3.16 Host range

EPFs are the diverse group of insect pathogens that contains a large number of genera

and species, the exact figure is unidentified but according to some estimation there are

approximately 700 species in 100 genera of entomopathogenic fungi, however, Onofre et al.

(2001) described it as 90 genera. These insect pathogens have broader host range and have been

found naturally occurring in various populations of insect pests. The more extensive work

regarding the natural host identification in order to recognize the biological activity of the

entomopathogenic fungi is done on B. bassiana and M. anisopliae. The host range for B.

bassiana is stated as 700 species including beneficial insects too (Goettel et al., 1990).

15

2.5.3.17 Effect of abiotic factors

2.5.3.17.1 Temperature

Among different abiotic factors affecting the fungal propagation and survival (Roberts

and Campbell, 1977; Fuxa, 1995); temperature is very important in determining the germination,

growth rate and viability of the fungal conidia not only in the host but in the environment as

well. Furthermore; the efficacy of fungal biopesticides has been highly influenced by the

environmental temperatures (Inglis et al., 1996; Klass et al., 2007). The awareness about the

fungal growth in association with the prevailing temperature is the preliminary step in selection

of any fungal strain (Fargues et al., 1992). Ferron (1978) described that optimum values for

different entomopathogenic fungi are between 20-30 ºC, which has been verified also by certain

other researchers as well such as Ekesi et al. (1999), Dimbi et al. (2004) and Kiewnick (2006).

The ability to tolerate different temperature profiles not only varies between the strains but the

thermal tolerance between the isolates is also significant (Parker et al., 2003; Dimbi et al., 2004).

Bugeme et al. (2008) observed the variable responses of B. bassiana and M. anisopliae isolates

and preeminent germination was seen at 25 and 30 ºC whereas 30 ºC was best for the radial

growth of the colony. The strains of M. anisopliae had better adoptability to tolerate high

temperatures for germination (Inglis et al., 1997; Milner, 1997).

The temperature is also considered as a factor which influences the virulence of different

fungal isolates (Tefera and Pringle, 2003). As far as the stored grain insect pest management is

concerned, mostly the virulence of different entomopathogenic fungi is evaluated under the

mutual effect of temperature and relative humidity (Sheeba et al., 2001; Batta, 2004;

Athanassiou et al., 2008). The geographical location of the isolates also account for their thermal

tolerance as studied by Vidal et al. (1997) who found that isolates of I. fumosorosea (P.

fumosoroseus) collected from Europe depicted growth at temperatures between 8-30 ºC

(optimum growth rates 20-25 ºC), the isolates from southern United States and West Asia

tolerated 8-35 ºC (optimum growth rates 25-28 ºC) whereas Indian isolates showed optimum

growth at relatively high temperature range i.e. 32-35 ºC.

2.5.3.17.2 Relative humidity

Humidity is also a key abiotic factor which greatly influences the efficacy and viability of

entomopathogenic fungi. The role of relative humidity (r.h.) is more significant in spore

germination and post mortal sporulation of the entomopathogens (Inglis et al., 2001). The

viability of the conidia over a period of time is the important parameter which is mainly

influenced by the interaction of relative humidity with different temperature levels

(Zimmermann, 2007). The prolonged stability of fungal conidia is reported mostly under cool

and dry conditions (Hedgecock et al., 1995; Hong et al., 1997). High relative humidity is the

prerequisite for the mycosis on dead cadavers (Fernandes et al., 1989) as optimum sporulation on

dead locusts (Schistocera gregaria) at >96% r.h. was observed by Arthurs and Thomas (2001).

After the treatment of different stored grain beetle’s eggs with B. bassiana among different

relative humidity levels, 92% r.h. reduced eggs hatchability up to 83 and 87% in R. dominica and

T. castaneum, respectively (Lord, 2009). Some studies have also revealed that entomopathogenic

fungi can germinate and infect the host even at low relative humidity levels (Inglis et al., 2001).

2.5.3.18 Effect of EPFs on non-target organisms

EPFs are developed as commercial formulations (biopesticides) to combat the insect

pests of agriculture and veterinary importance (Goettel et al., 1990; Kooyman et al., 1997;

16

Thungrabeab and Tongma, 2007; Reddy et al., 2008; Mahmoud, 2009). Different laboratory

trials had also been conducted to test the biological activity of formulated conidia against various

insect pests both under laboratory and field conditions (Ibrahim et al., 1999; Inglis et al., 2002;

Batta, 2003; Ugine et al., 2005). The effect on non-targeted organisms is one of the basic

principles for the evaluation of biopesticides. The broad spectrum activity of fungal

entomopathogens (Zimmermann, 2007) is the key point for their successful adaptation as

biological control agents but at the same time it might have some effects on non-target or

beneficial insects. Peveling and Demba (1997) recommended the mycopesticide as economically

sound and ecologically safe control measure of desert locust in date palm as M. flavoviride was

safe to the natural enemy (Pharoscymnus anchorago F.) of scale insects of date palm. Cottrell

and Shapri-Ilan (2003) also found that exotic Asian lady beetle (Harmonia axyridis) was less

susceptible to GHA strain of B. bassiana. On the other hand the negative effect of EPFs has also

been reported by several authors. The pathogenicity of eight EPFs isolated from natural

populations of Coccinellids was revealed against Coccinella septempunctata (Kubilay et al.,

2008). Some previous studies indicated the pathogenicity of EPFs to C. septempunctata (Manjula

and Padmavathamma, 1996; Haseeb and Murad, 1997; Cagan and Uhlik, 1999) and other

coccinellids as well (James and Lighthart, 1994; Pell and Vandenberg, 2002; Ashouri et al.,

2003).

2.5.3.19 Integration of EPF with other control measures

The publication “Silent Spring” by Rachel Carson in 1962, not only realized the side

effects of chemical insecticides to human health (Purwar and Sachan, 2006) and environment but

also lead a way to search for eco-friendly control strategies for the insect pest management. The

continuous search for biologically safe and ecologically sound control measures identified some

natural agents as potential candidates for insect control. The microbial control of insect pests

using viruses, bacteria, EPNs and EPFs (Bhattacharya et al., 2003; Sabbour and Sahab, 2005)

has become an important element of integrated pest management (Inglis et al., 2001). Among

these microbial agents, EPFs are known as promising alternative to conventional insecticides

which can effectively be used against a large number of pest species (McCoy et al., 1988;

Zimmermann, 1993; Kaur and Padmaja, 2008). The potential role of EPFs as microbial control

agents has been reviewed by various researchers (Evan, 1989; Ferron et al., 1991; Tanada and

Kaya, 1993; Boucias and Pendland, 1998; Inglis et al., 2001). One of the best strategies is the

combining EPFs with low lethal doses of chemical insecticides (Anderson et al., 1989). As

pesticides may have a variety of effects on EPFs (Alves and Leucona, 1998), therefore, care

should be taken while selecting the chemical substance that it should enhance the efficacy

without any adverse impact on the fungal strain (Inglis et al., 2001).

The first report of carbofuran being effectively combined with B. bassiana against

Ostrinia nubilalis (European corn borer) came from Lewis et al. (1996). In another study from

two carbamate insecticides (carbosulfan and carbofuran), carbosulfan inhibited the growth of B.

bassiana and B. brongniartii but overall effect revealed the possibility of using these insecticides

in IPM of Melolontha melolontha L. (Bednarek et al., 2004). Among organophosphates,

chlopyriphos 20 EC was less toxic while triazophos 40 EC and profenophos 50 EC were

moderately toxic to B. bassiana (Amutha et al., 2010). In addition to the chemical stressors

various EPFs have also been evaluated in combination with botanicals (phytoproducts); the most

prominent of them is neem and neem based insecticides. Akbar et al. (2005) evaluated the

effectiveness of B. bassiana for T. castaneum in integrated manner with plant essential oils and

17

organosilicone carriers. Azadirachtin was combined with P. fumosoroseus against Bemisia

argentifolii (James, 2003). The interaction and compatibility of different insect growth regulators

(IGRs) and herbicides with EPFs is in vague (Inglis et al., 2001). The fungus Lecanicillium

muscarium when simultaneously applied with buprofezin a higher mortality of B. tabaci 2nd

instar larvae was seen (Cuthbertson et al., 2010).

Triflumuron, a benzoylphenyl urea (BPU) which is a chitin synthesis inhibiter acted as

“general stressor” and made the Lepidopteran larvae more susceptible to fungal infection by M.

anisopline (Hassan and Charnley, 1989). Along with other agrochemicals; the herbicides are also

considered as potential inhibitor to entomopathogenic fungi (Inglis et al., 2001). While studying

the effects of four commonly used herbicides on vegetative growth and sporulation of six EPFs

under laboratory conditions. Poprawski and Majchrowicz (1995) found totally impaired fungal

growth at all tested temperatures. The EPFs have also been integrated with some other microbial

pathogens as an alternative use of chemical substances (Zimmermann, 1993). The interaction of

B. bassiana and B. thuringiensis var. israelensis for the control of Musca domestica in poultry

houses was studied in field trials (Mwamburi et al., 2009). In the same scenario the B.

thuringiensis has been incorporated with EPFs against a number of insect pests under different

agro ecosystems (Kryukov et al., 2009; Lawo et al., 2008; Wraight and Ramos, 2005; Lacey et

al., 2001; Brousseau et al., 1998; Molina et al., 2007).

2.5.3.20 EPF against RPW

EPFs are commonly found in the nature and cause epizootics in insect populations, thus

play a significant role in regulating insect population. Mostly, Entomophthorales and

Hyphomycetes attack on terrestrial insects. EPFs from various strains of B. bassiana and M.

anisopliae have been found in association with RPW. EPFs are among the most relevant

biological agents suggested to control RPW (Faleiro, 2006). Some of these EPF strains were

tested against RPW, M. anisopliae being more effective than the latter one (Gindin et al., 2006).

However, no strain was originally isolated from RPW in this study. A number of studies were

carried out to investigate the effectiveness of EPFs against RPW, the investigations led to the

detection of more active isolates against RPW individuals in laboratory and field trials (Gazavi

and Avand-Faghih, 2002; Shawir and Al-Jabr, 2010; Shaju et al., 2003; El-Sufty et al., 2007;

Tarasco et al., 2007; El-Sufty et al., 2009; Sewify et al., 2009; Dembilio et al., 2010).

Usually, EPFs infect their host through contact action which makes them superior than

the other entomopathogens (Butt and Goettel, 2000). In RPW infection mostly occur via direct

contact to the inoculum, transmission from diseased to the healthy ones (horizontal transmission)

and transmission from the subsequent developmental stages (vertical transmission) via new

generation of spores (Lacey et al., 1999; Quesada-Moraga et al., 2004). Thus, EPFs must be put

forward as potential bio-control agents in IPM to control RPW current outbreaks (Faleiro, 2006;

Murphy and Briscoe, 1999).

2.5.3.21 Natural incidence of EPFs on RPW

In the beginning, intentions were focused on isolation of fungal strains from RPW;

different strains of B. bassiana and M. anisopliae were recovered from pupae and adults of RPW

(Ghazavi and Avand-Faghih, 2002). Very first natural infection of M. anisopliae was recorded

from R. bilineatus as a result of accidental infection when treatments were applied against

Scapanes australis (Bedford, 1974) with M. anisopliae spore based commercial formulation

(Prior and Arur, 1985). Latter on different researchers found naturally infected RPW specimens

18

with B. bassiana and M. anisopliae (Salama et al., 2001; Shaiju-Simon and Gokulapalan, 2003;

Salama et al., 2004; Gindin et al., 2006; Güerri-Agulló et al., 2007, unpublished; El-Sufty et al.,

2007; Güerri-Agulló et al., 2008; Merghem, 2011). In 2007, RPW fungal infected pupae were

reported from date palm garden in Spain (Dembilio et al., 2010b). Colonies of B. bassiana,

Aspergillus sp., Metarhizium sp., Fusarium sp., Trichothecium sp. and Penicillium sp. were also

recovered from different developmental stages of RPW in Italy (Torta et al., 2009; Tarasco et al.,

2008). A B. bassiana isolate (B-SA3) isolated from Al-Qatif province (Saudi Arabia) from dead

RPW by Hegazy et al. (2007) which latter on was used against RPW in laboratory study.

Lo-Verde et al. (2014) isolated B. bassiana from RPW adults collected from Villagrazia

and Cinisi (Palermo Province, Sicily). Recently, M. pingshaense recovered from RPW in

Vietnam which kill the adults in a very short time (Cito et al., 2014). Thanks to an efficient

enzymes and toxin production.

2.5.3.22 Susceptibility of RPW to EPFs infections under laboratory conditions

Laboratory studies found that M. anisopliae strains caused 80-100% mortality in RPW

larvae and adult. Adults withstand for 4-5 week against spore suspension, while dried

formulation took 2-3 weeks to kill 100% RPW adults. Moreover, M. anisopliae caused 100%

larval mortality within 6 and 7 days, but this took longer time to B. bassiana for getting the same

level of mortality (Gindin et al., 2006). Similar findings were recorded in Egypt (Merghem,

2011) and Italy (Francardi et al., 2012, 2013) against RPW larvae and adults. Research studies

suggested that effectiveness of EPFs against RPW was about 85% under controlled and semi-

natural conditions (El-Sufty et al., 2009; Dembilio et al., 2010). Significantly higher mortality

was recorded for the bio-control of RPW by Vitale et al. (2009) when treated with the

commercial formulations of using a commercial product of B. bassiana and M. anisopliae alone

and an integrated manners, whereas sole application of B. bassiana recovered from dead RPW

cadavers did not gave promising results against adults. This might be attributed to the fact that

polar extracts of adult may inhibit adhesion and germination of pores.

In contrast efficient results were recorded by deploying B. bassiana as a bio-control agent

recovered from naturally infected RPW (Sewifi et al., 2009; Dembilio et al., 2010; Güerri-

Agulló et al., 2010). Thus, we cannot deny its importance in bio-controls of RPW. Dembilio et

al. (2010b) performed laboratory experiments to check the vulnerability of RPWs to B. bassiana.

The strain was infective to eggs, larvae and adult stages. They further reported adult lifespan was

reduced from 1/2 to 1/10 and adults of either sex transmitted 55 and 60% disease to the healthy

adults during courtship. B. bassiana not only induced mortality but also significantly affect the

fecundity (approximately 62.6%) and egg hatchability (32.8%). Likewise, larvae obtained from

infected female eggs exhibited 30-35% more mortality than the healthy ones, overall 78% less

progeny were recorded as compared to the check treatment (Dembilio et al., 2010a). These

finding are in accordance with the findings of Torta et al. (2009) and El-Sufty et al. (2009) who

reported significantly higher mortality of RPW small larvae and adults to indigenous strains of B.

bassiana.

They further revealed that susceptibility was more evident in young larvae than old ones

that might be due to the scarcity of antimicrobial cuticular compounds in younger larvae (Mazza

et al., 2011a). Most recently Hussain et al. (2014) reported varied susceptibility level of different

larval instars to four different isolates of B. bassiana. Other scientists also revealed the same

results with EPFs against different developmental stages of RWP (Ghazavi and Avand-Faghih

2002; Shaiju-Simon and Gokulapalan, 2003; Gindin et al., 2006; Dembilio et al., 2010; Cito et

19

al., 2014). Lo-Verde et al. (2014) evaluated B. bassiana against egg, larvae and adult, the strain

was isolated from infected adults and were found quite effective against all the tested stages. For

the very first time 3 isolates of another different EPF Isaria fumosorosea showed promising

results against RPW (Sabbour and Abdel-Raheem, 2014).

2.5.3.23 Field and Semi-field assessment of fungi for RPW management

The efficacy of EPFs depends to a great extent on formulation. Sewify et al. (2009)

reported successful reduction in RPW incidence in Egypt with indigenous strain of B. bassiana

isolated from a RPW cadaver under field conditions. In field trials El-Sufty et al. (2009) reported

13-47% mortality in adult RPW population using a strain of B. bassiana isolated in UAE. Later

on this strain was effectively deployed in auto-dissemination traps in date palm groves (El-Sufty

et al., 2011). Field studies were conducted using two formulations of local strains of B. bassiana

at UAE. The oil-based formulation of EPF exhibited 13.7-19.2% adult mortality, while dust

formulations imparted only 8.9% mortality (El-Sufty et al., 2007). However, Abdel-Samad et al.

(2011) observed little effect of oil based commercial formulation of B. bassiana on RPW, hence

not recommended for formulation and field application, since it was quite expensive as compared

to the other formulations. Moreover, polar extracts from adults were found to inhibit the spore

germination of B. bassiana commercial formulation (Mazza et al., 2011a).

Besse et al. (2011) reported high pathogenic potential of an indigenous strain of B.

bassiana against RPW, hence recommended as promising agent for bio-control. A preventive

and curative treatment with solid formulation of highly virulent strain of B. bassiana with high

persistence was applied against RPW under semi filed conditions on 5 year old P. canariensis

palms. The efficacies were >85.7%, confirming the pathogenic potential of this strain as a bio-

control agent against RPW (Güerri-Agulló et al., 2011). However, the few field studies carried

out so far lacked adequate experimental designs, used fewer replication, had high infection rates,

etc. (Güerri-Agulló et al., 2011). Lecanicillium (Verticillium) lecanii significantly affected the

mortality of various larval instars and adults and the egg hatching percentages of adult females.

Moreover, yield losses in date production decreased from 56 and 60% to 22 and 22% in El-Esraa

(Nobarya) and El-Kassaseen (Ismailia) respectively (Sabbour and Solieman, 2014). Solid state

formulations of two B. bassiana isolates deployed against RPW under field condition exhibited

100% mortality even after 30 days post application and the efficacy persisted for 3 months

(Ricaño et al., 2013).

Sabbour and Abdel-Raheem (2014) applied Iseria fumosorosea in date palm plantations

and reported significant reductions in date palm weight loss. Results revealed that palm weight

significantly increased in El-Kassaseen compared to El-Esraa to 5341±40.30 kg Feddan-1 as

compared to 1981±80.54 kg Feddan-1 in the control during season 2012. During 2012 season

yield losses were 59 and 62% in El-Esraa and El-Kassaseen which decreased to 28 and 27% in

these respective regions; the same results obtained during 2013 season. These results called for

expanded open field trials with I. fumosorosea strains to explore their bio-control potential.

Most recently, Jalinas et al. (2015), using acoustic recording methods, found greatly reduced

feeding activity after infesting palm trees with B. bassiana treated larvae. Retarded larval

movement and feeding noises suggested that B. bassiana infection weakened RPW larvae, which

reduced detectable feeding activity. The main difficulty in the implementation of acoustical

detection methods is accessibility of these bio-control agents to the pest insects. Given the

concealed nature of RPW, a systemic distribution of the agent, while highly desirable, is difficult

to achieve practically. Consequently, adults are the targeted stage for fungal delivery because

20

this is the only free living stage and future research should be focused at attracting-and-infecting

RPW adults could be most effective in managing this pest (Dembilio et al., 2010b).

2.5.4 Endophytic fungi

Fungal endophytes may be beneficial in preventing disease by induction of host defense

mechanisms (Sivasithamparam, 1998) or by directly affecting plant pests (Arnold et al., 2003).

Reports on endophytic colonization of EPFs in date palms have been published which may be

useful in the future (Gómez-Vidal et al., 2006). They inoculated B. bassiana, Lecanicillium

dimorphum and L. c.f. psalliotae within young and adult date palms petioles and exhibited the

fungal survival even after 30 days of inoculation; fungi were detected inside the parenchyma and

sparsely within vascular tissue using microscopy techniques without any detrimental effect on

date palm. Arab and El-Deeb (2012) applied endophytic fungi on date palm seedlings after 6

months the date palm pulp was offered to the larvae in laboratory which inflicted 80.3%

mortality after 14 days. Ben Chobba et al. (2013) provided the report on 13 different fungal

isolates from date palms from roots and leaves in Tunisia, although these might be affiliated to

some fungal diseases in date palms but provided the way to the researchers towards endophytic

colonization of EPFs. Nevertheless, the deployment of EPF strains with endophytic behavior is

not well understood for systemic protection of palms against RPW. Field trials with B. bassiana

strains to explore their bio-control potential are urgently needed.

2.5.5 Future prospects of entomopathogenic fungi

The successful utilization of EPFs is well recognized in biocontrol programs but the

inconsistent performance of these control agents is attributed by a variety of factors and the

major one is their great dependency on the environmental conditions. Different fungal strains

have limiting temperature ranges for germination, infection and post mortal sporulation. Same is

the case for humidity conditions as many strains require high humidity for spore germination and

sporulation (therefore oil formulations have been developed). The other restricting factors for the

broad spectrum application of the mycoinsecticides include; the limited production of toxins

(from the view point of registration authorities, the production of toxins is a hurdle for

registration and practical use) by the fungi, the slow rate of activity of the fungal conidia, the

higher doses of the conidia required for the effective control which ultimately yields inconsistent

results compared to the chemical insecticides (Gressel, 2001), and the pathogenicity of various

fungal strains to non-target organisms. Therefore, different approaches have been proposed to

tackle these limitations especially with regard of decreasing application rates and increasing

virulence of the fungal pathogens. The most significant among them is the integrated use of EPFs

with other biological control agents.

The second and the most promising approach is the implementation of biotechnology

which has great potential to play a vital role in the EPFs development process from the

identification of virulent strains to final formulation (Glare, 2003). Moreover, the high

production rates of commercialized fungal formulations can be compensated if the equivalent

control is attained at lower concentrations. The transgenic and genomic recombinant approaches

yielding the reduced median lethal concentration (LC50) of the pathogens and shorter survival

time of the target species (St. Leger et al., 1996) not only tend to improve the infection rate but

also reduce the cost of the applied formulation. The herbicide and fungicide resistant genes have

also been induced to various fungal strains (Bernier et al., 1989; Fang et al., 2004) which

21

ultimately make use of the fungal pathogens in combination with certain herbicides and

fungicides.

2.5.6 Entomopathogenic Nematodes (EPNs)

Interest in the use of EPNs as bio-control agents against a variety of pests has increased

in last two decades (Dolinski and Lacey, 2007; Lacey and Shapiro-Ilan, 2008). Researchers are

expanding the pathogenic potential of EPNs against a variety of plant nematodes, harmful

insects, soil-borne plant pathogens and mollusks (Grewal et al., 2005). So far more than 30

families of nematodes, associated with insects have been reported, but because of the bio-control

potential concentrations are focused on seven families of nematode including Sphaerularidae,

Rhabditidae, Allantonematidae, Mermithidae, Heterorhabditidae, Steinernematidae and

Neotylenchidae. For the biological control of RPW, Steinernematidae and Heterorhabditidae,

received the most attention. These nematodes carry species-specific pathogenic bacteria,

Photorhabdus by Heterorhabditidae and Xenorhabdus by Steinernematidae, which are released

into the insect hemocoel when infective juveniles (IJ), penetrate into the insect host body. The

third infective juvenile (IJ) stage of these EPNs actively searches for a suitable host, invades it,

and releases symbiotic bacteria into the insect hemocoel. This process kills the invaded insect via

bacterial septicemia and/or toxemia (Kaya and Gaugler, 1993).

2.5.6.1 Natural incidence

Few species of EPNs have been recorded as naturally infecting RPW. The efforts to

infect RPW with EPNs started with the recovery of parasitic species, Praecocilenchus

rhaphidophorus Poinar, from Rhynchophorus bilineatus in Papua New Guinea and New Britain

(Poinar, 1969) and Praecocilenchus ferruginophorus, isolated from infected RPW in India (Rao

and Reddy, 1980). P. ferruginophorus was recovered from hemocoel and hemocoel of adults,

and fat tissue, trachea and intestine of RPW larvae. Usually, the nematodes are released out of

the body from infected insect during oviposition or may also be released with the feces via

intestine. As a consequence of being released from the body the ovaries of infected weevils are

harmed due to the production of eggs (Triggiani and Cravedi, 2011). The practical importance of

nematode fauna associated with the Rhynchophorus palmarum lead researchers to study the

Bursaphelenchus cocophilus (Cobb) Baujard a causative agent of red ring disease in palms in

neo tropics (Giblin-Davis, 1993). Morover, Bursaphelenchus gerberae, Caenorhabditis angaria

and Mononchoides sp. have also been reported from R. palmarum (Gerber and Giblin-Davis,

1990; Giblin-Davis et al., 2006; Kanzaki et al., 2008; Sudhaus et al., 2011). But none beyond

Rhynchophorus spp. has been effectively surveyed.

Other non-pathogenic nematodes inflicted no harmful effects on RPW are known from

three Rhynchophorus spp.: Acrostichus rhynchophori (named Diplogasteritus in older

publications; Kanzaki et al., 2009) and Teratorhabditis palmarum were isolated from R.

palmarum and R. cruentatus respectively (Gerber and Giblin-Davis, 1990), while

Teratorhabditis synpapillata Sudhaus was recovered from RPW in India and Japan (Kanzaki et

al., 2008). Salama and Abd-Elgawad (2001) isolated Heterorhabditis spp. from five sites in

Egypt. However, only two out of the five isolated nematode strains survived for 24 hours

exposure in RPW infested palm tissue, nematode had a low viability of only 14-19%. The

retarded growth of nematodes is thought to be due to the generation of acetic acid, ethyl acetate

and ethyl alcohol from the infested palm tissue that limit the use of EPNs especially

Heterorhabditis indica (Monzer and El-Rahman, 2003) in addition to the concealed nature of

22

RPW (Abraham et al., 2002). On the other hand, El-Bishry et al. (2000) reported that the host

finding ability of juveniles decreased when the palm tissues were washed and sterilized.

Anti-desiccants such as Leaf Shield and Liqua-Gel were reported to improve the efficacy

of EPNs isolated from date plantations in Saudi Arabia (Hanounik et al., 2000). Steinernema sp.

was isolated from naturally infected field collected adult of RPW inform the eastern province in

Saudi Arabia (Saleh et al., 2011). Recently, Mononchoides sp., Teratorhabditis sp. and

Koerneria sp. were found infecting pupae and adults of RPW in southern Italy, but their species

identification, clarification on their biological parameters and type of association between RPW

and these nematode species are still in progress (Oreste et al., 2013).

2.5.6.2 Susceptibility of RPW to EPNs infections under laboratory conditions

Laboratory studies revealed that both larvae and adults of RPW were infected by

Steinernema riobrave, S. carpocapsae and Heterorhabditis sp. (Abbas and Hononik, 1999).

Similar results were reported by Salama and Abd-Elgawad (2001) when tested five strains of

Heterorhabditis, were more virulent to RPW than other tested entomophilic nematode species

(Salama and Abd-Elgawad, 2001). Laboratory studies showed that RPW larvae were suitable

host for H. indicus (Banu et al., 1998). Similarly, Elawad et al. (2007) reported high mortality of

H. indicus against RPW in UAE under laboratory conditions. Laboratory studies performed in

Italy H. bacteriophora Poinar was reported to be the most effective against RPW larvae and

adults (Triggiani and Tarasco, 2011). Additionally, exposure of RPW larvae to genetically

modified strains of Heterorhabditis and Steinernema exhibited 95-100% and 50% mortality

under and laboratory and field conditions respectively (Hanounik, 1998).

Similar findings were reported from Turkey in which H. bacteriophora inflicted 69% and

80% larval and pupal mortality respectively in RPW (Atakan et al., 2009). This might be

attributed to the fact that S. carpocaspsae was not encapsulated by RPW hemocytes, thus it is

necessary to discover the phenomenon which contributed to the lack of reproduction in larvae

and adult of RPW (Manachini et al., 2013). Shahina et al. (2009) evaluated seven Pakistani

strains of EPNs against eggs, first, third, sixth and final larval instar, and adults of RPW under

laboratory conditions. Significant differences were observed in the mortality of various life

stages of the weevil, while the highest egg mortality was found from S. siamkayai and H.

bacteriophora (95±2.1 and 97±2.2% at 150 infective juveniles (IJs ml-1). Recently, Atwa and

Hegazi, 2014 evaluated 12 EPNs, that all were infective against first instars of RPW larvae.

Some species were selective for a specific host stage while others were effective against all

stages.

2.5.6.3 Field and semi-field assessment of EPNs for RPW management

For EPNs applications in the field a preliminary agarose assay is preferred. This is a

simple and rapid laboratory test for measuring chemo-attraction of nematodes to host diffusates

and host recognition as a predictive screening tool for field testing of new Heterorhabditis

isolates (Monzer, 2004). Earlier field studies with EPNs such as Steinernematid and

Heterorhabditid in date palms did not exhibit efficacious results due to the environmental

constraints and RPW ecology (Hanounik, 1998; Abbas et al., 2001). Similarly, Koppenhöfer and

Fuzy (2004) reported gradually decreased susceptibility of RPW larvae to S. carpocapsae IJs.

However, later studies in the date palms from Middle East reported adult and larval mortality

from the same nematode spp. isolated from respective hosts. The soil treatment of these

nematodes around palms with 8×106 IJs palm-1 led to 33-87% adult mortality, while spraying the

23

palm trunk with the same nematode suspension resulted in only 8-13% adult mortality (Abbas et

al., 2000).

Similar results were recorded by Santhi et al. (2015) who evaluated S. carpocapsae and

H. bacteriophora against the RPW under simulated natural conditions and reported that pupae in

cocoons and adults exhibited a high susceptibility to S. carpocapsae. These findings are

important when considering optimal use of EPNs for the control of RPW under natural

conditions. Studies in curative and preventive assays of S. carpocapsae with chitosan by

spraying the product at a dose of 3.6×106 IJs + 36 ml chitosan palm-1 in about 2 l of water on

trunk and the bases of the fronds of each palm until the run off with the help of a manually

operated backpack compact sprayer. The treatment showed efficacies of around 80% in curative

assays and 98% in preventive assays in various palms.

Accordingly, Llácer et al. (2009) and Dembilio et al. (2010a) performed experiments on

P. canariensis and P. theophrasti respectively by using S. carpocapsae with chitosan, efficacies

ranged from 83.8-99.7% and palm survival significantly increased as compared to the check

treatments. From these experiments it was proved that chitosan as adjuvant can be effectively

used with EPNs, particularly S. carpocapsae, which extend their period and protect them from

environmental conditions (Llácer et al., 2009; Dembilio et al., 2010a). Apart from weather

factors, other organisms associated with RPW can interfere with the efficiency of EPNs. The

RPW associated organisms can interfere with the effectiveness of entomopathogens such as

RPW predatory mites Centrouropoda almerodai Wisniewski and Hirschmann can reduce the

efficacy of the nematode S. carpocapsae (Morton and Garcia-del-Pino, 2011; Mazza et al.,

2011b).

In Egypt, encouraging results were also recorded with another Steinernema sp. recovered

from pupae and adults of RPW; this strain along with two other indigenous strains of the same

genus inflicted considerable mortality against larvae and adults of RPW both under laboratory

and field conditions (Shamseldean and Atwa, 2004). Shamseldean (2002) performed field studies

on date palm trees with Egyptian isolates H. indicus (strain EGBB) H. bacteriophora (strain

EKB20) and Steinernema sp. (strain EBNUE). He reported no symptoms of old or new

infestation in treated palm trees at all treatments. The Saudi Arabian strain of H. indica induced

60 and 46% larval and adult mortalities, respectively, when the nematode was applied at the base

of tree into the soil (Saleh and Alheji, 2003). Similar finding were observed from the field trials

in France but raised an important point regarding defining optimal application standards (Chapin

and André, 2010; Pérez et al., 2010). Because the efficacies in the field study of Dembilio et al.

(2010a) were not significantly different when S. carpocapsae was applied singly or integrated

manner with imidacloprid.

Two successive application of Steinernema sp. by trunk injection resulted in significant

reduction in RPW population after 3 weeks. Efficacies ranged from 48-88% in the curative assay

and significant increase in palm survival was recorded as compared to the control treatment

(Atwa and Hegazi, 2014). Recently, 42 billion worms were imported from Germany to Israel to

combat voraciously feeding RPW spp. in Israel (Anonymous, 2015). The use of EPNs should be

considered when designing integrated management strategies against RPW.

2.5.6.4 Interactions between EPNs and pesticides

Sole or the integrated application S. carpocapsae and imidacloprid under natural

conditions were not significantly different from each other with RPW mortalities ranging from

73-95% and significant increase in plant growth (Dembilio et al., 2010a). Similar results were

24

also observed by Tapia et al. (2011) and suggested application of S. carpocapsae and

imidacloprid after every 60 days as preventive measure during field studies in Southern Spain. In

the light of above findings the results suggested that the combined effect of S. carpocapsae in

chitosan formulation and imidacloprid greatly enhanced the efficacy against RPW under field

conditions and significantly reduced the reproductive potential of the RPW (Tapia et al., 2011).

2.5.7 Entomopathogenic Bacteria

2.5.7.1 History

Existence of bacteria is as old as the history of life on earth. Evidence of bacterial fossils

dates back to the Devonian period (416-359.2 million years ago) and considerable signs depict

their presence from Precambrian time, about 3.5 billion years ago. The fossils found in north-

west Australia's Pilbara region are thought to be nearly 3.5 billion years old and considered the

oldest ones on earth planet. In Proterozoic Eon (about 1.5 billion years ago), when the activity of

cyanobacteria resulted in oxygen production, bacteria became widespread (Anonymous, 2013).

The gradual evolution of the bacteria made them able to survive under a wide range of

environmental conditions with several descendent forms. As a result of this, today an

uncountable and immeasurable diversity in morphology, physiology and taxonomy of bacteria

prevails. Bacteria have been found living very close to every living organism including human

beings. Both beneficial and harmful forms of bacteria have been thriving in various climates like

soil, water, air and hot water springs etc.

Confirmatory evidence of using entomopathogens for the control of insect pests are not

known in ancient times, however, human interest in exploiting microbes particularly bacteria

rose to its extreme after the discovery and the commercial availability of microscope in late 19 th

and early 20th century. Scientific efforts for the survival of the famous Japanese silk industry

against sudden death of caterpillars proved fruitful resulting in the discovery of a spore forming

bacterium, Bacillus sotto, by Sigetane Ishiwata (1868-1941) (Aizawa, 2001). This discovery lead

to the world’s first ever demonstration of toxins when many other scientists including Aoki and

Chigasaki (1915) and Mitani and Watari (1916) found enhanced lethal actions of bacterial

cultures on silk worms when they were applied in alkaline solution (Aizawa, 2001). Doors of

discoveries were opened for man and a German scientist Ernst Berliner in 1909 isolated a

bacterium named by him as B. thuringiensis that killed the flour moth, Ephestia kuhniella

Guenée (Lepidoptera: Pyralidae).

Within the Prokaryotes, bacteria are the microorganisms that lack a nuclear membrane

which separates genetic material from cytoplasmic contents and other membrane bounded

organelles. Bacteria surround us all around and thus, can be isolated from any environment and

hence their enriched flora can be given the name of metabolic strategy which they use to earn

energy such as phototrophs (gain energy from sunlight), lytotrophs (obtain energy form

inorganic material) and organotrophs (receive energy from organic material). The variation in

their size is from one to few microns, and depending upon the morphologies, they can be

grouped as cocci (spherical), bacilli (rod shaped) and spirochetes (spiral shaped). Propagation in

bacteria is carried out through binary fission, a mode of asexual reproductions in which daughter

cells are produced from mother cell as clonal copies (Jurat-Fuentes and Jackson, 2012).

2.5.7.2 Classification

Entomopathogenic bacteria mostly belong to the families Bacillaceae,

Enterobacteriaceae, Pseudomonadaceae, Micrococaceae and Streptococcaceae (Tanada and

25

Kaya, 1993). Although many bacteria are beneficial and essential but members from families

Eubacteriales, Bacillus and Serratia have been registered against insect pests (Tanada and Kaya,

1993). For the successful control of RPW, bacterium has been exclusively isolated from different

developmental stages of RPW and deployed under laboratory conditions and field conditions.

The recognized factor for classifying bacteria involves the sequence of 16S ribosomal RNA.

Two important groups of bacteria are Eubacteria (true bacteria) and Archaea containing bacteria

having similar features of DNA replication, transcription and translation as exhibited by

eukaryotes. Three major divisions within Eubacteria are primarily based on the presence or

structure of cell wall: Gracilicutes (gram negative typed cell wall bacteria), Firmicutes (gram

positive typed cell wall bacteria) and Tenericutes (Eubacteria which are devoid of cell wall).

Most recent classification within Eubacteria mostly relies on the use of polyphasic taxonomy that

includes analysis of nucleotide sequence of RNA (16S rDNA), DNA-DNA hybridization,

genotypic, phenotypic and phylogenetic aspects (Brenner et al., 2005).

Entomopathogenic bacteria; greatly concerned with entomological studies are grouped in

Eubacteria. The cell wall of bacteria greatly serves the purpose to classify, support the molecules

and organelles. In gram-positive bacteria, the cell wall is formed of cross-linked peptidoglycan

while on the other hand, cell wall in gram-negative bacteria is formed of rather complex thin

layer of peptidoglycan and lipoproteins and an outer polysaccharide membrane. Gram-negative

bacteria are distinguished from gram-positive bacteria by lacking the ability to retain crystal

violet dyes. Gram-positive are endospore forming, rod and cocci shaped bacteria often

undergoing sporulation. Gram-negative bacteria on the other hand appear to be in rod or

cocciform. They are much more diverse in their distribution and hence isolation can be

successful from diseased and dead insect specimens (Jurat-Fuentes and Jackson, 2012).

2.5.7.3 Life cycle

Life cycle of B. thuringiensis can be divided into different phases for convenience in

understanding; Phase-I (vegetative growth); Phase-II (transition to sporulation); Phase-III

(sporulation); and Phase-IV (spore maturation and cell lysis) (Berbert-Molina et al., 2008).

Specific insecticidal (Cry) proteins lying deposited in crystals within mother cells starts to form

with the onset of sporulation (Pérez-García et al., 2010). There are some evidences of the

production of the insecticidal proteins within culture medium during vegetative growth (Singh et

al., 2010; Abdelkefi-Mesrati et al., 2011). Distinctive characteristic insecticidal properties to Bt

are conferred by another additional virulence factor phospholipase C, proteases and hemolysins

(George and Crickmore, 2012) which are under control of pleiotropic regulator plc R. Removal

of plc R gene results in drastic reduction in the virulence of Bt in orally infected insects

(Salamitou et al., 2000). Sporulation leads to the production of two types of insecticidal proteins

(cry toxins and cyt-toxins) within crystalline bodies. A single Bt strain is naturally provided with

one or more toxins packaged into a single or multiple crystals (de Maagd et al., 2001). The Cry

toxin is named for its production within crystals whereas Cyt-toxin got the mnemonic Cyt due to

their in vitro cytolytic activity (Crickmore et al., 1998). The Cry toxins acquired the mnemonic

Cry from the fact that they are found in the crystal while the Cyt-toxins acquired the mnemonic

Cyt because of their in vitro cytolytic activity (Crickmore et al., 1998).

2.5.7.4 Ecology

Earlier, B. thuringiensis was thought to have confined to soil only, but advanced isolation

techniques in ‘Insect Pathology’ discovers its various sources of origin (Chaufaux et al., 1997).

26

Now this school of thought has expired and Bt has been isolated from dust, stored grain and silos

materials (Iriarte et al., 1998). The probability of isolation of Bt from a site varies with its

climate, geographical and environmental conditions. In much of the findings till today reveal

most common shapes of crystals as bipyramidal and spherical. More often, Bt is regarded as soil

inhabiting, but it lacks the capacity to multiply in soil or water that offers healthy environment

for other bacteria to compete (Furlaneto et al., 2000). Reproduction in regular practice is carried

within host insects. Most of the commercial Bt formulations are isolation from infected insects,

so it would be obvious to say that soil acts as a reservoir for bacterium instead of multiplication

site.

2.5.7.5 Mechanism of action

The infection cycle start with the ingestion of Bt spores by insects making its way to

alkaline environment of midgut (pH >9.5). The exposure to higher pH of the gut solublizes the

inactive proteins that on the other hand remain insoluble. This activation results in the release of

crystal proteins that produces δ-endotoxins. Insecticidal activities of δ-endotoxins get magnifies

as a result of proteolytic activation and activated toxin readily get bound to specific receptors

present at apical brush border of the midgut microvillae in target insects (Hofmann et al., 1988).

The toxic action of proteins is because of N-terminal half consisting of seven anti-parallels α-

helices. Loss of integrity of insect’s gut is the outcome of Bt activity that ultimately leads to

death of insect due to starvation and septicemia (Kumar et al., 2013).

2.5.7.6 Commercial formulations

A rapid development of interest in bio-pesticides has led to the commercial preparation of

several Bt products. Now a day, wide variety of commercial products is accessible to farmers

infecting a wide range of host insects. While developing commercial products, Bt strains used

against lepidopterous insects belong to subspecies; thuringiensis, kurstaki, morrisoni and

aizawai. For Dipterous insects, Bt strains include subspecies israelensis while coleopterans pests

infecting products include subspecies tenebrionis. The total bio-pesticides used in agriculture

globally, Bt products contribute the share of about 80% (Whalon and Wingerd, 2003). A series of

complex changes are involved in Preparation of Bt products require standard fermentation batch

process including vegetative phase, a sporulation phase release of spores and sporangia in final

phase. Fermented solids at this phase are concentrated and mixed with inert material for packing

as finished product (JuratFuentes and Jackson, 2012). Recently, over 400 of Bt originated

commercial products are marketed over the world in different names registered against pests in

different formulations (solid and liquid). These products contain in them various insecticidal

proteins and viable spores, yet some products are also available with inactivated spores

(Ahmedani et al., 2008). Several valuable products have been prepared from B. thuringiensis var.

israelensis (Gnatrol, Aquabee, Bactimos, LarvX, Teknar AND Mosquito Attack etc.), B.

thuringiensis var. kurstaki (Bioworm, Bactur, Dipel, Topside, Caterpillar Killer, Javelin, Futura,

Thuricide, Worthy Attack and Tribactur), B. thuringiensis var. tenebrionis (M-One, Foil,

Novardo, M-Track and Trident), B. sphaericus (Vectolex WDG and Vectolex CG etc.), B.

thuringiensis var. aizawai (Certan) B. lentimorbus and B. popilliae (Japidemic, Milky Spore

Disease, Doom and Grub Attack etc.).

27

2.5.7.7 Methods of applications of Bt products

B. thuringiensis is undoubtedly regarded as biologically active pesticide effective against

important insect pests well suited to IPM strategies. Based on the ecological aspects of target

pest and infection cycle of active insecticidal proteins, Bt products in solid form as well as liquid

form are applied. Bt products in solid form are either dust or granules (spread over the area of

infection) while in liquid form (foliar sprays), Bt products are sprayed directly (Ali et al., 2010)

to point of infection. A more persistent and biotechnologically advanced way of supplying toxins

to target insect is to genetically express toxin-encoding genes within transgenic plants (Walter et

al., 2010; Chen et al., 2011). This practice leaves no chances of escape of insect from active Bt

components as toxin remained concealed along with the preferred diet of insects. Moreover, the

presence of toxin-encoding genes leaves no harm to non-target fauna, no obvious changes in the

physiology of host plant and no chemical change in the products and byproducts from host plant.

2.5.7.8 Superiority of Bt products over synthetic insecticides

Environmental concern of pesticides has ever remained the hot issue. The hazards posed

with the use of insecticides predominantly broad spectrum nature, quick onset of resistance in

insects (Ahmad et al., 2008), environmental persistence, effect to non-target individuals, and

phenomenon of bio-magnification have declared pesticides an evil for mankind. Another peculiar

reason for the considering bio-control agents as key weapon against notorious insect pests is the

selective (Stevens et al., 2011) and environmental friendly nature (Chen et al., 2011). Several

laboratory and field studies have declared Bt toxins as a necessary component of insect control

strategies and a widely preferred tool over the synthetic chemistries. Bt toxins have specific

insecticidal impacts on insect pests of order coleopteran (Sharma et al., 2010), Diptera (Roh et

al., 2010), Hymenoptera (Sharma et al., 2008), Lepidoptera (Baig et al., 2010) and non-insect

hosts like nematodes (Hu et al., 2010). Although no such reports of harm to non-target individual

has been reported, yet some studies provide insight into reduction in reproduction capacity in

bumblebee (Bombus terrestris) workers after using commercial Bt aizawai strain (Mommaerts et

al., 2010). Beside of this, Bt products still rule over bio-pesticide market and remains hub of bio-

control policies launched against insect pests.

2.5.7.9 Concerns to use of Bt

Bt has several advantages over chemical insecticides: it is host specific and highly toxic

to the target insects highly toxic to insects and yet highly specific. Bt toxins are safer to

environment, animals, human beings and vast array of non-target pests. Therefore, Bt can be

considered an ideal component for IPM programs (Nester et al., 2002). Besides these

advantages, Bt formulations have some reservations to be considered (McGaughey and Whalon,

1992). Most of the microbial based products need repeated application for effective control of

insect pests and sometimes officious only against immature stages immature stages feeding

externally. This seems to be a concern limiting its worth for internal plant feeding insects. This

can be overcome by incorporation of Bt gene (s) in transgenic plants (Krattiger, 1997). Some

recent studies are reporting the development of resistance and cross resistance developed after

the continuous use of Bt toxins is one of the emerging concern about the future of biological

control. Even the laboratory investigations are confirming the incidence of resistance

development in some insects (Pereira et al., 2010) and field populations (Sayyed et al. 2004).

The term ‘cross resistance’ is used by some researchers (Gong et al., 2010; Xu et al.,

2010) for the previously resistant insects (for a specific toxin) showing resistance to other

28

toxin/toxins they have not been exposed yet. Different routes and mechanism of resistance have

been discovered, the most important explanation includes the reduced binding of toxins to

receptors in midgut lining of insects, reduction in solubilisation of protoxin, precipitation by

proteases, toxin degradation and or alteration in proteolytic processing of protoxins (Bruce et al.,

2007). Several mode of resistance has been verified about the development of resistance, the

‘Mode 1’ being the most accepted that hypothesizes that reduction is the outcome of reduction in

binding of Cry1A toxin to specific receptors (Heckel et al., 2007). The alteration of protease

profile in the midgut of Cry1Ac resistant American boll worm is due to proteolytic processing of

Cry1Ac ultimately producing 95 and 68 kDa toxins normally producing active 65 kDa toxins by

midgut protease in vulnerable insects (Rajagopal et al., 2009). Apart from resistance, another

constraint in use of Bt is the narrow host range and limited efficacy. Bt toxins are very limited in

regard of host infection (Shu et al., 2009) and in most of Bt strains, infections remain limited to a

single specie only a few Bt strains exhibit activity span against two or more insect orders (Zhong

et al. 2000).

2.5.7.10 Interaction of Bt products and other toxins

Narrow spectrum of activity in case of Bt is one of the constraint making biopesticides a

second choice after synthetic insecticides. Most of Bt isolates show rather poor control over

insects but their pathogenicity can be magnified by using in synchrony with some other suitable

toxin. For instance, Cyt1Aa is a weak toxin to mosquitoes but synergistic action is found when

combined with toxins like Cry4Ba and Cry11Aa (Fernandez-Luna et al., 2010). Combining Bt

insecticidal toxins in combination with other proteins not only boosts their pathogenic effect but

also helps to lower the resistance developed by insect pests. Proteins like cadherin fragments

have been found to be successfully synergizing the efficacy of several insecticidal toxins (Peng

et al., 2010). The simultaneous use of Cry1Ac and Cry2Ab results in powerful synergistic

interaction against H. armigera (Ibargutxi et al., 2008). Mixing of spores and crystal proteins

from same strain also yields synergistic insecticidal action (Johnson et al., 1998). A dose

dependent interaction of Bt was also recorded against H. armigera in Pakistan from soil isolated

M. anisopliae which integrated synergistically as well as antagonistically at lower and higher

doses (Wakil et al., 2013). Kalantari et al. (2013) interpreted synergistic effect of Bt and

HaSNPV by combining a suitable dose among several tested doses against H. armigera. Future

research will open the horizon of success in integrating Bt with several agents to boost up its

efficacy against a wide host range.

2.5.7.11 Effect of Bacillus thuringiensis on non-target invertebrates

From the last few decades, Bt pesticides are being studied to control crop, forest and

aquatic insect pests. Most of Cry toxins are specific to insects belonging to one of the insect

orders either Lepidoptera, coleopteran and Diptera. Cry2 is an exception to this fact as it exhibits

insecticidal activity against several families of Diptera and Lepidoptera (Schnepf et al., 1998).

Many of the Bt formulations containing purified Cry toxins registered against Lepidopteran

orders show no harm to non-lepidopterous insects (MacIntosh et al., 1990; Sims, 1997).

Conversely, there is an exception as non-target Lepidoptera are not necessarily secure from Bt

treated plants especially in forests (Sample et al., 1996; Herms et al., 1997). The drifting of

aerially applied Bt subsp. kurstaki (Bt-k) to control gypsy moth was also found to be lethal to

non-target Lepidoptera 3000 m away from treated site as demonstrated by Whaley et al. (1998).

However no or negligible effect was found for aquatic habitats in Bt treated sites when

29

Kreutzweiser et al. (1992) demonstrated high concentrations of Bt-k on drift and mortality of

Ephemeroptera, Plecoptera, and Prichoptera. Predators that preyed upon Bt treated hosts were

not found susceptible except the Chrysoperla carnea. So in this regard, it would rather be

justified statement to declare Bt toxin rather safe, specific in action and compatible to non-target

individuals.

2.5.7.12 Mode of infection

The ingestion of B. thuringiensis compounds by insects follows the route of midgut to

expose it to alkaline environment of gut (pH >9.5). Here the higher pH of the gut solublizes the

inactive, otherwise insoluble proteins resulting in the release of crystal proteins that produces δ-

endotoxins. This proteolytic activation of δ-endotoxins offers an extraordinary insecticidal

activity to insects and this activated toxin readily gets bound to specific receptors present at

apical brush border of the midgut microvillae in target insects (Hofmann et al., 1988). The toxic

action of proteins is attributed to N-terminal half consisting of seven anti-parallels α-helices.

These α-helices offers potential gradient by penetrating the membrane and forming an ion

channel in apical brush border membrane allowing rapid flux of ions. Loss of integrity of insect’s

gut is the outcome of B. thuringiensis activity causes starvation and septicemia which leads to

the death of insects (Kumar et al., 2013). The penetration of α-helices in the apical brush border

membrane forms an ion channel (Knowles and Dow, 1993). As a result, rapid flux of ions takes

place because of toxin-induced pores formation (Wolfersberger, 1989). Consequently the gut

integrity gets lost that resultant starvation and/or septicemia leads to insect death.

A wide array of B. thuringiensis products formulated for commercial uses have an

extended spectrum of action effective to secure food crops, forest trees, stored grains and

ornamentals (Meadows, 1993). Contrary to hazards associated with chemical pesticides, B.

thuringiensis formulation offers a wide range of benefits. Although it is highly virulent to target

insects, yet it is harmless to non-target insects due to its specificity. In spite of decades of use in

field, B. thuringiensis toxins are still reported as non-hazardous to animals, human beings and

other non-target pests. All these characteristics render it highly suited to include IPM programs

(Nester et al., 2002). Besides these benefits, B. thuringiensis formulations have some associated

limitations (McGaughey and Whalon, 1992). One of the limitations is its effectiveness against

specific stage of insect especially immature stage. For this reason, an effective control of targeted

insect requires repeated application. B. thuringiensis products perform better against insect

exposed pests than insects concealed within plant parts or some other structures. But the

expression of B. thuringiensis gene (s) using transgenic cultivars (Krattiger, 1997) may be able to

address such concerns.

2.5.7.13 Important entomopathogenic bacteria

2.5.7.13.1 Bacillus thuringiensis

Bacillus thuringiensis (Bt) holds a prominent position among commercial chemical

compounds important for agricultural insect pests. It is a naturally occurring spore forming,

gram-positive bacterium. It has been found as a source and reservoir of several important

insecticidal proteins like δ-endotoxins, vegetative insecticidal proteins (vip) and cytolytic

proteins etc. Among these proteins, δ-endotoxins have a vital role in protecting number of

important crops from various insect pests. B. thuringiensis based insecticides have proved their

worth as a bio-pesticide to protect food crops, cash crops, ornamentals, forest trees and stored

commodities (Meadows, 1993). For convenience, life cycle of B. thuringiensis can be divided

30

into different phases; Phase-I: vegetative growth; Phase-II: transition to sporulation; Phase-III:

sporulation; and Phase-IV: spore maturation and cell lysis (Berbert-Molina et al., 2008). More

than 150 genes of exhibiting insecticidal nature have been identified from Bt δ-endotoxins family

of proteins (Crickmore et al., 1998). These crystalline (cry) proteins remain inactive until the

exposure to alkaline contents (pH >9.5) of insect mid gut, solubilize them (Milne and Kaplan,

1993) and ultimately liberating δ-endotoxins proteins.

2.5.7.13.1 Paenibacillus popilliae

Paenibacillus popilliae previously known as B. popilliae is a gram-positive spore-

forming bacterium which was initially isolated from infected Japanese beetle (Popillia japonica)

(Coleoptera: Scarabaeidae) larvae in the late 1930s and then named after the name of its first

host. The spore forming capability of bacterium protects it from heat, cold, drying and other

harsh environmental regimes. P. popilliae plays a major role in biologically managing scarabs,

particularly Japanese beetle (Petterson et al., 1999). B. popilliae has been reported from at least

29 scarabs, mostly from Melolonthinae and Rutelinae. P. popilliae causes milky spore disease in

P. japonica and it is the first pathogen registered as insect biological control in USA.

2.5.7.13.2 Brevibacillus laterosporus

Brevibacillus laterosporus is a gram-positive, rod-shaped, endospore-forming bacterium

and is considered an important entomopathogenic and antimicrobial agent. It is morphologically

distinguished by producing characteristic canoe-shaped parasporal body (CSPB) firmly attached

at one end of the spore imparting it lateral position in the sporangium. Ubiquitous existence of

this bacterium has enabled its isolation from various reservoirs particularly soils, insect bodies,

fresh and sea water, leaf surfaces, compost, milk, honey, factory effluents, animal hide, wool and

many other materials (Ruiu, 2013). It was discovered by White (1912) during 20 th century

associated with honey bees determined during an investigation on European foulbrood.

2.5.7.13.3 Bacillus subtilis

German botanist Ferdinand Cohn in 1877, while working on hay Bacillus, discovered two

new forms of Bacillus strain named Bacillus subtilis; one of them was heat sensitive (without

endospore) while other was heat tolerant (endospore). A significant genomic diversity in the

bacterium has been publicized using genomics analysis based on microarray-based techniques. It

is competent for growth in many environmental conditions and is often considered as soil

dweller. Most common sources of its isolation are air, soil, water and decomposing plants.

However in most of the cases, it is not found naturally in biologically active but occurs in spore

forms. Bacillus subtilis is scientifically fabulous for its ability to produce a number of antibiotics

especially bacitracin and iturin. It regulates the development of adult mosquitoes by inhibiting

their growth (Ramathilaga et al., 2012).

2.5.7.13.4 Bacillus sphaericus

Bacillus sphaericus is a naturally occurring spore-forming gram positive bacterium that

exhibits strong insecticidal properties. It possesses efficient larvicidal properties against

mosquito by producing delta-endotoxins via sporulation that binds strongly to receptors in

midgut epithelial lining of mosquito larvae. The bacterium has narrow spectrum and quite

specific activity that sometimes decreases its demand for use in field. Enhanced time of lethal

action against some mosquito species and recycling of toxin within dead mosquito sometimes

http://animaldiversity.ummz.umich.edu/accounts/Scarabaeidae/classification/#Scarabaeidae

31

works as limiting factors for its use. One of the advantages exhibits over B. thuringiensis var.

israelensis is its longer persistence that provides long lasting control (Filha et al., 2008).

2.5.7.13.5 Wolbachia

Wolbachia are α-protobacteria, the members of the order Rickettsiales; a varied group of

intracellular bacteria that comprises species exhibiting parasitic, mutualistic and commensal

associations with their hosts. With its pathogenic nature extended to arthropods and filarial

nematodes, it is regarded as the most common endosymbiotic bacterial species on the globe. The

only member contained with genus Wolbachia in family Anaplasmataceae and order

Rickettsiales is Wolbachia pipientis; the rest of the species; W. melophagi and W. persica have

been recently declared as unrelated (Dumler et al., 2001). An insight into the intracellular life

study of the bacterium ensures its obligate nature of infection to hosts and it has been found

successfully infesting about 66% of the insect species (Hilgenboecker et al., 2008). Wolbachia

being intracellular bacterium are vertically transmitted through the egg. Wolbachia sometimes

manipulate the reproduction of host insects by cytoplasmic incompatibility. One of the vital

reasons behind the successful propagation of Wolbachia in arthropods is its inherent ability to

take control of the host’s reproductive cycle by providing nutrients and protecting host from

other pathogens (Hosokawa et al., 2010).

The genera closely related to Wolbachia; Anaplasma, Ehrlichia and Neorickettsia during

their life stages include an invertebrate ‘vector’ and mammalian ‘host’ and in some cases

invertebrate associations in some species have also been found. But contrary to unlike members,

Wolbachia does not necessarily affect vertebrates. One of the important reasons behind increased

interest for Wolbachia is their immense diversity, interesting phenomena shown while infecting

their hosts such as reproductive manipulation, and their possible exploitations for pest and

disease vector control (Bourtzis, 2008).

2.5.7.14 Host range of B. thuringiensis

Different commercial products of B. thuringiensis for use in crops, forests and aquatic

system do not necessarily contain β-exotoxin, but most of the B. thuringiensis products

registered against insect pests contain Cry toxins (also known as δ-endotoxins). Normally, a

single Cry protein works perfectly against a single order and sometimes against several families

within an order. The Cry2 is an exception to this fact as it exhibits insecticidal nature against

several families of Diptera and Lepidoptera (Schnepf et al., 1998). Most of the commercial B.

thuringiensis products or purified Cry toxins formulated for lepidopterous insects are non-

hazardous to a vast variety of non-target organisms (Sims, 1997). However, non-target

Lepidopterans are mostly at risk in B. thuringiensis treated plants particularly in forests (Herms

et al., 1997). For instance, the aerial spraying of B. thuringiensis subsp. kurstaki (Bt-k) to control

gypsy moth was found to be lethal to non-target Lepidoptera 3000 m away from treated site

(Whaley et al., 1998). However, no or a negligible effect was found for aquatic habitats in Bt

treated sites when Kreutzweiser et al. (1992) demonstrated high concentrations of Bt-k on drift

and mortality of Ephemeroptera, Plecoptera, and Trichoptera. Predators that preyed upon B.

thuringiensis treated hosts were not found susceptible except the Chrysoperla carnea. So in this

regards, it would rather be justified statement to declare B. thuringiensis toxin rather safe,

specific in action and compatible to non-target individuals.

32

2.5.7.15 Natural incidence

Effect of different bacterial spp. on RPW has been reported by many scientists. Dangar

and Banerjee (1993) discovered some bacteria species belonging to Serratia sp., Bacillus sp. and

the coryneform group from adult and larval stages of RPW in India, while Bacillus sphaericus

Meyer and Neide and B. thuringiensis Berliner were isolated from larvae and adult of RPW in

Egypt (Alfazairy et al., 2003; Alfazariy, 2004). Later on (Banerjee and Dangar, 1995) isolated

Pseudomonas aeruginosa from larvae infected with this agent from Kerala, India. In Egypt,

Salama et al. (2004) recovered three potential spore-forming bacilli from RPW larvae. The three

bacteria belonged to the genus Bacillus and were identified as variants of B. laterosporus

Laubach (strain 27), B. sphaericus (strain 73) and B. megaterium de Bary (strain 15).

Under in vitro conditions, B. sphaericus caused 40% and 60% mortality in 2nd instar

larvae of RPW when use different isolates of Bt. B. sphaericus considered being the most active

culture which produces crystalline endotoxins and spherical endospores which are responsible

for disease production in RPW. In Italy (Sicily), B. sphaericus B. thuringiensis and B.

megaterium were recovered from RPW cadavers, but these isolates exhibited weaker pathogenic

effect against eggs of RPW (Francesca et al., 2008), although lacking antimicrobial compounds

(Mazza et al., 2011a). Most recently Francesca et al. (2015) isolated distinct strains of seven

species from RPW beetle cadavers (B. cereus, B. amyloliquefaciens, B. pumilus, B. licheniformis,

B. subtilis, B. megaterium and Lysinibacillus sphaericus) from Sicily.

2.5.7.16 Susceptibility of RPW to entomopathogenic bacteria under laboratory conditions

Regarding the pathogenicity of Bt strains against different developmental stages of RPW

very few studies has been conducted so for. In laboratory study application of P. aeruginosa

suspension, either by inoculation by forced feeding, injecting and wading RPW larvae in the

suspension. Complete mortality occurred eight days post inoculation in case of forced feeding

and wading, while injection took 6 days, moreover, younger larvae were more vulnerable than

the older ones (Banerjee and Dangar, 1995). This might be attributed to the fact that younger

larvae probably lacking antimicrobial cuticular compounds (Mazza et al., 2011a). Alfazariy

(2004) revealed successful control of RPW in laboratory conditions by infecting with B.

thuringiensis var kurstaki (Bt-k). Albite this, other scientists revealed different susceptibility of

RPW to the same bacterium (Bauce et al., 2002; Sivasupramaniam et al., 2007; Birda and

Akhursta, 2007; Manachini et al., 2008a, b; Manachini et al., 2009).

Evidences suggested that feeding cessation and midgut damage were observed amongst

surviving larvae. Manachini et al. (2009) integrated commercially available B. thuringiensis into

RPW larvae diet and revealed moderate pathogenicity against RPW larvae. Similar results were

also recorded by Dembilio and Jacas (2013). Under laboratory conditions this bacterium can be

effective against RPW larvae when ingested but their commercial application does not give

satisfactory control (Manachini et al., 2009). However, retarded larval growth and its effect on

hemocytes was primarily described, exhibiting that the bacterium is capable of growing in the

hemolymph when uptake by the larvae (Manachini et al., 2011).

2.5.7.17 Field and Semi-field assessment of bacteria for RPW management

Sequentially, several investigators tested certain commercial products based on Bt against

RPW and reported the difficulty of using such products as a good control agent against the insect

due to different reasons (Dembilio and Jacas, 2013). So for no solid evidence regarding

susceptibility of RPW to entomopathogenic bacteria and confirmation of successful control

33

under field or semi-field conditions has been reported. The reduced susceptibility might be due

to the host defense mechanism. Manachini et al. (2011) conducted preliminary study by

deploying B. thuringiensis and Saccharomyces cerevisiae against the cellular immune response

of RPW and exhibited that Bt is a stress factor for RPW. Future experiments to control RPW by

deploying microorganisms must be designed to explore the molecular mechanism of disease

resistance among RPWs and interaction between them and RPW immune system. Most recently

Francesca et al. (2015) tested nine distinct stains of bacterium which significantly reduced the

egg hatching, while B. licheniformis exhibited significant insecticidal activity against RPW

larvae.

Dangar (1997) evaluated the bio-control potential of free living unidentified yeast

recovered from RPW haemolymph. The calculated lethal dose was 8,000,000 yeasts insect-1,

while lethal time was recorded 4 days. Latter on Salama et al. (2004) isolated yeast from infected

pupae of RPW in Egypt which caused 20-35% mortality in 2nd instar larvae of RPW after 7 days

of application.

2.5.8 Microbial control agents as a component of RPW IPM

As components of RPW IPM, entomopathogens can provide significant and selective

control. An integrated approach is needed which can provide maximum effectiveness when

combined with other control practices (Edwards, 1989). In the near future efforts are being made

to study the synergistic interaction between entomopathogens and other pest control tactics

(integration with soft chemical pesticides, semiochemicals, resistant plants, other natural

enemies, remote sensing and chemigation etc.). These efforts will enhance the efficacy and

sustainability of entomopathogens.

Different formulations of EPNs have been employed in order to enhance the bio-control

potential of EPN against RPW. A commercial formulation (Biorend-R® Palmeras) S.

carpocapsae has been found effective both for preventive and curative measure under semi-filed

conditions (Llácer et al., 2009). EPNs exhibit varying degrees of dispersal to find and invade the

insect host in their habitat, and this affects the fitness traits of parasitism and infectivity

(Koppenhöfer and Fuzy, 2008).

Nematodes disperse by following long-distance cues to search for hosts. The IJs use

distinctive foraging patterns to discover potential prey; either vigorously hunting for insect hosts

(cruisers), or standing on their tails in an upright position over the surface and waiting for the

host insect to pass by (ambushers) (Lewis et al., 2006). These foraging and dispersal strategies

have a significant impact on effective traits of EPNs and on determining their relationship with

the host, an important aspect in predicting EPN efficacy. Moreover, EPFs particularly B.

bassiana and M. anisopliae have been reported to be effective against RPW but promising

results under field conditions are not recorded except with solid formulation of B. bassiana

which exhibited high RPW pathogenicity and persistence for preventive and curative treatments.

The integration of microbial control agents with suitable control measures can be effectively

used to combat RPW both under laboratory and field conditions.

2.5.9 Ecological engineering and agricultural practices to conserve microbial control agents

Ecological Engineering (EE) in IPM of pest control is a comprehensive strategy

integrating modern technologies with traditional cultural techniques to promote

entomopathogens as a cornerstone for sustainable agricultural productivity (Gurr et al., 2004). It

is argued that these ecological based approaches for managing insect populations of agricultural

34

importance can be safer and more sustainable than sole dependence on conventional chemical

insecticides, hence the need to examine the concept and practice of ecological engineering as a

component of modern agricultural activity. Ecological based pest control strategies are more

appealing to the researchers, conservation bio-control in particular that increase the number and

activity of natural enemies in the natural conditions by manipulation of habitat (Gurr et al.,

2004). The conservation of antagonistic organisms in date palm gardens mostly targets

indigenous strains with knowledge of local natural microbe communities instead of inundative

applications of microbial antagonists.

The idea behind conservation is to increase the number of entomopathogens and protect

refuges in the orchards which resultantly will encourage entomopathogens to reduce pest

infestation. Unluckily, in date palm pest control intention are mostly focused to inundative

applications rather than conservation. The enhanced efficacy of EPNs can be achieved by

providing supplementary food sources such as organic amendments in the soil enhance the

efficacy and persistence of EPNs. Contrarily, some amendments with manures and plants

containing allelopathic compounds can exert also hazardous effects to EPNs. In case of

endophytic colonization of entomopathogens plant genotype can interfere with rhizosphere

colonization and antagonist’s metabolites production, as well as the expression of induced

resistance by plants. Ultimately, indicators will need to be identified, such as the presence of

particular antagonists, which can guide decisions on where it is practical to use conservation

biological control. Combination of entomopathogens with conservation practices can be helpful

in improving the effectiveness and persistence of entomopathogens. Future research must be

focused on the greater use of bioassays that accounts for the RPW suppression as effectiveness

of a particular entomopathogen against RPW is not significantly affected by their abundance.

2.5.10 Biotechnological approaches to enhance virulence of microbial control agents

Biotechnology provides magnificent opportunities to enhance the virulence of microbial

control agents through incorporation of gene of interest exhibiting excellent control against

insect pests e.g. the transformation of M. anisopliae by Aspergillus nidulans. To understand the

virulence mechanism efforts are being made, emphasizing the cuticuler area where mostly

penetrations occur and possessing a key enzyme, an endoprotease (St. Leger et al., 1986). In the

future, insect killing speed of EPFs may be enhanced by inserting delta-endotoxin genes from B.

thuringiensis into fungi which certainly will achieve improved strains. Except from the Bt delta-

endotoxin there are several other proteins of insecticidal properties such as alpha-endotoxin,

Vegetative Insecticidal Proteins (VIP) and numerous secondary metabolites that are prone

genetic modification a (Attathom, 2002).

The introduction of gene coding for proteinaceous insect toxins (scorpion toxin, mite

toxin, trypsin inhibitor), hormones (eclosion hormone, diuretic hormone) or metabolic enzymes

(juvenile hormone esterase) into nucleopolyhedroviruses genome are some approaches to

increase speed of kill, enhanced virulence and extend host specificity of the virus (Attathom,

2002). Future, attention should be focused on formulation development and targeting RPW

populations. Advantages of this approach include reduced risk for development of resistance and

greater safety to the environment, and lack of effect on non-target and beneficial organisms. We

believe that biotechnology and genetic engineering will come up with the effective use of insect

antagonists as an integral part of integrated pest management program worldwide.

35

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CHAPTER 3

Genetic variation among populations of Red Palm Weevil Rhynchophorus ferrugineus

(Olivier) (Coleoptera: Curculionidae) from the Punjab and Khyber Pakhtunkhwa

provinces of Pakistan

Abstract

The red palm weevil (RPW) Rhynchophorus ferrugineus is a voracious pest of various palm

species. In recent decades its geographic range has expanded greatly, particularly impacting the

date palm industries in the countries of the Middle East. This has led to conjecture regarding the

origins of invasive RPW populations. For example, in parts of the Middle East, RPW is

commonly referred to as the “Pakistani weevil” in the belief that it originated there. We sought

evidence to support or refute this belief. The first reports of the weevil in Pakistan were from the

Punjab region in 1918, but it is unknown whether RPW is native or invasive there. We estimated

genetic variation across 5 populations of RPW from the Punjab and Khyber Pakhtunkhwa

provinces of Pakistan, using sequences of the mitochondrial cytochrome oxidase subunit I gene.

Four haplotypes were detected, of which, two (H1, H5) were abundant (accounting for >88% of

specimens) across all five sampled populations. There was no geographic overlap in the

distribution of the remaining “rare” haplotypes (H51 and H52) which were restricted to three

(Bahawalpur, Muzaffargarh and Dera Ismail Khan) and two (Dera Ghazi Khan and Layyah)

populations respectively. Levels of mitochondrial haplotype diversity reported herein were much

lower than those previously recorded in accepted parts of the native range of RPW, suggesting

that the weevil may indeed be invasive in Pakistan. In a “global” analysis, the close affinity of

Pakistani haplotypes to those reported from India (and of course the geographical proximity of

the two countries), make the latter a likely “native” source. With regards the validity of the name

“Pakistani weevil”, we found little genetic evidence to justify the name.

Key words: Rhynchophorus ferrugineus, Middle East, Pakistani weevil, Punjab, Khyber

Pakhtunkhwa

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3.1 Introduction

The Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier) (Coleoptera:

Curculionidae: Rhynchophorinae) has been recognized as a major economically important pest

of palm species for more than a century. It has been found devastating > 40 different commercial

and ornamental palm tree species, belonging to 23 different genera and 3 families (Faleiro et al.

2012; Giblin-Davis et al., 2013). These include date palm Phoenix dactylifera L. (Mukhtar et al.,

2011), oil palm Elaeis guineensis (Murphy and Briscoe, 1999), coconut Cocos nucifera (Faleiro,

2006) and Canary Island date palm P. canariensis, (El-Mergawy and Al-Ajlan, 2011). The larval

stages of RPW typically reside within the trunk of an infested palm tree, destroying the vascular

system and boring into the heart of the host. Voracious feeding by these larvae may subsequently

lead to tree collapse (Ju et al., 2011). In favorable climates, the reproductive biology of RPW is

such that an infestation of just a single female has the potential to turn into about five million

weevils in just four generations (about one year) (Nirula, 1956; Rahalkar et al., 1972; Avand-

Faghih, 1996; Esteban-Durán et al., 1998; Cabello, 2006). In India, yield loses of 10-25% have

been reported in coconut plantations (Murphy and Brisco, 1999). In the Arabian Peninsula (an

area that accounts for 30% of global date production), RPW is estimated to damage up to 5% of

the date palm plantations, resulting in losses of 5-20 million US dollars (El-Sabea et al., 2009).

In Pakistan, production losses of 10-20% have been reported in different varieties of dates

(Baloch et al., 1992).

Around the world, RPW is also variously referred to as the Asiatic palm weevil, coconut

weevil, sago palm weevil, and red stripe weevil (although the actual specific identity of many

reported populations, particularly those in SE Asia, is likely to be wrong; Rugman-Jones et al.,

2013). Furthermore, because of the cryptic, internal nature of the beetle’s attack, and the

resulting slow death of the palm tree, it has also been referred to as “the hidden enemy” and even

date palm AIDS (Khamiss and Abdel-Badeea, 2013). In the Middle East, RPW is often referred

to as the “Pakistani weevil” in the belief that it invaded the former from Pakistan. However, this

is somewhat controversial nomenclature, since there is little empirical evidence supporting a

causative link (Rugman-Jones et al., 2013). Furthermore, RPW is generally considered to be

invasive in Pakistan, although it was first formerly reported in what are now the Multan,

Muzaffargarh and Dera Ghazi Khan Districts of the Pakistani province of Punjab, and the

neighboring Indian state of Punjab, almost a century ago (Lal, 1917; Milne, 1918).

Wattanapongsiri (1966) has since defined the native range of RPW as an area stretching east

from India throughout SE Asia (although its occurrence in Indonesia has recently been thrown

into doubt; Rugman-Jones et al., 2013), and in his detailed revision of the genus Rhynchophorus,

based on several extensive museum collections, he recorded only a single un-dated specimen

from modern day Pakistan. However, it remains a possibility that RPW was “always” present in

Pakistan.

The objective of this study was to characterize genetic diversity within and between

different RPW populations in Pakistan with the hope of identifying: 1) whether Pakistan forms

part of the native range of RPW; or if not, 2) the most likely origin of Pakistani RPW

populations; and finally, 3) whether there is any conclusive evidence that Middle Eastern

populations of RPW originated from Pakistan. We used sequences of the mitochondrial

cytochrome oxidase I (COI) gene to investigate genetic variation in RPW from 5 different

geographically isolated populations in the Punjab and Khyber Pakhtunkhwa (KPK) provinces of

Pakistan, and also compared these with publically available sequences from RPW populations

from around the world. If Pakistan is part of the native range, we expect to find relatively high

63

levels of diversity (i.e. a large number of haplotypes; see Rugman-Jones et al., 2013). In contrast,

relatively low levels of diversity are typical of invasive populations and by comparison with

other populations, can be used to make inferences about their potential origins.

3.2 Materials and methods

3.2.1 Specimen collections

During February and March, 2015, RPW were collected from five districts spread across

the Punjab and KPK provinces of Pakistan (Table 3.1; Figure 3.1). Live insects were collected

from infested or fallen date palm plants, with the permission of the orchard’s owners or farmers,

and permission from the Director of the Regional Agricultural Research Institute (RARI)

(Bahawalpur). A total of 80 RPW adults were collected (Table 3.1), stored collectively in plastic

jars containing 95% ethanol (one per location), and maintained at -20 °C in the Microbial

Control Laboratory, University of Agriculture, Faisalabad. At the end of March 2015, the

ethanol-preserved insects were transported to the laboratory of Dr. Richard Stouthamer

(University of California Riverside, USA [UCR]). During transport, the insects were not kept

under controlled temperatures. At UCR each weevil was transferred to an individual plastic vial,

labelled accordingly, and kept in the freezer at -20 °C until processing.

3.2.2 DNA extraction and amplification

Each specimen was extracted using the protocol detailed in Rugman-Jones et al. (2013).

Specifically, a small piece (2-5 mm3) of muscle tissue was dissected from a single tibia using

flame-sterilized scissors and forceps, and allowed to air dry for 1 min on sterile filter paper. The

tissue was then transferred to a sterile 0.6 ml microcentrifuge tube and ground up in 6 µl

proteinase-K (>600mAU/mL; Qiagen, Valencia, CA) using a glass pestle. To this was added 120

µl of a 5% (w/v) suspension of Chelex® 100 resin (Bio-Rad Laboratories, Hercules, CA) and the

reaction was incubated at 55 °C for 1 h followed by 10 min at 99 °C. After this the tubes were

centrifuged for 4 minutes at 14000 rpm to pellet the Chelex. Subsequently, 80 µl of supernatant

was carefully transferred to a new eppendorf tube.

The polymerase chain reaction (PCR) was used to amplify a section of the mitochondrial

gene (mtDNA) cytochrome oxidase subunit 1 (COI) from each specimen. PCR was performed in

25 µl reactions containing 2 µl of DNA template (concentration not determined), ddH2O,1X

ThermoPol PCR Buffer (New England BioLabs, Ipswich, MA), an additional 1 mM MgCl2, 400

µM dUTP, 200 µM each dATP, dCTP and dGTP, 10 µg BSA (NEB), 1 U Taq polymerase

(NEB), and 0.2 µM of each PCR primer. Initial reactions utilized the primers C1-J-1718 and C1-

N-2329 (Simon et al., 1994). Reactions were performed in a Mastercycler® ep gradient S

thermocycler (Eppendorf North America Inc., New York, NY) programmed for an initial

denaturing step of 2 min at 94 °C; followed by five cycles of 30 s at 94 °C, 1 min 30 s at 45 °C,

and 1 min at 72 °C; followed by a further 35 cycles of 30 s at 94 °C, 1 min 30 s at 51 °C, and 1

min at 72°C; and, a final extension of 5 min at 72 °C. Amplification was verified by standard

agarose gel electrophoresis, and samples that failed to amplify were subject to two further

attempted amplifications, this time using the primer sets SIMON and BRON (El-Mergawy et al.,

2011) or BRON and C1-N-2329 (see Rugman-Jones et al., 2013). The integrity of the DNA

extracted from any specimen that still failed to yield a COI amplicon was tested in a 4 th PCR, this

time targeting the 28S rRNA region with the primers (28sF3633 and 28sR4076) and protocol

detailed in Rugman-Jones et al. (2013).

64

3.2.3 Cleaning and sequencing

PCR products were purified using ExoSAP-IT® (Affymetrix, Santa Clara, CA), and

sequenced in both directions at the Institute for Integrative Genome Biology core instrumentation

facility (UCR). Sequences were aligned manually and trimmed to 528 bp (removing the primers

and ambiguous tails) using BioEdit version 7.0.9.0 (Hall, 1999), and then translated using the

EMBOSS-Transeq website (http://www.ebi.ac.uk/Tools/emboss/transeq/index.html) to confirm

the absence of nuclear pseudogenes (Song et al., 2008). All sequences were deposited in

GenBank (Benson et al., 2008) (accession numbers KU696489-KU696537).

3.2.4 Genetic analysis

Sequences of the COI gene generated in this study were collapsed into haplotypes, and

the number and nature of polymorphic sites was characterized, using DnaSP v5.10.01 (Librado

and Rozas, 2009). Genetic variation within each Pakistani population (Table 3.2), was

characterized by calculating the number of COI haplotypes, haplotype diversity (Hd; the

probability that two randomly sampled haplotypes are different), and the average number of

nucleotide differences in pairwise comparisons among COI sequences (k) using DnaSp. Since

most estimators of population differentiation can be highly unreliable when using a single locus

and relatively small sample sizes, genetic variation between topographical populations was also

investigated simply by obtaining population-pairwise estimates of k, again in DnaSP. In order to

put variation within Pakistan into a global context, we then combined our sequences with those

from three earlier studies (El-Mergawy et al., 2011; Rugman-Jones et al., 2013; Wang et al.,

2015; GenBank accessions GU581319-GU581628, KF311358-KF311740, KF413063-

KF413073, respectively). All sequences were trimmed to a uniform length, resulting in a matrix

of 539 sequences, each 528bp long. Sequences were again collapsed into haplotypes using

DnaSP v5.10.01 and a “global” haplotype network was constructed using the statistical

parsimony method of Templeton et al. (1992) in the software program TCS, version 1.21

(Clement et al., 2000).

3.3 Results

Using various combinations of four PCR primers, sequences of the COI gene were

successfully obtained from 50 of our 80 RPW specimens collected from the Punjab and KPK

provinces of Pakistan. For the remaining 30 “failed” specimens, attempts to amplify the highly

conserved 28S rRNA also failed, suggesting that our extractions from those specimens had

yielded no amplifiable DNA. Among the 50 specimens successfully sequenced, we found four

haplotypes. The four haplotypes were very closely related, with only three polymorphic

nucleotides (positions 63, 156, and 174) (see GenBank accessions). All substitutions were

synonymous. Just two haplotypes accounted for the majority (88%) of the specimens and

corresponded to haplotypes H1 (n=20) and H5 (n=24) previously encountered by El-Mergawy et

al. (2011) and Rugman-Jones et al. (2013). These haplotypes were common in all five Pakistani

populations (Table 3.2; Figure 3.2). The remaining two haplotypes had restricted, and non-

overlapping distributions, being found in three and two populations, and our “global” haplotype

analysis revealed that neither had been encountered in the earlier studies of El-Mergawy et al.

(2011), Rugman-Jones et al. (2013), or Wang et al. (2015). Hereafter they are referred to as H51

(n=4) and H52 (n=2), respectively.

Given the very similar nature of the four Pakistani haplotypes, and the prevalence of just

two of those haplotypes across the five Pakistani RPW populations, estimates of genetic

65

variation within and between populations were very low (k was <1 in all comparisons; Table

3.3). In global terms, the haplotypes detected in our Pakistani samples were most similar to

native haplotypes from other parts of the Indian sub-continent; haplotypes H9-16 from Rugman-

Jones et al. (2013) (Figure 3.3). Outside of the native range, the most common Pakistani

haplotypes (H1 and H5) were also invasive in the United Arab Emirates, Oman, and Syria

(Figure 3).

3.4 Discussion

In the Middle East, RPW is often referred to as the “Pakistani weevil” in the belief that it

invaded from Pakistan. However, strong empirical evidence to justify this belief has not been

forthcoming (e.g., Rugman-Jones et al., 2013). Furthermore, there is some doubt as to the actual

status (invasive or native) of RPW in Pakistan, although it has typically been considered an

invasive pest there. In this study we found four haplotypes across 50 specimens, from five

sampled populations located in the Pakistani provinces of Punjab and KPK. Of these haplotypes,

two (H1 and H5) were common in all the populations sampled. The other two were relatively

rare, and non-overlapping, with one (H51; Fig 3.3) represented by only four specimens (two

from Bahawalpur and one form Muzaffargarh and DI Khan), and the other (H52; Fig 3.3) by two

specimens (one from Layyah and another from DG Khan). In global terms, the four Pakistani

haplotypes were very similar to native haplotypes in the remainder of the Indian sub-continent

(Fig 3.3), and H1 and H5 were also common in invasive populations in parts of the Middle East

(UAE, Oman, and Syria).

Is the red palm weevil native to Pakistan?

Low levels of genetic diversity are atypical of RPW populations across its described

native range, but very characteristic of invasive populations of this species around the globe (see

Rugman-Jones et al., 2013). Levels of genetic diversity detected in our study lay somewhere

between the two extremes. If we consider RPW to be an invasive, this suggests that the RPW

populations in the Punjab and KPK provinces of Pakistan have resulted from: a) the influx of a

large number of weevils during a single invasion event; and/or b) multiple invasions from one or

more sources. The genetic similarity between our Pakistani haplotypes and those previously

reported from the Indian state of Goa suggests that India would have been the most likely source

of any invasion. This is also the most likely scenario in a biogeographical sense. Commercial

cultivation of dates in India is focused largely in the western states of Gujarat, Rajasthan and

Punjab, adjacent to the border with Pakistan, and (to a much lesser extent) the southernmost

states of Tamil Nadu and Kerala.

RPW is a strong flyer and it is easy to imagine that Pakistan may have been invaded by

weevils from one or more of these Indian states. However, convincing support for such a

hypothesis will require a much bigger sample from India, which sadly remains a genetic “black

hole” since the country does not allow researchers to collect and export specimens, due to Indian

claims of intellectual property rights over genetic resources. In contrast to the invasive

“argument”, our results could also be interpreted as evidence that the Punjab and KPK provinces

of Pakistan actually fall within the native range of the weevil. At least three known hosts of RPW

are native to Pakistan [Nannorhops ritchiana, Phoenix loureirii and P. sylvestris (Champion et

al., 1960; Mughal, 1992; Malumphy and Moran, 2007; Malik, 2015), and the date palm Phoenix

dactylifera has been cultivated in the Sindh province of Pakistan for more than a thousand years

(http://edu.par.com.pk/wiki/dates/).

http://edu.par.com.pk/wiki/dates/

66

Furthermore, the presence of RPW in the Punjab province of Pakistan, and what is now

the neighboring Indian state of Punjab, was first documented almost a century ago (Lal, 1917;

Milne, 1918). Therefore, we must consider the possibility that small populations of RPW have

always been present in Pakistani Punjab, but have simply gone ignored, or unnoticed, because of

the relative isolation of the region, and/or because their economic impact was (at that time) not

significant. In light of this information, it is perhaps surprising that Wattanapongsiri (1966), in

his revision of the genus Rhynchophorus, considered only a single R. ferrugineus specimen from

anywhere in Pakistan (a specimen from the Kalat District of the modern Balochistan province,

held in the Bavarian State Collection of Zoology, Munich). Prior to the Partition of India in

1947, the two “Punjabs” were considered a single province under the governance of the British

Raj, and British collectors described vast numbers of insects from the entire Indian sub-continent

(including Pakistan), depositing the bulk of their specimens at the British Museum of Natural

History, London. Had RPW been abundant at that time, it seems unlikely that such a conspicuous

insect would have escaped collection. However, despite having access to the BMNH collections

(among many others), Wattanapongsiri (1966) included only the single “Balochistan” specimen,

in his work. Unfortunately, that specimen was without a collection date, and so sheds little

further light on the history of RPW in Pakistan.

Whether native or invasive, RPW has certainly been present in Pakistan for some time.

The recent “rise” of RPW in the Punjab and KPK provinces has likely been exacerbated by

anthropogenic movement of date palm germplasm from the neighboring provinces of Sindh and

Baluchistan where date palms have been cultivated for centuries, and/or the rise of date

cultivation in neighboring Indian states. Again, this is difficult to substantiate without sampling

of those areas, and that should be a priority for further genetic work.

The Pakistani weevil?

It has been claimed by some Middle Eastern countries that RPW originally crossed into

Arabia in ornamental plants imported from Pakistan in 1985 (Dawn News 2003). While our data

cannot completely refute this hypothesis it cannot fully support it either. Although both of the

abundant Pakistani haplotypes detected in our study have been recorded in the Middle Eastern

countries of UAE and Oman (and H1 only also in Syria), a third haplotype H8, has not been

detected in Pakistan, but was found to be widespread in Saudi Arabia (El Mergawy et al. 2011;

Rugman-Jones et al., 2013). Indeed, El Mergawy et al. (2011) detected three further haplotypes

from Oman and UAE. If the Middle East was invaded solely by RPW from Pakistan it is hard to

explain why there are additional haplotypes in the Middle East that have not been detected in

Pakistan.

One answer, originally put forward by Rugman-Jones et al. (2013), is that whether or not

the Middle East has been invaded from Pakistan, it has also been invaded from somewhere else

in the native range of RPW (most likely Thailand). It should also be noted that RPW-like

damage was recorded in Iraq around the same time RPW was first recorded in the Punjab,

although no specimens were collected to confirm this (Buxton, 1920). There is currently no

genetic data available for Iraqi populations of RPW, but given its relative proximity to the

Middle East, it is possible that the latter (and indeed Pakistan) were invaded by RPW from Iraq.

Intensive sampling of Iraq should be a priority for future genetic work.

67

Conclusions The present study showed that the red palm weevil is native to Pakistan and had been

present in Pakistan for a long time. It may have been invaded from India and Sri Lanka. The

population present in KSA is called the "Pakistani Weevil’’ but in my study I have indicated that

this weevil might have been invaded from Thialand or Vietnam instead of Pakistan. In Pakistan

four different groups of haplotypes are present which are commonly found in al the collection

areas of the country. Only the haplotype H52 was the rare haplotype that was only present in the

populations collected from Layyah and Dera Ghazi Khan districts of Punjab.

Acknowledgements

This research work was supported by the scholarship from Higher Education

Commission (HEC), Islamabad, Pakistan (112-30536-2AV1-263) under Indigenous Ph.D.

Fellowship Program.

68

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70

Table 3.1 Sampling information for RPW populations collected from date palm Phoenix

dactylifera in Punjab and KPK provinces of Pakistan

Population Location Collection date

No. of specimens

Province Geographical Characteristic

Alt. (m) Lat. Long.

LY Layyah 27-Feb-2015 8 Punjab 143 30°58'N 70°56'E

BWP Bahawalpur 14-Mar-2015 17 Punjab 252 29°59′N 73°15′E

DGK D.G. Khan 10-Feb-2015 21 Punjab 150 29°57'N 70° 29'E

MG Muzaffargarh 8-Mar-2015 18 Punjab 114 30°50'N 71°54'E

DIK D.I. Khan 1-Feb-2015 16 KPK 166 31°49'N 70°52'E

Table 3.2 Genetic characterization of five RPW populations from the Punjab and KPK

provinces of Pakistan based on a 528 bp section of the mitochondrial COI gene.

For population abbreviations, see Table 3.1.

Population N No. of haplotypes Haplotypes Haplotype diversity (Hd)

LY 9 3 H1, H5, H52 0.556

BWP 11 3 H1, H5, H51 0.691

DGK 8 3 H1, H5, H52 0.464

MG 15 3 H1, H5, H51 0.590

DIK 7 3 H1, H5, H51 0.668

Table 3.3 Variation in a 528 bp segment of the cytochrome c oxidase subunit I (COI) region

of mitochondrial DNA (mtDNA) of Rhynchophorus ferrugineus. Average number

of pairwise nucleotide differences (k) within (diagonal element) and between

(below diagonal) populations in the Punjab and KPK provinces of Pakistan. For

population abbreviations, see Table 3.1.

LY BWP DGK MG DIK

LY 0.722 - - - -

BWP 0.747 0.836 - - -

DGK 0.833 0.909 0.500 - -

MG 0.689 0.733 0.667 0.667 -

DIK 0.778 0.792 0.714 0.686 0.857

71

Figure 3.1 Map of collection sites in Punjab and KPK provinces of Pakistan.

72

Figure 3.2 Distribution of mitochondrial haplotypes across five populations of RPW from the

Punjab and KPK provinces of Pakistan.

73

Figure 3.3 Relationships between four Pakistani COI haplotypes and 48 others occurring

around the world. Haplotype network constructed from 539 COI sequences (each

528 bp long) generated by the present study and three earlier studies (see text).

Each haplotype is represented by an oval or for that with the highest outgroup

probability, a rectangle. Size of each haplotype is indicative of the number of

specimens sharing that haplotype; also given inside each haplotype. H1-43 are

numbered according to El Mergawy et al. (2011) and Rugman-Jones et al. (2013);

H44-50 correspond to additional haplotypes from Wang et al. (2015); and H51-52

are new to this study.

74

CHAPTER 4

Resistance to commonly used insecticides and phosphine (PH3) against Rhynchophorus

ferrugineus (Olivier) (Coleoptera: Curculionidae) in Punjab and Khyber Pakhtunkhwa,

Pakistan

Abstract In the first ever survey of insecticide resistance in field populations of Red Palm Weevil

Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) in Pakistan were collected

from seven date palm growing areas across Punjab and Khyber Pakhtunkhwa (KPK), Pakistan,

and assessed by the diet incorporation method against the formulated commonly used chemical

insecticides profenophos, imidacloprid, chlorpyrifos, cypermethrin, deltamethrin, spinosad,

lambda-cyhalothrin and a fumigant phosphine (or hydrogen phosphide) (PH3). Currently, there is

no IRAC approved bioassay method for R. ferrugineus, so this study aimed to develop a suitable

susceptibility test. Elevated levels of resistance were recorded for cypermethrin, deltamethrin

and PH3 in R. ferrugineus after a long history of use in Pakistan. Resistance Ratios (RRs)

documented for PH3 were 63- to 79-fold for cypermethrin 16- to 74-fold for deltamethrin 13- to

58-fold for profenophos 2.6- to 44-fold for chlorpyrifos 3- to 24-fold for lambda-cyhalothrin 2-

to 12-fold and for Spinosad 1- to 10-fold as compared to the control. Resistant populations of R.

ferrugineus mainly belonged to southern Punjab and to some extent from the KPK populations.

The populations from Bahawalpur, Vehari, Layyah and Dera Ghazi Khan were found most

resistant to chemical insecticides, while all populations exhibited high levels of resistant to

phosphine. Of the eight agents tested, lower LC50 and LC90 values were recorded for spinosad

and lambda-cyhalothrin. Resistance levels were very low to low against imidacloprid, very low

to moderate against profenophos and chlorpyrifos, low to high against cypermethrin and

deltamethrin and high against phosphine. These results suggest that spinosad and lambda-

cyhalothrin exhibit unique modes of action and given their better environmental profile could be

used in insecticide rotation or assist in discarding the use of older insecticides.

Keywords: Rhynchophorus ferrugineus, insecticide resistance, profenophos, imidacloprid,

chlorpyrifos, cypermethrin, deltamethrin, spinosad, lambda-cyhalothrin,

phosphine

75

4.1 Introduction

The Red Palm Weevil’s (RPW), Rhynchophorus ferrugineus (Olivier) (Col.,

Curculionidae) invasive potential is a consequence of the elevated female fecundity (Faleiro,

2006), the ability to complete several generations in a year even in the same tree (Rajamanickam

et al., 1995; Avand Faghih, 1996). It is one of the most destructive pests of ornamental and

economically important palms, which is currently present in 50% of date growing countries and

15% coconut producing countries of the world. The weevil is concealed in nature and all their

life stages remain inside the tree, usually found up to 1m in the tree trunk (Azam et al., 2001).

The beetles quite often interbreed and reproduce within the same plant and continue devastating

their host to death.

The aboriginal home of this pest is South and Southeast Asia and Melanesia where it has

been found destructing coconut palms (Lefroy, 1906; Brand, 1917; Viado and Bigornia, 1949;

Nirula, 1956). Lately in 1918 the beetle was found inflicting date palm in India, during the same

year weevil was found from some southern districts of Punjab, Pakistan (Multan, Muzaffargarh

and Dera Ghazi Khan) (Milne, 1918). Two year latter Buxton (1920) reported this pest on date

palm plantation from Mesopotamia (Iraq). It was only during the mid-1980s that RPW attained a

major pest status on date palms, in the Middle Eastern region (Abraham et al., 1998).

Subsequently, the weevil moved from North Africa into Europe, where it was reported for the

first time in the South of Spain (Cox, 1993; Barranco et al., 1995). So far pest has been

distributed to many areas worldwide; its range now includes much of Asia, regions of Oceania,

the Middle-East and North Africa, southern Europe, the Caribbean, and most recently it has been

found in southern California in 2010 from the canary Island.

To combat this voracious pest synthetic pesticide remained the mainstay since decades

but this offers a challenge due to the cryptic nature of the pest, moreover insecticidal treatments

with fumigants, soil treatments with insecticides, frond axil filling, trunk injections, wound

dressing and crown drenching remains the main strategy for R. ferrugineus control (Hussain et

al., 2013). Different scientists evaluated insecticidal potential of various chemical insecticides

against this pest successfully through different application methods and their combinations

(Cabello et al., 1997; Azam et al., 2000; Ajlann et al., 2000; Khalifa et al., 2001; AboEl-Saad et

al., 2001; Abdul-salam et al., 2001; Al-Rajhy et al., 2005; Kaakeh, 2006; Llácer and Jacas,

2010).

In Pakistan, the application of insecticides on date palms has an ancient history to combat

R. ferrugineus infestation because more than hundred year history of R. ferrugineus presence, but

their published records was not available. Shar et al. (2012) evaluated ten different insecticides

against R. ferrugineus under field conditions and found fipronil, spirotetramat, chlorpyrifos and

methidathion the most effective among all the tested insecticides. Al-Jabr et al. (2013) tested ten

different chemical insecticides against R. ferrugineus midgut cell line, emamectin benzoate was a

highly potent that significantly reduce the growth inhibition and increased the mortality. In

Pakistan effectiveness of commercially used insecticides has complained by the date palm

grower, reduced effectiveness may be because of the development of resistance. Although,

resistance mechanism in R. ferrugineus against commonly used insecticides is poorly

understood. The limited exploration of resistance mechanism in R. ferrugineus is focused in the

design of the current investigation. We selected seven commonly used insecticides and

phosphine (PH3), and checked their effectiveness against different distinct populations of R.

ferrugineus collected from seven different areas of the Pakistan.

76

4.2 Materials and methods

4.2.1 RPW collection and rearing

Different developmental stages of R. ferrugineus were collected from fallen and infested

date palm trees from various areas of Punjab and KPK during 2014-2015 (Table 1). The areas

were selected on the basis of ancient history of date palm cultivation and long term use of

insecticides and phosphine. During collection adults, larvae and pupae were kept in separate

plastic jars for each location until brought to the laboratory. In the laboratory larvae were

provided with sugarcane (Saccharum officinarum L.; Poales: Poaceae) stems for feeding and

pupation, while adults were offered shredded sugarcane pieces for both feeding and as an

oviposition substrate. Pupae were kept in separate boxes for adult emergence in incubators

(Sanyo Corporation Japan) at 27±2 oC, 60±5 RH and photoperiod of 12: 12 (D: L) hours. On

emergence adults were transferred to jars for feeding and mating. The colonies were maintained

in plastic boxes (15×30×30 cm) having a lid whose center (8 cm diameter covered with mesh

wire gauze (60 mesh) for aeration. Rearing was carried out in IPM laboratory, Department of

Entomology, University of Agriculture, Faisalabad, Pakistan. The adult food was changed after

every three days and replaced sugarcane pieces were kept in separate jars for egg hatch. After

egg hatching neonate larvae were transferred to sugarcane pieces for feeding until freshly molted

4th instar larvae were recovered. The laboratory strain was used as reference strain which was

reared in the IPM laboratory since year 2009. The strain was maintained for more than 25

generations in the laboratory without any insecticidal exposure before these tests commenced.

Preliminary laboratory bioassays showed high susceptibly of tested insecticides, being as

susceptible as used by Ahmad et al. (2003).

4.2.2 Test chemicals

For bioassay, commercial formulations of Curacron® (profenophos, 500 g/liter, 500 EC;

Syngenta Pakistan Ltd., Karachi, Pakistan); Confidor® (jmidacloprid, 700g/kg, 70 WG; Bayer

Crop Sciences, Pakistan Pvt. Ltd., Karachi, Pakistan); Lorsban® (chlorpyrifos, 400 g/liter, 40 EC;

Arysta Life Science Pakistan Pvt. Ltd., Karachi, Pakistan); Arrivo® (cypermethrin, 100 g/liter,

10% EC; FMC United Pvt. Ltd., Lahore, Pakistan), Deltamethrin® (deltamethrin, 25 g/ liter,

2.5% EC; Target Agro Chemicals, Lahore, Pakistan); Tracer® (spinosad, 240 g/liter, 240 SC;

Arysta LifeScience, Pakistan Pvt. Ltd.), Karate® (lambda-cyhalothrin 50g/liter, 5 EC; Syngenta

Pakistan Ltd., Karachi, Pakistan). Phosphine (PH3) was generated by using aluminum phosphide

(Celphos 56%; Jaffer Brothers (PVT) Ltd., Lahore, Pakistan) tablets.

4.2.3 Generation of phosphine gas

The PH3 gas was generated using the FAO method (Anonymous, 1975). The apparatus

for generation of PH3 gas consisted of a 5 liter beaker, a collection tube (cylinder), an inverted

funnel, Aluminum phosphide tablets and muslin cloth. The tube for collection of gas was sealed

from one side with an air-tight rubber stopper and then was filled with 5% sulphuric acid

(H2SO4) solution. Half of the beaker was also filled with 5% H2SO4 solution. The gas collecting

tube was placed carefully into the beaker over the inverted funnel in such a way that there is no

loss of H2SO4 solution from the collection tube, while dipping into the beaker. Before generating

PH3 gas all air in collection tube was removed within collection tube. Then aluminum phosphide

tablets (wrapped in muslin cloth) were placed under inverted funnel. PH3 gas was then collected

in the gas collecting tube inverted over the funnel. As the funnel filled with generated gas, the

77

level of solution went down. When the collecting tube was filled, 5 ml gas were sucked out with

the help of an air tight syringe and was injected into sealed desiccators of known volume, then 50

ml of gas was taken out from the desiccators and injected into a Phosphine meter to measure gas

concentration allowing the required concentrations of PH3 gas to be obtained.

4.2.4 Bioassay

From each population freshly molted F1 fourth-instar (L4) larvae of R. ferrugineus were

challenged with the test insecticides. The F1 generation was obtained by mass mating of the field

collected beetles and the beetles emerged from field-collected larvae and pupae. Toxicity

bioassays were performed using artificial diet (Martín and Cabello, 2006). For each bioassay

artificial diets were prepared by diluting the respective concentrations of commercial products in

distilled water (mg a.i./liter of water) previously determined for each bioassay. In the control

treatment diet was prepared using distilled water. A piece of artificial diet from each diet was

offered to ten freshly molted L4 larvae of R. ferrugineus individually in plastic cups measuring

(6×6 cm) with 40 mesh/inch screen lids for aeration and the avoid insects escape. All the

treatments were replicated three time and incubated at 27±2 °C with 65±5% RH and a

photoperiod of 12: 12 (L: D) hours.

FAO Method No. 16 (FAO 1975) was used on 4th instar larvae of R. ferrugineus with

slight modifications. For each population, 10 larvae were placed in each of 10 glass cups and

were placed in 4 liter air tight glass boxes (serving as fumigation chambers) before PH3

fumigation. A small quantity of artificial diet (10 g) was added to each cup. The boxes were

centrally equipped with a port on the metal screw on lid which was fitted with a rubber injection

point which served as an entry point for PH3. Before the lid was screwed onto the box, a rubber

gasket was placed in it, and a thin layer of vacuum grease applied for a tight seal between the

metal lid and the top edge of the box to increase gas tightness. Five phosphine doses (mg l -1)

were measured in preliminary toxicity assays against 4th instar larvae of R. ferrugineus. Another

4 liter box without any treatment served as control. The gas was introduced into each box

containing R. ferrugineus larvae through the rubber septum by using a gas tight syringe after first

removing an equivalent volume of air from the jar using a syringe. Two drops of water were

added to each box using a syringe in order to maintain 70% RH inside the boxes. Boxes were

then placed in an incubator maintained at 27±2 °C. The boxes were opened 24 hours after

application, and all larvae from each treatment were placed on damp filter paper and maintained

at 27±2 °C and 65±5% RH for an extra 5 days to allow recovery.

4.3 Statistical Analysis

For insecticidal treatments three days post-release of insects to treated diets, mortality

counts were made in each treatment and at 24h +(5 recovery days) after PH3 application. Results

for mortality were converted into percentages, corrected using mortality in the untreated check

using Abbott’s (1925) formula and analyzed statistically in Probit analysis by Polo-Plus (LeOra,

2003). LC50, LC90 and 95% fiducial limits were estimated for each insecticide at each location.

Resistance ratios (RRs) were determined at LC50 and LC90 by dividing the lethal concentration

(LC) values of each insecticide by the respective LC values determined for the laboratory

susceptible strain. The distinction devised by Ahmad and Arif (2009) was used for categorizing

the RRs, which were summarized as “no resistance” if (RR≤ 1), “very low” (RR = 2–10), “low”

(RR = 11-20), “moderate” (RR = 21-50), “high” (RR = 51-100), and “very high” (RR> 100).

78

4.4 Results

4.4.1 Imidacloprid

Resistance levels varied among the seven distinct field populations of R. ferrugineus. The

degree of resistance varied from very low to low levels i.e RR 2.10- to 17.68-fold for all the

tested populations (Figs. 4.1 and 4.2; Table 42). A very low resistance level was recorded in

Bahawalpur (2.10-fold), Dera Ismail Kahn (2.86-fold), Vehari (4.20-fold) and Muzaffargarh

(5.60-fold) populations at LC50 level. Low level of resistance were observed in populations of R.

ferrugineus from Rahim Yar Khan (11.70-fold), Dera Ghazi Khan (15.12-fold) and Layyah

(17.68-fold) at LC50 values.

4.4.2 Spinosad

Very low level (1.02- to 9.77-fold) resistance ratios were recorded to spinosad among

different field populations of R. ferrugineus (Fig. 4.2; Table 4.2). The resistance levels were

detected in Dera Ghazi Khan (1.02-fold), Bahawalpur (2.03-fold), Layyah (2.37-fold), Rahim

Yar Khan (3.41-fold), Muzaffargarh (4.40-fold), Vehari (7.15-fold) and Dera Ismail Khan (9.77-

fold) populations of R. ferrugineus at LC50 values.

4.4.3 Lambda cyhalothorin

Very low to low levels (1.96- to11.88-fold) RR to Lambda-cyhalothrin were recorded

(Fig. 4.2; Table 4.2). A very low level of resistance was found in Bahawalpur (1.96-fold), Dera

Ghazi Khan (2.01-fold), Layyah (2.77-fold), Rahim Yar Khan (3.76-fold), Muzaffargarh (4.72-

fold) and Vehari (9.21-fold) populations, while low level of resistance was recorded in Dera

Ismail Khan (11.88-fold) populations of R. ferrugineus at LC50 values.

4.4.4 Chlorpyrifos

Very low and low to moderate levels (3.03- to 24.47-fold) RR to chlorpyrifos were

recorded among tested populations of R. ferrugineus (Fig. 4.2; Table 4.2). A very low level of

resistance was found in Dera Ghazi Khan (3.03- fold) and Bahawalpur (5.45-fold) populations,

while low level of resistance was recorded in Layyah (10.65-fold), Muzaffargarh (17.91-fold)

and Rahim Yar Khan (15.98-fold), and moderate level of resistance was recorded in Dera Ismail

Khan (21.84-fold) and Vehari (24.47-fold) populations of R. ferrugineus at LC50 values.

4.4.5 Porfenophos

Very low and low to moderate levels (RR 2.65- to 44.10-fold) were recorded for

profenophos among different field populations of R. ferrugineus (Fig. 4.2; Table 4.2). A very

low level of resistance was found in Bahawalpur (2.65-fold), Dera Ismail Khan (4.57-fold) and

Vehari (6.90-fold) populations, while low level of resistance was recorded in Muzaffargarh

(10.50-fold), and moderate level of resistance was recorded in Rahim Yar Khan (30.31-fold),

Dera Ghazi Khan (35.22-fold) and Layyah (44.10-fold) populations of R. ferrugineus at LC50

values.

4.4.6 Deltamethrin

Low and moderate to high levels (RR 12.90- to 57.97-fold) of resistance to deltamethrin

were recorded among different field populations of R. ferrugineus (Fig. 4.2; Table 4.2). A low

level of resistance was found in Dera Ghazi Khan (12.90-fold) and Dera Ismail Khan (16.42-

79

fold) populations, while moderate level of resistance was recorded in Layyah (26.76-fold),

Rahim Yar Khan (34.52-fold), and high level of resistance was recorded in Muzaffargarh (51.37-

fold), Bahawalpur (53.81-fold) and Vehari (57.97-fold) populations of R. ferrugineus at LC50

values.

4.4.7 Cypermethrin

Low and moderate to high levels (RR 15.89- to 73.82-fold) of resistance to cypermethrin

were recorded among different field populations of R. ferrugineus (Fig. 4.2; Table 4.2). A low

level of resistance was found in Dera Ghazi Khan (15.89-fold) population, while moderate level

of resistance was recorded in Dera Ismail Khan (22.59-fold), Layyah (31.11-fold), Rahim Yar

Khan (39.41-fold), and high level of resistance was recorded in Muzaffargarh (64.29-fold),

Bahawalpur (69.49-fold) and Vehari (73.82-fold) populations of R. ferrugineus at LC50 values.

4.4.8 Phosphine

High levels of resistance were recorded against PH3 in all the tested populations of R.

ferrugineus, ranging from 63.09- to 79.46-fold (Fig. 4.2; Table 4.2) with very little difference

between populations. The highest levels of resistance were recorded in Rahim Yar Khan (63.09-

fold), Muzaffargarh (63.78-fold), Bahawalpur (68.76-fold), Dera Ghazi Khan (72.29-fold), Dera

Ismail Khan (73.64-fold), Vehari (76.30-fold) and (79.46-fold) in Layyah populations of R.

ferrugineus at LC50 values. The slope of regression line was >2 in all the insect populations,

indicating significant differences in RR from the reference strain.

4.5 Discussion

Knowledge of the resistance status of pests of economic importance is imperative for

researchers to guide the farming community in combating pest problems. It could be helpful for

the farming community in partially reduce or completely suspend the use of particular chemical

in their farming system. In our study we considered seven populations of R. ferrugineus collected

from different areas of Punjab and Khyber Pakhtunkhwa provinces of Pakistan and tested

resistance to seven commonly used chemical insecticides and PH3 against laboratory reared F1 of

these populations. These areas are reported to be the major date producing areas of the country

and contribute the major share of the in country’s date production. Among these areas most have

the history of R. ferrugineus infestation stretching back almost 100 years (Milne, 1918). To

combat this voracious pest, farmers have mainly used conventional insecticides and fumigants,

particularly PH3, throughout the country over decades, resulting in R. ferrugineus.

This is the very first report of a resistance investigation in R. ferrugineus against

conventional insecticides and PH3 in Pakistan. Seven laboratory populations of R. ferrugineus

were established from field collections and all tested strains exhibited significantly different

susceptibility levels to all tested insecticides through dose-mortality bioassays. In our study

laboratory population was considered reference strain which exhibited no resistance to the tested

chemicals. Among the tested chemicals spinosad and lambda-cyhalothrin were the most effective

and revealed very low level of resistance in any tested population. The LC50 value of spinosad

and lambda-cyhalothrin was significantly lower for all the tested populations as compared to rest

of the insecticides. The chemical insecticides fed with artificial diet to R. ferrugineus larvae

caused mortality in a dose-dependent manner. The most consistent resistance across seven

populations was recorded for deltamethrin and cypermethrin. High level of resistance against

PH3 were observed for all the seven populations. The current study provides a base line of

80

resistance to cypermethrin, deltamethrin and PH3 against R. ferrugineus. In the strains examined

low and moderate to high level of resistance was due to the excessive application of these

chemical insecticides to manage R. ferrugineus in Pakistan.

The cryptic habit of R. ferrugineus facilitates almost year-round activity in date palm

plantations which has forced the farmers to expose field populations to different chemical

insecticides and fumigants in order to successfully control pest infestations. The repeated

application of these chemical insecticides could then lead to the resistance in R. ferrugineus

populations. Resistance to cypermethrin and deltamethrin is common amongst the arthropod

pests worldwide (Mueller-Beilschmidt, 1990). In Pakistan resistance against these commonly

used insecticides in various crop pests such as in Spodoptera exigua, Brevicoryne brassicae,

Spodoptera litura, Helicoverpa armigera and Bemisia tabaci has been reported by many

scientists (Ahmad 2008, 2009; Ahmad and Akhtar, 2013; Ahmad and Mehmood, 2015; Ahmad

et al., 2001; Sayyed et al., 2008; Ishtiaq et al., 2012; Qayyum et al., 2015). So far only one

published record of cypermethrin resistance against R. ferrugineus is reported from Saudi Arabia

by Al-Ayedh et al. (2015). Resistance to PH3 is not surprising because deployment of PH3 in the

form of aluminum phosphide tablets is common practice among date palm farming community

in the country. Resistance to PH3 is common in stored grain insets such as Tribolium castaneum,

Rhyzopertha dominica and psocid species worldwide e.g. Opit et al. (2012) reported 119 fold

and 1500 fold resistance in T. castaneum and R. dominica strains respectively, collected from

Oklahoma. Many other researchers have reported PH3 resistance in stored product pests from all

over the world.

It is advised not to use cypermethrin and deltamethrin against this pest and alternative

chemicals such as spinosad and lambda-cyhalothrin should be utilized in control strategies. So

far, there is no understanding of the molecular mechanisms in this species not any understanding

of cross-resistance pattern. This gap should be filled in order to design strategies to mitigate the

resistance problem. Spinosad and lambda-cyhalothrin possesses high efficacy against the

devastating stage of R. ferrugineus in laboratory assays (Abo-El-Saad et al., 2001). Treatment

strategies with chemical insecticides which include spinosad in control programs would be worth

considering. Moreover, integrated use of microbial control agents with newer chemistry

insecticides with novel modes of action could successfully replace the resisted chemistries in

field control programs.

Conclusions The present study showed that the population of red palm weevil in Pakistan has

resistance against commonly used chemical insecticides and phosphine. The unwise use of these

insecticides can lead to this problem. Almost all the insecticides exhibited resistance which was

varied form very low to, low and moderate to high for all the population tested. Delatmethrin,

cypermethrin and phosphine exhibited moderate to high resistance to almost all the populations.

Populations from Layyah Dera Ismail Khan and Vehari showed most resistance as compared to

the other populations tested.

Acknowledgements



Fellowship Program.

81

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84

Table 4.1 Geographical characteristics of the localities where R. ferrugineus populations

were collected in the Punjab and Khyber Pakhtunkhwa, Pakistan

Location Province Host plant Geographical Characteristic

Alt. (m) Lat. Long. Layyah Punjab Date palm 143 30°58'N 70°56'E Bahawalpur Punjab Date palm 252 29°59′N 73°15′E Dera Ghazi Khan Punjab Date palm 150 29°57'N 70°29'E Muzaffargarh Punjab Date palm 114 30°50′N 71°54′E Dera Ismail Khan KPK Date palm 166 31°49'N 70°52'E Vehari Punjab Date palm 135 29°58'N 71°58'E Rahim Yar Khan Punjab Date palm 83 27°40'N 60°45'E

85

Table 4.2 Resistance to commonly used insecticides and phosphine against susceptible

strains and field-collected populations of R. ferrugineus

Insecticide Localities LC50 (mg liter-1)

(95% fiducial limits)

LC90 (mg liter-1)

(95% fiducial limits)

Slope Resistance

Ratio (RR)

Imidacloprid

Bahawalpur 16.59 (12.09-23.41) 250.87 (197.42-314.98) 1.12±0.18 2.10

Muzaffargarh 44.22 (36.31-51.39) 669.08.16 (581.35.793.45) 1.42±0.19 5.60

Layyah 139.52 (108.54-195.02) 2112.86 (1984.81-2358.44) 2.50±0.22 17.68 Dera Ismail Khan 22.64 (18.76-29.78) 341.52 (285.16-459.49) 1.34±0.20 2.86

Dera Ghazi Khan 119.37 (94.62-137.87) 1806.54 (1685.43-2132.38) 2.21±0.23 15.12

Vehari 33.19 (26.14-42.81) 501.51 (435.32-651.32) 1.23±0.19 4.20

Rahim Yar Khan 92.35 (79.37-117.69) 1397.43 (1258.15-1606.42) 1.36±0.21 11.70

Laboratory 7.89 (5.81-12.25) 119.48(106.03-148.11) 0.74±0.14 -

Spinosad

Bahawalpur 7.98 (3.03-10.08) 128.22 (114.34-165.10) 0.92±0.16 2.03

Muzaffargarh 17.26 (14.96-22.82) 276.34 (204.28-385.40) 1.14±0.18 4.40

Layyah 9.32 (4.67-13.92) 150.22 (101.34-236.10) 1.06±0.15 2.37

Dera Ismail Khan 38.30 (30.57-46.74) 620.75 (502.22-844.95) 1.42±0.19 9.77

Dera Ghazi Khan 3.98 (2.80-5.55) 64.41 (42.16-97.84) 0.61±0.13 1.02

Vehari 28.06 (24.87-33.91) 454.23 (357.81-648.35) 1.12±0.17 7.15

Rahim Yar Khan 13.37 (10.75-17.21) 216.72 (141.38-321.18) 1.17±0.15 3.41

Laboratory 3.92 (2.89-5.63) 63.56 (49.34-88.32) 0.69±0.14 -

Lambda cyhalothrin

Bahawalpur 13.85 (10.51-18.83) 221.59 (165.41-352.82) 0.72±0.17 1.96

Muzaffargarh 32.29 (26.54-42.12) 535.51 (448.32-679.32) 1.21±0.19 4.72

Layyah 19.64 (15.31-24.44) 313.82 (217.46-416.10) 1.07±0.17 2.77

Dera Ismail Khan 83.44 (73.39-95.72) 1347.16 (1081.84-1564.82) 1.61±0.21 11.88 Dera Ghazi Khan 14.15 (10.97-19.21) 228.76 (151.39-318.30) 0.72±0.15 2.01

Vehari 64.76 (56.34-71.72) 1045.11 (803.22-1281.35) 1.41±0.21 9.21

Rahim Yar Khan 26.44 (20.31-75.63) 425.93 (319.32-620.47) 1.33±0.20 3.76

Laboratory 7.02 (5.21-11.27) 113.48(102.03-130.10) 0.90±0.15 -

Chlorpyrifos

Bahawalpur 44.21 (37.57-53.40) 611.92 (499.40-881.55) 1.22±0.19 5.45

Muzaffargarh 145.36 (117.57-174.38) 2011.35 (1861.65-2351.88) 2.42±0.21 17.91

Layyah 86.42 (73.70-105.08) 1195.43 (996.15-1440.42) 1.37±0.20 10.65

Dera Ismail Khan 177.25 (145.86-197.65) 2452.54 (2275.47-2865.36) 2.31±0.21 21.84

Dera Ghazi Khan 24.61 (20.48-30.22) 340.55 (249.42-481.64) 1.72±0.19 3.03

Vehari 198.52 (146.54-237.53) 2747.67 (2505.43-3018.38) 2.55±0.20 24.47

Rahim Yar Khan 129.64 (97.54-182.38) 1794.75 (1598.89-2121.52) 2.31±0.18 15.98

Laboratory 8.11 (4.01-11.92) 112.29 (78.34-208.10) 0.97±0.16 -

Profenophos

Bahawalpur 23.31 (19.18-27.71) 307.01 (261.52-377.90) 1.25±0.18 2.65

Muzaffargarh 92.19 (78.12-115.08) 1220.43 (1161.15-1383.42) 1.30±0.21 10.50

Layyah 387.26 (330.65-424.59) 5125.41 (4871.21-5561.27) 3.22±0.23 44.1

Dera Ismail Khan 40.20 (32.98-46.87) 531.87 (414.23-680.45) 1.41±0.18 4.57

Dera Ghazi Khan 309.37 (286.17-353.53) 4093.08 (3814.56-4405.82) 2.53±0.18 35.22 Vehari 60.64 (52.34-67.72) 801.21 (719.22-1029.35) 1.41±0.17 6.90

Rahim Yar Khan 266.28 (224.23-305.29) 3522.54 (3368.83-3917.76) 2.56±0.20 30.31

Laboratory 8.78 (4.44-12.35) 116.22 (90.45-174.59) 0.99±0.16 -

Deltamethrin

Bahawalpur 348.28 (296.65-374.59) 5729.41 (5204.21-6480.27) 3.61±0.23 53.81

Muzaffargarh 332.43 (283.65-365.59) 5469.41 (5108.21-5911.27) 3.36±0.21 51.37

Layyah 173.29 (144.54-199.65) 2849.54 (2617.47-3227.36) 2.31±0.20 26.76

Dera Ismail Khan 106.34 (96.12-121.05) 1748.22 (1553.08-2092.29) 1.73±0.19 16.42

Dera Ghazi Khan 83.54 (73.24-92.11) 1373.16 (1202.84-1591.82) 1.54±0.17 12.90

Vehari 375.95 (329.65-411.59) 6172.41 (5728.21-6798.27) 3.52±0.23 57.97

Rahim Yar Khan 223.41 (194.36-284.66) 3675.76 (3339.78-4105.55) 2.72±0.21 34.52

Laboratory 6.47 (4.97-9.12) 106.48(93.03-122.13) 0.71±0.14 -

Bahawalpur 503.12 (454.22-583.48) 8127.65 (7875.63-8587.39) 3.72±0.23 69.49

Muzaffargarh 565.54 (391.43-605.66) 7519.76 (7256.32-7991.18) 3.83±0.22 64.29

86

Cypermethrin

Layyah 225.39 (201.57-267.29) 3638.02 (3425.25-4058.32) 2.59±0.20 31.11

Dera Ismail Khan 163.65 (139.23-204.32) 2641.58 (2470.43-2911.43) 2.48±0.19 22.59

Dera Ghazi Khan 115.18 (94.12-128.11) 1859.64 (1715.76-2119.64) 2.16±0.21 15.89

Vehari 534.47 (489.22-622.48) 8633.65 (7247.63-9211.39) 3.65±0.23 73.82

Rahim Yar Khan 255.42 (233.23-311.23) 4609.54 (4266.83-5165.76) 2.81±0.21 39.41

Laboratory 7.24 (5.39-11.23) 116.96(103.77-134.12) 0.71±0.19 -

Phosphine

Bahawalpur 4008.70 (3280.23-

4955.42)

51177.95 (43719.73-

59088.76) 5.46±0.27 68.76

Muzaffargarh 3718.37 (3011.23-4604.56)

47679.37 (42165.65-55248.54) 5.82±0.24 63.78

Layyah 4632.51 (3846.43-

5517.72)

59402.62 (51484.42-

64804.32) 6.37±0.34 79.46

Dera Ismail Khan 4293.21 (3618.43-

5180.41)

55048.84 (50342.23-

62268.54) 6.20±0.30 73.64

Dera Ghazi Khan 4214.50 (3529.43-

5065.54)

54039.67 (5045534-

621180.23) 5.82±0.28 72.29

Vehari 4448.33 (3750.43-

5309.52)

57038.53 (50118.76-

63308.37) 6.02±0.31 76.3

Rahim Yar Khan 3678.14 (2972.23-

4545.56)

47163.47 (42540.65-

54540.84) 4.83±0.23 63.09

Laboratory 58.3 (52.45-64.54) 747.56 (692.54-894.89) 1.48±0.21 -

87

Figure 4.1 Map of collection sites in Punjab and Khyber Pakhtunkhwa provinces of Pakistan

(1. Bahawalpur 2. Rahim Yar Khan 3. Vehari 4. Dera Ghazi Khan 5.

Muzaffargarh 6. Layyah 7: Dera Ismail Khan)

1. Bahawalpur

2. Rahim Yar Khan

3. Vehari

4. Dera Ghazi Kahn

5. Muzaffargarh

6. Layyah

7. Dera Ismail Khan

88

Figure 4.2 Resistance ratios (RRs) of chemical insecticides and phosphine against

susceptible strains and field-collected populations of R. ferrugineus populations of

R. ferrugineus from various localities in Punjab and Khyber Pakhtunkhwa,

Pakistan

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CHAPTER 5

Insecticidal potential of Beauveria bassiana and Metarhizium anisopliae isolates against

Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

Abstract

Entomopathogenic fungi are amongst the most common microbial control agents in nature

against insect pests. Their efficacy against a variety of arthropod pests had been witnessed since

for many years. The aim of this study was to screen 19 different isolates of Beauveria bassiana

s.l. and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales), recovered from different soil

samples (field crops, fruit orchards, vegetable fields and forests) and insect cadavers at two

different spore concentrations (1×107 and 1×108 conidia ml-1). Three isolates of B. bassiana

(WG-41, WG-42 and WG-43) and two isolates of M. anisopliae (WG-44 and WG-45) exhibited

˃88% larval and ˃75% adult mortality of Rhynchophorus ferrugineus (Olivier) (Coleoptera:

Curculionidae) on their highest dose rate. On the other hand more sporulating cadavers were

observed at high dose rate compared to low dose on both life stages of R. ferrugineus. The

current study confirmed the lethal action of B. bassiana, and M. anisopliae isolates with

differential mortality levels, usually directly proportional to the conidial concentration. This

study further confirmed that the isolates recovered from R. ferrugineus dead cadavers exhibited

more mortality compared to the other sources. In virulence assay WG-41 and WG-42 caused

highest percentage of both larval and adult mortality at all the exposure intervals which suggest

that these two isolates may be the most promising for their use in sustainable management

programs aimed at microbial control in date palm plantation.

Key words: Entomopathogens, Beauveria bassiana, Metarhizium anisopliae, Red Palm

Weevil sporulating

90

5.1 Introduction

The red palm weevil (RPW) Rhynchophorus ferrugineus (Olivier 1790) (Coleoptera:

Curculionidae) is a devastating palm pest that has caused large economic losses in palm farming

worldwide (Murphy and Briscoe, 1999; Faleiro, 2006). This beetle can affect a wide range of

palms (Barranco et al., 2000) including economically important species such as the date palm

(Phoenix dactylifera L.), Canary Islands date palm (P. canariensis Hort), coconut (Cocos

nucifera L.), African oil palm (Elaeis guineensis Jacq.) and chusan palm (Trachycarpus fortunei)

(Sabbour and Solieman, 2014). The weevil has been found devastating palm plantations almost

50% of the date palm growing countries of the world (Faleiro, 2006) resulting yield losses from

0.7-10 tons hac-1 (Singh and Rethinam, 2005), while in Pakistan it caused 10-20% production

losses to different varieties of dates (Baloach et al., 1992). Presently its distribution is reported in

Oceania, Asia, Africa and Europe and was found in Curaçao and Marruecos, in 2008, and USA,

in 2010 (EPPO 2006, 2007, 2009a, 2009b, 2010).

The females lay eggs at the base of the fronds in separate holes made with their rostrum.

Neonate larvae bore into the palm core and upon completion of development move back to the

base of the fronds to pupate. A new generation emerges and adults may remain within the same

host and reproduce until the palm eventually dies and after that adults emerge out of the plant for

infection to the new plants (Dembilio et al., 2010). Furthermore, R. ferrugineus is a strong flyer

that increases the weevil’s ability to disperse, colonize and breed at new sites (Murphy and

Briscoe, 1999). However, longevity, activity and behavior of adult weevils are greatly affected

by humidity.

The most commonly used control treatments for this voracious pest are chemical

insecticides such as Diazinon, Imidacloprid, Phosmet and phosphine (Llácer and Jacas, 2010;

Llácer et al., 2010). These pesticides are applied with numerous application methods, including

wound dressing, frond axil filing, fumigation, injection, and spraying, are being tried for the

control of RPW infestations (Cabello et al., 1997; Abraham et al., 1998; Al-Rajhy et al., 2005).

Undoubtedly, the use of synthetic pesticides will continue to reduce RPW infestations. However,

several factors, including the evolution of resistance, residue persistence, applicator safety,

environmental hazards and harms non-target organisms have urged researchers to explore

alternatives to control RPW which can be compatible to human health and environment friendly

(Gindin et al., 2006; Hussain et al., 2013; Jalinas et al., 2015).

Alternatively, a number of biological control agents like predators, parasites, parasitoids,

and microbial control agents (bacteria, fungi and nematodes) are deployed to combat this pest. In

laboratory studies of the infection of RPW with different microorganisms, including

entomopathogenic nematodes (EPNs) (Gerber and Giblin-Davis, 1990; Llácer et al., 2009;

Dembilio et al., 2010a), entomopathogenic bacteria (Banerjee and Dangar, 1995; Salama et al.,

2004; Manachini et al., 2009) and entomopathogenic fungi (EPFs) (Ghazavi and Avand-Faghih,

2002; Shaiju-Simon and Gokulapalan, 2003; Gindin et al., 2006; Dembilio et al., 2010b; Cito et

al., 2014) have shown variable results in terms of larval and adult mortality.

Among these microorganisms, EPFs are considered promising microbial control agents

against RPW due to their epizootic potential, transmitted horizontally, natural dispersion and safe

to non-target organisms, environment friendly and ability to maintain lasting control once

established in the environment (Van Driesche et al., 2007; Hussain et al., 2015). Microbiological

treatments with Beauveria bassiana s.l. (Ascomycota: Hypocreales) and Metarhizium anisopliae

s.l. (Ascomycota: Hypocreales) offer an alternative and ecologically compatible pest

management strategy (Inglis et al., 2001). Several research programs have also been initiated for

91

studying the biological control of RPW. Specifically, by deploying these two agents, have been

detected on the RPW and tested under laboratory and field conditions (Deadman et al., 2001;

Gindin et al., 2006; El-Sufty et al., 2007; 2009; 2011; Sewify et al., 2009; Torta et al., 2009;

Vitale et al., 2009; Dembilio et al., 2010; Güerri-Aguilló et al., 2010; 2011; Merghem, 2011;

Francardi et al., 2012; Ricaño et al., 2013; Cito et al., 2014). The main advantage of using EPFs

is their unique mode of action. Unlike other insect pathogens, fungi infect the host by contact,

penetrating the insect cuticle. The host can be infected by direct treatment or by transmission of

inoculum from treated insects/cadavers to untreated insects or to subsequent developmental

stages via the new generation of spores (Quesada-Moraga et al., 2004).

A number of studies were carried out to isolate the EPFs from different developmental

stages of RPW. Entomopathogenic M. anisopliae was isolated from Rhynchophorus bilineatus

(Montrouzier) (Coleoptera: Curculionidae) in New Guinea, after treatment of young palms

against the Scapanes australis Boisduval (Coleoptera: Scarabaeidae: Dynastinae) with a

formulation based on M. anisopliae spores (Murphy and Briscoe, 1999). Other published report

(Ghazavi and Avand-Faghih, 2002) from Iran; showed the recovery of M. anisopliae and B.

bassiana from RPW adults and pupae. The pupae of R. ferrugineus presumed to be infected with

entomopathogenic fungi were collected in a date palm grove in Spain during 2007 which later on

proved to be infected with the entomopathogenic fungus B. bassiana (Dembilio et al., 2010b).

The aim of this study was to screen and identify pathogenicity of 19 isolates of M. anisopliae and

B. bassiana recovered from different soils, stored grain insects and cadavers of infected R.

ferrugineus. This study also aimed to determine the exposure time dose mortality relationships of

virulent fungal isolates, and to confirm infection against RPW under laboratory conditions.

5.2 Materials and Methods


Different life stages of RPW were collected from fallen and infested date palm trees with

the permission of farmers (owners) from date palm growing areas of west Punjab and Khyber

Pakhtunkhwa (KPK), Pakistan. During collection adult, larvae and pupae were kept in 2 liter

plastic jars until brought to the laboratory. After arriving to the laboratory larvae were provided

with sugarcane (Saccharum officinarum L.; Poales: Poaceae) stems for feeding and pupation,

while adult were offered with the shredded sugarcane pieces as both for feeding and substrate for

oviposition. Pupae were kept in separate boxes (15×30×30 cm) for adult emergence in incubator.

As the adults were emerged, they were transferred to the adult’s jars for feeding and mating.

Colony was developed in plastic boxes (15×30×30 cm) having a lid covered with mesh wire

gauze (60 mesh size) in the middle (7 cm diameter) for aeration. Rearing was carried out in

Microbial Control Laboratory, Department of Entomology, University of Agriculture,

Faisalabad, Pakistan. The rearing conditions were maintained at 25±2oC and 65±5% RH and

12:12 (D: L) h photoperiod in an incubator. Adults diet was changed after every three days and

replaced sugarcane pieces were kept in separate jars for egg hatching. After egg hatching neonate

larvae were transferred to the new sugarcane pieces for feeding and pupation.

5.2.2 Culture collection

The virulence of 19 isolates of entomopathogenic fungi B. bassiana and M. anisopliae,

14 of them (WG-2, WG-3, WG-4, WG-5, WG-6, WG-7, WG-10, WG-11, WG-12, WG-13, WG-

15, WG-16, WG-17, WG-18) belonging to the culture collection of Microbial Control

92

Laboratory, Department of Entomology, University of Agriculture, Faisalabad, Pakistan. All

these isolates were recovered from soils of different origin (field crops, fruit orchards, vegetable

fields and forest) and stored grain insect pests (Wakil et al., 2013; 2014). Other five isolates,

three of B. bassiana (WG-41, WG-42 and WG-43) and two of M. anisopliae (WG-44 and WG-

45) were isolated from infected RPW cadavers collected from Layyah, Bahawalpur, Dera Ismail

Khan and Muzaffargarh districts (Table 4.1).

5.2.3 Isolation from RPW cadavers

The B. bassiana and M. anisopliae isolates were obtained from naturally infected RPW

adults collected from different areas of Punjab, Pakistan. All the dead cadavers were washed

with 75% ethanol for 30 sec, rinse with distilled water and transferred to 3% NaClO for 1 min,

followed by rinse in distilled water for 2 times. Cadavers were then dried on filter paper and

placed in petri dishes containing either Sabouraud Dextrose Agar (Merck, Germany) or Potato

Dextrose Agar (BD, France) supplemented with 0.1 g liter-1 of streptomycin sulfate. Plates were

sealed with parafilm and incubated at 25°C for 7 days. Insects were examined under a

microscope every 24 h for the appearance of any fungal outgrowth on the medium (Wakil et al.

2014). Where more than one fungal colony was present on the medium, the culture was purified

following single spore method (Choi et al. 1999). The identification of the isolated fungi was

done with the taxonomic keys (Barnett & Hunter 1999; Domsch et al. 2007). The culture was

sub-cultured and stored in petri dishes at 4°C in refrigerator. Spore concentration was determined

with an improved Neubauer haemocytometer and the conidial viability was confirmed <90%

before each assay.

5.2.4 Screening assay

Nineteen fungal isolates (Table 4.1) were assayed against larvae and adults of R.

ferrugineus. Two conidial concentrations (1×107 and 1×108 conidia ml-1) were employed against

4th instar larvae and adult by dipping method. The larvae were immersed in conidial suspension

for 60s and adults for 90s (Dembilio et al., 2010b). After treatment insects were transferred to

the moistened filter paper for 24 h and shifted individually to 150 ml plastic cups (6×6 cm). The

cups were covered with screening lids and treated larvae were offered with artificial diet (Martín

and Cabello 2006) and adults with shredded sugarcane pieces (3×3 cm). The control individuals

were treated with 0.01% Tween-80 solution. Each treatment consisted of 10 individuals and

three replicates in each treatment, a total of 30 insects per treatment were used. Mortality was

checked daily for 12 days and dead individuals were transferred to SDA medium for 10 days to

observe mycosed individuals. The whole experiment was repeated twice.

5.2.5 Virulence assay

Potential strains which showed high pathogenicity in preliminary screening assays (WG-

41, WG-42, WG-43, WG-44 and WG-45) were further tested against both larvae and adult of R.

ferrugineus at 1×106, 1×107, 1×108 and 1×109 conidia ml-1. Ten 6th instar larvae were dipped in a

conidial suspension for 60s and adults for 90s and air dried in sterile petri dish lined with damp

filter paper for 24 h and transferred to the plastic cups individually (Dembilio et al., 2010b). The

conditions in an incubator were adjusted to a photoperiod 12:12 h (L: D) and 65±5% RH at

25±2°C. Then the larvae were allowed to feed on artificial diet (Martín and Cabello, 2006) in

150 ml plastic cups (6×6 cm) individually and adults with shredded sugarcane pieces (3×3 cm).

The larval mortality was recorded after 7, 14 and 21 days. For control treatment the individuals

93

were immersed in distilled water containing 0.01% Tween-80. Three replicates were used for

each treatment and entire experiment was repeated thrice independently. Median lethal

concentration LC50, LC90 and lethal time LT50, LT90 were calculated for different exposure

intervals.

5.2.6 Statistical analysis

Mortality for each treatment was corrected for control mortality using Abbott's (1925)

formula and subjected to one way analysis of variance (ANOVA) in Minitab (Minitab, 2002)

using Tukey’s Kramer test (HSD) (Sokal and Rohlf, 1995) at 5% significance level. Probit

analysis was used to estimate the LC50, LC90, LT50 and LT90 of isolates using Confidence Limit

95% (CL).

5.3 Results

5.3.1 Screening assay

The results revealed that all the tested isolates of B. bassiana and M. anisopliae were

pathogenic to larvae and adult of R. ferrugineus under laboratory conditions. Overall both fungal

isolates inflicted greater mortality of R. ferrugineus larvae (Table 5.3) compared to the adults

(Table 5.4). For example, five isolates caused highest mortality ranging 65.55-88.33% at 1×108

conidia ml-1 after 12 days post incubation, whereas adult mortality was ranged between 46.03-

75.95% at the same concentration. All the fungal isolates differ significantly in their virulence

against larvae (F18, 113 = 31.2, P≤0.05) at 1×107 conidia ml-1 and (F18, 113 = 56.8, P≤0.05) at 1×108

conidia ml-1 and for beetles (F18, 113 = 41.4, P≤0.05) at 1×107 conidia ml-1 and (F18, 113 = 46.8, P≤0.05)

at 1×108 conidia ml-1. More numbers of mycosed individuals were recorded from the treatments

where lower spore concentration (1×107 conidia ml-1) was applied than higher concentration

(1×108 conidia m l-1). Mycosis in larvae was also significantly different (F18, 113 = 757, P≤0.05) at

1×107 conidia ml-1 and (F18, 113 = 433, P≤0.05) at 1×108 conidia ml-1 and for beetles (F18, 113 = 763,

P≤0.05) at 1×107 conidia ml-1 and (F18, 113 = 423, P≤0.05) at 1×108 conidia ml-1. Main effects and

their associated interactions for mortality and mycosis were significantly different (Table 5.2).

Among five most virulent isolates three belonged to B. bassiana (WG-41, WG-42 and WG-43)

and two to M. anisopliae (WG-44 and WG45). These five isolates were selected for further

virulence assays.

5.3.2 Virulence assay

In virulence assay the mortality of larvae and adult was recorded after 7, 14 and 21 days

after application. Until 7th day of application no isolate caused 100% mortality in either larvae or

adult, highest activity was recorded for WG-41 76.95% and 63.96% in larvae and adult

respectively at highest spore concentration (Table 5.6). After last count all isolates caused 100%

mortality both in larvae and adult at highest dose rate used except WG-43 and WG-45 which

caused 98.41and 91.83 % adult mortality respectively. Overall WG-41 was the most virulent

isolate followed by WG-42, WG-44, WG-43 and WG-45. The mortality of larvae were

significantly affected by the dose rates of fungal isolates at 5days exposure (WG-41: F3, 35 = 71.1,

P≤0.05; WG-42: F3, 35 = 101, P≤0.05; WG-43: F3, 35 = 63.3, P≤0.05; WG-44: F3, 35 = 105, P≤0.05

and WG-45: F3, 35 = 60.8, P≤0.05). Similarly significant difference was also recorded in case of

adults at this exposure (WG-41: F3, 35 = 133, P≤0.05; WG-42: F3, 35 = 108, P≤0.05; WG-43: F3, 35 =

106, P≤0.05; WG-44: F3, 35 = 56.6, P≤0.05; WG-45: F3, 35 = 58.3, P≤0.05). After 14 days post

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application, only WG-41 exhibited 100% larval mortality at highest dose rate used (Table 5.7),

while highest adult mortality (93.22%) was also recorded for the same isolate at highest fungal

dose rate (Table 5.8). Significant differences have been observed between the mortalities of the

larvae at all the dose rates (WG-41: F3, 35 = 220, P≤0.05; WG-42: F3, 35 = 193, P≤0.05; WG-43: F3,

35 = 120, P≤0.05; WG-44: F3, 35 = 170, P≤0.05; WG-45: F3, 35 = 203, P≤0.05). Significant

differences were also recorded in case of adults at this exposure (WG-41: F3, 35 = 166, P≤0.05;

WG-42: F3, 35 = 162, P≤0.05; WG-43: F3, 35 = 112, P≤0.05; WG-44: F3, 35 = 172.0, P≤0.05; WG-45:

F3, 35 = 135, P≤0.05). After last count, all tested isolates caused 100% mortality both in larvae and

adult except WG-43 and WG-45, and significant differences were observed between all the

tested isolates for larvae (WG-41: F3, 35 = 138, P≤0.05; WG-42: F3, 35 = 245, P≤0.05; WG-43: F3, 35

= 179, P≤0.05; WG-44: F3, 35 = 168, P≤0.05; WG-45: F3, 35 = 223, P≤0.05) and adults (WG-41: F3,

35 = 164, P≤0.05; WG-42: F3, 35 = 312, P≤0.05; WG-43: F3, 35 = 261, P≤0.05; WG-44: F3, 35 = 165,

P≤0.05; WG-45: F3, 35 = 271.0, P≤0.05). Factorial analysis shows that the main effects were

significant, while their associated interactions Isolate × Treatment and Isolate × Interval for

larvae were non-significant and Isolate × Interval were also non-significant for adults (Table

5.5).

The results of our study revealed that all isolates of B. bassiana and M. anisopliae are

virulent to larvae and adults of R. ferrugineus at the dose rate of 106, 107, 108, and 109 conidia

ml-1. The lethal concentrations LC50 and LC90 at the 15th day after treatment were assessed

(Table 5.9). The isolates (WG-41 and WG-42) exhibited half of the larval mortality with a

concentration of 105 conidia ml-1 and isolates (WG-43, WG-44 and WG-45) revealed LC50

values at 106 conidia ml-1. While half of the adult mortality for all the tested isolates were

recorded for a concentration of 106 conidia ml-1. The isolates WG-41 and WG-42 caused a high

percentage of adult mortality, thus requiring a lower concentration of fungal conidia to cause an

average percentage of adult or larval mortality; hence, these isolates were considered the most

virulent among all the isolates investigated in this study.

The increased virulence, shown by the reduction in LT50, increased mortality and

proportion of mycosed cadavers were closely related to the conidial concentration. LT50

decreased when conidial concentration increased from 106-109 conidia ml-1. Estimated LT50 of

all fungal isolates against R. ferrugineus larvae varied from 17.75 to 27.92 days at 106 conidia

ml-1, while less time 5.27 to 7.82 days were recorded in case of highest dose rate used (Table

5.10). In case of estimated LT50 of all fungal isolates against R. ferrugineus adults varied from

20.41 to 30.06 days at 106 conidia ml-1, while less time 4.93 to 9.57 days were recorded in case

of 109 conidia ml-1 (Table 5.11). Overall WG-41 was considered most virulent isolate against

both larvae and adult which inflicted highest adult and larval mortality at almost all the dose rate

used within short period of time as compared to the other isolates used.

5.4 Discussion

Fungal entomopathogens are important biological control agents against insect pests

worldwide and have been the subject of intense research for more than 100 years (Vega et al.,

2012). Some of the advantages offered by the use of entomopathogenic fungi in microbial

control programs are their specificity, contact transmission, natural dispersion, safety for non-

target organisms and the ability to maintain lasting control once established in the environment

(Van Driesche et al., 2007). Laboratory screening of fungal isolates bring down to manageable

number is a vital step in identifying virulent strains prior to field use (Cherry et al., 2005). The

two stage approach to screening adopted in this study proved a robust mechanism that has been

95

used effectively by many other workers (Moino et al., 1998; Kassa et al., 2002). In our studies, a

panel of 19 isolates that were never screened were selected and selected 5 isolates based on their

pathogenicity potential for critical assays in vivo which exhibited ˃ 85% and ˃ 75% larval and

adult mortality respectively. Via this hierarchical approach we ultimately identified three B.

bassiana (WG-41, W-42 and WG-43) and two M. anisopliae (WG-44, WG-45) isolates,

exhibited shortest LC50 of 105 conidia ml-1 with potential as a novel bio-pesticide for use against

R. ferrugineus.

The conidial viability is very important in the host infection process because it permits

success at the beginning of the early stages of fungal infection process of the host cuticle,

followed by conidial germination and the formation of a germ tube (Schrank and Vainstein,

2010). In the present study, the conidia viability of the 19 isolates was 100%, which ensured the

quality of conidia present in the fungal suspensions used to treat R. ferrugineus larvae. The re-

isolation of fungal isolates after treatment confirmed the infection capacity of the studied

isolates. Our results indicated the B. bassiana isolates (WG-41, WG-42) exhibited high larval

and adult mortality, suggesting an enormous potential for this fungal species to be used for pest

control. Moreover, five isolates presented the better results, with average almost 100% larval and

adult mortality by the 21st day post treatment at highest dose rate used and they should be

considered as ideal isolates to be used in formulations for field studies, exhibited LC50 of high

virulence range within 6.47×105-3.66×106 conidia ml-1 for larvae and 1.04×106-9.30×106 conidia

ml-1. These LC50 values are practical for the development of a myco-insecticide aimed to control

R. ferrugineus in integrated management programs.

The individuation of virulent strains of entomopathogenic fungi towards the R.

ferrugineus in the countries of introduction represent a precious opportunity to increase studies

on the microbiological control efficacy in view of a possible field applications. Within fungal

taxa, individual isolates can exhibit substantially restricted host range (Inglis et al., 2001) and

isolates recovered from a target host and closely related species are generally more virulent than

isolates from non-related species or from soil. Ricaño et al. (2013) reported high efficacy of B.

bassiana isolates recovered from RPW as compared to the other sources (Insects and soil

samples). Moreover, B. bassiana strains recovered from 30 day old RPW were most pathogenic

strains among all tested isolates. These isolates were also the most virulent on RPW adults. Lo

Verde et al. (2014) also reported the significantly more pathogenic action of RPW isolated B.

bassiana strains. El-Sufty et al. (2009) obtained a mortality of 12.8-47.1% in adult R.

ferrugineus population in field assays using a strain of B. bassiana isolated in the United Arab

Emirates. These published reports are supportive to our findings as our results showed that B.

bassiana isolates (WG-41, WG-42) recovered from RPW cadavers inflicted highest mortality.

Further corroborating our results, an indigenous strain of B. bassiana obtained from mycosed

RPW collected in field showed good results in laboratory and field tests (El-Sufty et al., 2007;

Sewify et al., 2009). Our results are in accordance with Cherry et al. (2005) who reported,

indigenous isolates that had been recovered from C. maculatus were more virulent in laboratory

bioassays against C. maculatus than exotic isolates from other insects. Similarly Goettel et al.

(1990) reported, a fungal isolate may be more pathogenic against the host from which it was

obtained than to other novel hosts.

Contrarily, Monteiro et al. (1998a, b) reported that an ant derived isolate was more

effective against ticks than a tick derived isolate. Ángel-Sahagún et al. (2010) indicate that

isolates with high pathogenicity against ticks could be found from samples derived from soil on

the last instars of the great wax moth, Galleria mellonella. Fernandes et al. (2011), observed that

96

several B. bassiana isolates obtained from naturally infected ticks were not significantly more

virulent against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) than isolates obtained

from other arthropod orders. With regard to entomopathogenic fungi important factors in

reaching these goals are: intra alia, the availability of an isolate highly virulent towards the

target insect, suitability for mass spore production on an appropriate contamination substratum,

an efficient delivery system and inoculum stability and germinability in field conditions (Ibrahim

et al., 1999; Zhang et al., 2011).

On contrary, Gindin et al. (2006) observed high mortality of R. ferrugineus adults treated

with dry spores of M. anisopliae than the B. bassiana treatments after two weeks. Tests carried

out with the experimental traps showed that M. anisopliae was the more virulent pathogen,

causing 75% cumulative mortality in R. ferrugineus adults, while B. bassiana gave a 45%

cumulative mortality (Francardi et al., 2103). Similar findings were reported by the Francardi et

al. (2012) who evaluated field collected strain of B. bassiana and M. anisopliae isolated in Italy

from naturally infected R. ferrugineus adults, and tested under laboratory conditions. M.

anisopliae obtained from R. ferrugineus showed the highest efficacy against RPW larvae and

adults which showed values of cumulative larval mortality of 100% and adult mortality of 90%.

Our study indicated that the indigenous strain of B. bassiana recovered from R.

ferrugineus were more infective to larvae and adult of R. ferrugineus. This study was further

supported by the findings of Dembilio et al. (2010b) who reported potential effect of indigenous

strain of B. bassiana against different developmental stages of R. ferrugineus. The use of

entomopathogenic fungi, in particular indigenous strains of B. bassiana and M. anisopliae,

obtained from naturally infected weevils, should be seriously considered for biological control

because both have provided encouraging results for the control of certain economic pests

(Jaronski, 2010). This suggests that the identification of indigenous entomopathogenic fungi

already active on the weevil may offer better prospects for its biological control.

Conclusion

The present study showed that B. bassiana and M. anisopliae isolates recovered from the

RPW used in laboratory bioassays caused high mortality in larvae and adults compared to the

other tested fungal isolates. More number of mycosed individuals was also observed from the

same isolates. For these reasons, the use of entomopathogenic fungi can be considered to be

useful tool as an integral part of successful IPM program. Moreover, the inoculum of

entomopathogenic fungi may be transmitted by R. ferrugineus due to their low mortality which

may further be helpful in reducing RPW population in date palm systems, further research is

needed to support this thought.

Acknowledgements

This research work was supported by Higher Education Commission, Islamabad

(Pakistan) (2AV1-263) under Indigenous Ph.D. Fellowship Program.

97

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103

Table 5.1 Characterization of B. bassiana and M. anisopliae isolates obtained from soils and insect cadavers

*Geographical attributes based on web source indicating the nearest point

Species Isolate

No.

Host/substrate Location Geographical attributes

Altitude (m)* Latitude* Longitude*

Metarhizium anisopliae WG-02 Soil (Vegetables) Changa Manga 191 31°08′N 73°96′E

Metarhizium anisopliae WG-03 Tribolium castaneum Murree 2300 33°56'N 73°28'E

Metarhizium anisopliae WG-04 Soil (Vegetables) Chichawatni 159 30°53′N 72°70′E

Metarhizium anisopliae WG-05 Rhyzopertha dominica Khanewal 128 30°71'N 71°55'E

Metarhizium anisopliae WG-06 Soil (Forests) Lal Sohanra 114 29°28′N 71°58′E

Metarhizium anisopliae WG-07 Soil (Forests) Bahawalpur 109 29°24'N 71°40'E

Metarhizium anisopliae WG-10 Soil (Crop fields) Rawalpindi 497 33°58′N 73°08′E

Beauveria bassiana WG-11 Soil (Crop fields) Lal Sohanra 114 29°28′N 71°58′E

Beauveria bassiana WG-12 Soil (Fruits) Chichawatni 159 30°53′N 72°70′E

Beauveria bassiana WG-13 Sitophilus oryzae Changa Manga 191 31°08'N 73°96'E

Beauveria bassiana WG-15 Soil (Forests) Faisalabad 184 31°30′N 73°05′E

Beauveria bassiana WG-16 Tribolium castaneum Sargodha 193 32°10'N 72°40'E

Beauveria bassiana WG-17 Callosobruchus maculates Gujranwala 223 32°10'N 72°12'E

Beauveria bassiana WG-18 Soil (Forests) Rawalpindi 497 33°58′N 73°08′E

Beauveria bassiana WG-41 Rhynchophorus ferrugineus Layyah 143 30°58'N 70°56'E

Beauveria bassiana WG-42 Rhynchophorus ferrugineus Dera Ismail Khan 166 31°49'N 70°52'E

Beauveria bassiana WG-43 Rhynchophorus ferrugineus Bahawalpur 109 29°24'N 71°40'E

Metarhizium anisopliae WG-44 Rhynchophorus ferrugineus Layyah 143 30°58'N 70°56'E

Metarhizium anisopliae WG-45 Rhynchophorus ferrugineus Muzaffargarh 114 30°50'N 71°54'E

104

Table 5.2 Factorial analysis of screening and mycosis of R. ferrugineus exposed to B. d M.

anisopliae isolates

S.O.V.

Df

Mortality Mycosis

Larvae Adult Larvae Adult

F P F P F P F P

Treatment 1 158.20 ≤0.05 733.72 ≤0.05 178.89 ≤0.05 733.72 ≤0.05

Isolate 18 146.69 ≤0.05 110.84 ≤0.05 18.28 ≤0.05 110.84 ≤0.05

Treatment ×

Isolate

18 29.90 ≤0.05 8.49 ≤0.05 1.87 0.02 8.49 ≤0.05

Error 185 - - - - - - - -

Total 227 - - - - - - - -

Table 5.3 Percentage pathogenicity (%±SE) and mycosis (%±SE) of 19 isolates of B.

bassiana and M. anisopliae isolates against R. ferrugineus larvae after 12 days

post incubation

Isolate 1×107 Conidia ml-1 1×108 Conidia ml-1

% Mortality % Mycosis % Mortality % Mycosis

WG-02 3.49±0.21j 0.00±0.00j 8.01±0.66k 17.8±0.87h

WG-03 9.90±0.89ghij 3.50±0.42ij 25.23±1.12hi 47.16±1.68g

WG-04 18.35±1.31fghi 12.50±0.56gh 48.25±1.48ef 87.33±1.42bc

WG-05 21.69±1.17 33.33±0.71d 55.23±2.16de 93.66±1.35ab

WG-06 9.90±0.85ghij 15.83±0.94g 27.61±1.55ghi 63.83±1.90e

WG-07 13.75±1.09fghij 21.33±1.22f 35.63±1.62fgh 54.50±1.54f

WG-10 15.88±0.98fghij 25.83±0.94e 41.50±1.19efg 73.66±2.04d

WG-11 5.46±0.34ij 4.50±0.42i 24.12±1.38hij 42.50±1.17g

WG-12 8.96±1.09ghij 10.66±0.84h 29.92±1.52ghi 56.66±1.42f

WG-13 16.99±1.08fghij 31.33±1.22d 49.28±1.43ef 88.50±1.70bc

WG-15 4.27±0.24j 3.50±0.42ij 9.04±0.39jk 15.33±1.04h

WG-16 26.21±1.40def 56.66±1.38c 56.42±2.16cde 95.50±1.78a

WG-17 19.46±1.21efgh 32.16±1.25d 46.98±1.70ef 93.33±1.71ab

WG-18 7.68±0.53hij 9.50±0.76h 19.44±1.17ijk 41.66±1.28g

WG-41 53.71±1.28a 72.50±2.78a 88.33±2.47a 96.50±1.99a

WG-42 46.80±1.07ab 65.33±1.45b 79.04±1.54ab 95.66±1.62a

WG-43 35.52±1.55bcd 56.66±1.88c 71.27±1.69bc 87.33±1.38bc

WG-44 41.33±1.21abc 61.83±1.67b 74.68±2.56ab 84.66±1.67c

WG-45 32.11±1.60cde 53.50±1.99c 65.55±1.47bcd 90.66±1.73abc

105

Table 5.4 Percentage pathogenicity (%±SE) and mycosis (%±SE) of 19 isolates of B. M.

anisopliae isolates against R. ferrugineus adults after 12 days post incubation

Isolate 1x107 Conidia ml-1 1x108 Conidia ml-1

% Mortality % Mycosis % Mortality % Mycosis

WG-02 0.00±0.00i 0.00±00m 5.63±0.65k 4.83±0.30l

WG-03 4.44±0.22ghi 2.33±0.33lm 20.65±1.12hij 47.50±1.23i

WG-04 16.94±1.09def 8.50±0.42jk 32.06±1.37efgh 72.50±1.88ef

WG-05 21.58±1.05de 29.66±1.26g 38.88±1.88ef 74.16±2.10rf

WG-06 5.55±0.64ghi 6.66±0.66kl 21.82±1.00ghij 43.33±1.40i

WG-07 8.96±1.06fghi 11.50±0.69j 23.96±1.63ghi 56.50±1.52h

WG-10 10.15±1.10fghi 16.33±1.08i 27.54±1.42fghi 65.83±1.90g

WG-11 0.00±0.00i 0.00±00m 17.06±1.09ijk 30.33±1.08j

WG-12 3.33±0.37ghi 1.66±0.22m 21.74±1.29ghij 48.50±1.76i

WG-13 11.34±0.81efgh 17.50±1.08hi 35.47±1.18efgh 71.33±1.54fg

WG-15 0.00±0.00i 0.00±00m 8.968±0.88jk 16.16±1.10k

WG-16 21.50±1.21de 36.66±1.14f 43.49±2.01de 78.66±2.52de

WG-17 13.65±1.03efg 21.50±0.98h 36.58±1.85efg 77.50±1.85def

WG-18 2.22±0.24hi 0.00±00m 13.65±1.07ijk 31.16±1.24j

WG-41 41.90±1.88a 78.16±1.19a 75.95±2.78a 95.33±1.62a

WG-42 36.19±1.36ab 62.83±1.42b 69.04±2.73ab 87.16±2.12bc

WG-43 27.22±1.16bcd 55.83±1.37c 55.00±1.37bcd 91.50±2.14ab

WG-44 32.93±1.65abc 49.66±1.54d 59.68±2.93bc 83.33±1.70cd

WG-45 24.92±1.04cd 41.50±1.20e 46.03±2.07cde 76.83±1.44ef

106

Table 5.5 Factorial analysis for virulence of B. bassiana and M. anisopliae isolates against

larvae and adult of R. ferrugineus

S.O.V. df Larvae Adult

F P F P

Isolate 4 153.27 ≤0.05 163.71 ≤0.05

Treatment 3 2192.69 ≤0.05 2202.71 ≤0.05

Interval 2 2700.31 ≤0.05 2852.28 ≤0.05

Isolate × Treatment 12 1.24 0.25 1.94 0.02

Isolate × Interval 8 0.66 0.72 1.92 ≤0.05

Treatment × Interval 6 90.92 ≤0.05 78.87 ≤0.05

Isolate × Treatment ×

Interval

24 6.26 ≤0.05 5.60 ≤0.05

Error 472 - - - -

Total 539 - - - -


exposure treated with B. bassiana and M. anisopliae isolates

Stage Isolates Dose (Conidia ml-1)

106 107 108 109

Larvae

WG-41 19.52±1.11Ca 26.57±1.36Ca 47.17±2.12Ba 76.95±2.41Aa

WG-42 16.56±1.27Cab 21.23±1.41Cab 40.29±2.19Bab 65.73±2.28Ab

WG-43 11.21±1.03Cab 15.82±1.25Cb 28.79±1.53Bcd 52.48±1.78Ac

WG-44 13.43±1.32Cab 19.75±1.37Cab 36.60±1.26Bbc 58.62±1.90Abc

WG-45 8.14±1.15Cb 13.49±1.20Cb 22.71±1.04Bd 43.54±1.50Ad

Adult

WG-41 12.69±1.32Ca 19.47±1.23Ca 38.30±1.11Ba 63.96±1.78Aa

WG-42 9.68±1.10Cab 15.02±1.28Cab 32.22±1.69Bab 54.76±1.66Ab

WG-43 5.92±1.03Cab 10.47±1.08Cbc 21.79±1.45Bcd 40.63±1.62 Acd

WG-44 8.14±1.20Cab 12.69±1.19Cabc 26.98±1.17Bbc 47.35±2.09Abc

WG-45 2.91±0.45Cb 7.46±0.76Cc 18.73±1.14Bd 35.23±1.56Ac

107


exposure treated with B. bassiana and M. anisopliae isolates


106 107 108 109

Larvae

WG-41 37.35±1.19Ca 58.83±2.22Ba 93.28±1.92Aa 100.0±0.00Aa

WG-42 31.95±1.37Dab 49.68±2.06Cab 84.70±2.34Bab 98.46±2.01Aab

WG-43 23.49±1.26Dbc 38.78±1.37Cc 73.12±2.58Bc 92.38±1.78Ab

WG-44 27.46±1.18Dbc 43.49±1.73Cbc 78.46±2.84Bbc 95.45±1.59Aab

WG-45 19.78±1.07Dc 35.02±1.26Cc 71.69±2.19Bc 84.60±1.34Ac

Adult

WG-41 28.04±1.14Ca 47.40±1.60Ba 84.76±2.90Aa 93.22±1.97Aa

WG-42 21.27±1.46Dab 41.16±1.34Cab 76.98±2.51Bab 86.87±2.26Aa

WG-43 14.44±1.08Cbc 35.82±1.34Bbc 67.19±1.98Abc 75.29±2.49Abc

WG-44 17.46±1.19Db 38.14±1.48Cbc 70.05±1.54Bb 83.12±2.10Aab

WG-45 8.30±0.78Dc 32.06±1.96Cc 57.24±1.46Bc 71.53±1.51Ac


exposure treated with B. bassiana and M. anisopliae


106 107 108 109

Larvae

WG-41 57.67±2.00Ca 88.50±2.15Ba 100.0±0.00Aa 100.0±0.00Aa

WG-42 52.16±1.56Cab 83.16±2.00Bab 100.0±0.00Aa 100.0±0.00Aa

WG-43 37.54±1.34Cc 65.93±2.16Bc 94.70±1.86Aa 100.0±0.00Aa

WG-44 46.88±2.09Cb 77.75±2.86Bb 98.41±1.64Aa 100.0±0.00Aa

WG-45 31.40±1.31Dc 59.10±2.00Cc 85.49±2.47Bb 100.0±0.00Aa

Adult

WG-41 49.78±2.05Ca 80.79±2.73Ba 100.0±0.00Aa 100.0±0.00Aa

WG-42 41.48±1.34Cab 72.32±2.62Bab 96.98±2.13Aab 100.0±0.00Aa

WG-43 30.74±1.94Dcd 59.15±1.93Cc 85.55±2.27Bc 98.41±1.94Aa

WG-44 38.30±1.55Dbc 68.62±2.32Cb 91.53±2.55Bbc 100.0±0.00Aa

WG-45 22.22±1.29Dd 43.86±1.61Cd 76.08±2.15Bd 93.81±2.40Ab

108

Table 5.9 LC50 and LC90 values of B. bassiana and M. anisopliae isolates tested against larvae and adult R. ferrugineus

Stage Isolate LC50 (Conidia ml-1) (CI) LC90

(Conidia ml-1) (CI) Slope Intercept ᵪ2 (df = 2) P

Larvae

WG-41 6.42×105 (3.61×105-9.49×105) 6.52×106 (4.51×106-1.03×107) 0.54±0.07 -6.84 0.53 <0.01

WG-42 9.46×105 (5.67×105-1.39×106) 1.34×107 (8.95×106-2.36×107) 0.47±0.05 -7.95 3.56 <0.01

WG-43 2.22×106 (1.50×106-3.11×106) 3.90×107 (2.52×107-6.81×107) 0.44±0.06 -10.11 6.98 <0.01

WG-44 1.24×106 (7.92×105-1.76×106) 1.81×107 (1.20×107-3.15×107) 0.46±0.05 -8.65 6.05 <0.01

WG-45 3.63×106 (2.41×106-5.22×106) 1.01×108 (6.21×107-1.80×108) 0.37±0.04 -10.81 4.07 <0.01

Adult

WG-41 1.04×106 (6.35×105-1.51×106) 1.53×107 (1.02×105-6.81×104) 0.47±0.05 -8.21 4.68 <0.01

WG-42 1.61×106 (1.02×106-2.35×106) 3.34×107 (2.16×107-5.95×107) 0.42±0.06 -9.44 3.27 <0.01

WG-43 3.92×106 (2.61×106-5.64×106) 1.23×108 (7.55×107-2.30×108) 0.37±0.04 -10.9 4.62 <0.01

WG-44 2.13×106 (1.33×106-3.15×106) 6.25×107 (3.90×107-1.15×108) 0.37±0.03 -9.88 2.16 <0.01

WG-45 9.28×106 (6.51×106-1.25×107) 2.63×108 (1.60×108-5.02×108) 0.38±0.03 -12.24 10.55 <0.01

109

Table 5.10 LT50 and LT90 values of B. bassiana and M. anisopliae isolates tested against larvae of R. ferrugineus

Isolate Dose LT50 (Conidia ml-1)

(CI)

LT90 (Conidia ml-1)

(CI)

Slope Intercept x2 (df =2) P

WG-41

106 17.75 (15.90-20.33) 35.70 (30.34-45.82) 0.07± 0.01 -7.07 0.00 ˂0.01

107 11.62 (10.41-12.69) 21.79 (20.04-24.26) 0.12± 0.01 -8.02 0.30 ˂0.01

108 7.14 (6.07-7.98) 12.89 (11.88-14.33) 0.22± 0.02 -6.15 0.16 ˂0.01

109 6.01* 7.66* 0.77±108.49 -0.01 0.00 0.99

WG-42

106 19.64 (17.55-22.96) 38.08 (32.03-49.87) 0.06±0.01 -7.43 0.03 ˂0.01

107 13.24 (12.09-14.35) 23.84 (21.86-26.69) 0.12±0.01 -8.70 0.57 ˂0.01

108 8.16 (7.10-9.03) 14.77 (13.66-16.28) 0.19±0.02 -7.01 1.39 ˂0.01

109 5.27 (3.61-6.26) 10.40 (9.45-11.95) 0.24±0.04 -3.74 0.00 ˂0.01

WG-43

106 25.22 (21.64-32.94) 47.08 (37.54-69.54) 0.05±0.01 -7.59 0.01 ˂0.01

107 16.35 (15.00-17.94) 29.66 (26.45- 34.79 0.09±0.01 -8.48 0.00 ˂0.01

108 10.07 (8.96-11.03) 18.41 (17.08-20.19) 0.15±0.01 -8.06 0.24 ˂0.01

109 6.53 (5.19-7.52) 12.95 (11.88-14.48) 0.19±0.02 -5.22 0.34 ˂0.01

WG-44

106 21.53 (19.11-25.73) 40.25 (33.55-53.72) 0.06±0.01 -7.77 0.04 ˂0.01

107 14.34 (13.15-15.57) 25.88 (23.53-29.39) 0.11±0.01 -8.67 0.94 ˂0.01

108 8.85 (7.72-9.79) 16.44 (15.23-18.08) 0.16±0.01 -7.33 0.44 ˂0.01

109 5.81 (4.30-6.84) 11.89 (10.85-13.42 0.21±0.02 -4.45 0.20 ˂0.01

WG-45

106 27.92 (23.52-38.35) 50.31 (39.43-77.72) 0.05±0.01 -7.80 0.11 ˂0.01

107 17.86 (16.35-19.85) 32.19 (28.33-38.66) 0.08±0.01 -8.46 0.02 ˂0.01 108 11.21 (9.98-12.29) 21.19 (19.54-23.50) 0.12±0.01 -7.87 7.41 ˂0.01

109 7.82 (6.66-8.75) 14.74 (13.61-16.30) 0.18±0.02 -6.51 1.66 ˂0.01

*Unable to estimate confidence limits from the data

110

Table 5.11 LT50 and LT90 values of B. bassiana and M. anisopliae isolates tested against adult of R. ferrugineus

Isolate Dose LT50 (Conidia ml-1)

(CI)

LT90 (Conidia ml-1)

(CI)

Slope Intercept x2 (df = 2) P

WG-41

106 20.41 (18.46-23.42) 36.38 (31.26-45.68) 0.08± 0.01 -8.43 0.00 ˂0.01

107 13.89 (12.77-15.00 24.38 (22.37-27.26) 0.12±0.01 -9.09 0.24 ˂0.01

108 8.47 (7.49-9.29) 14.79 (13.71-16.25) 0.20±0.02 -7.49 1.15 ˂0.01

109 4.93 (2.82-6.29) 12.35 (11.19-14.02) 0.17±0.02 -3.41 0.91 ˂0.01

WG-42

106 23.17 (20.65-27.56) 39.98 (33.70-52.11) 0.07±0.01 -8.61 0.031 ˂0.01

107 15.55 (14.40-16.81) 26.73 (24.35-30.27) 0.11±0.01 -9.34 0.00 ˂0.01

108 9.53 (8.46-10.45) 17.20 (15.97-18.83) 0.16±0.01 -7.97 0.04 ˂0.01

109 6.36 (4.71-7.53) 14.12 (12.91-15.82) 0.16±0.02 -4.72 1.88 ˂0.01

WG-43

106 26.96 (23.45-34.03) 44.57 (36.59-61.63) 0.07±0.01 -8.67 0.00 ˂0.01

107 17.90 (16.55-19.61) 30.46 (27.25-35.54) 0.10±0.01 -9.23 1.03 ˂0.01

108 11.81 (10.66-12.84) 21.50 (19.87-23.76) 0.13±0.01 -8.43 4.45 ˂0.01

109 8.68 (7.43-9.69) 16.89 (15.59-18.66) 0.15± 0.01 -6.82 1.93 ˂0.01

WG-44

106 24.25 (21.55-29.12) 40.79 (34.31-53.47) 0.07±0.01 -8.80 0.22 ˂0.01

107 16.38 (15.20-17.72) 27.69 (25.15-31.48) 0.11±0.01 -9.52 0.04 ˂0.01

108 10.72 (9.59-11.71) 19.63 (18.19-21.58 0.14±0.01 -8.20 0.98 ˂0.01

109 7.52 (6.21-8.54) 14.99 (13.80-16.64) 0.17±0.01 -5.97 2.61 ˂0.01

WG-45

106 30.06 (25.65-39.89) 47.23 (38.10-68.46) 0.07± 0.01 -8.60 0.03 ˂0.01

107 21.17 (19.18-24.31) 36.51 (31.45-45.68) 0.08±0.01 -8.67 4.74 ˂0.01 108 13.54 (12.32-14.72) 24.90 (22.70-28.12) 0.11±0.01 -8.34 3.70 ˂0.01

109 9.57 (8.268-10.66) 18.97 (17.48-21.02) 0.13±0.01 -7.02 0.00 ˂0.01

111

CHAPTER 6

Combined effectiveness of endophytically colonized Beauveria bassiana and Bacillus

thuringiensis against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

Abstract Research study was carried out to investigate the insecticidal properties of endophytically

colonized Beauveria bassiana and Bacillus thuringiensis var. kurstaki (Bt-k) against 2nd, 4th and

6th instar larva of red palm weevil (RPW) Rhynchophorus ferrugineus. Initially five isolates of B.

bassiana (WG-11, WG-40, WG-41, WG-42 and WG-43) were screened by inoculating

endophytically in date palm leaf petioles. Only one B. bassiana isolate (WG-41) recovered from

up to the 10 cm after 30 days during both years was considered to be effective. Both agents were

applied alone and in combination to tested instars and pupation, adult emergence and egg

eclosion was recorded from survivers. Moreover development, diet consumption, frass

production and weight gain were also observed. Mortality was low in sole treatments, while in

combined treatments increase in mortality, decrease in pupation, adult emergence and egg

eclosion found inversely correlated to toxic levels of both microbial agents. Second instar larvae

exhibited more susceptibility followed by 4th and 6th instar larvae. Synergistic effect (CTF≥20)

on the mortality was observed when larvae were exposed to simultaneous application of WG-41

with 40 µg ml-1 of Bt-k in case of all three larval instars tested. All the tested instars exhibited

varying level of growth and development when exposed to the sub-lethal doses of palm petiole

piece (6 cm away from inoculated point) inoculated with WG-41 and dipped in Bt-k

concentrations; moreover significant variations were recorded for larval duration, larval weight,

pupal duration, pupal weight, pre-pupal duration, pre-pupal weight, adult longevity (male and

female) and adult weight (male and female). The toxic nature of microbial agents also influenced

the frass production and diet consumption. Larvae treated with Bt-k gained more weight than the

WG-41 and their combined application. Initial weight of larvae exerted its impact on the weight

gain and diet consumption and the trend was found linked to pathogenicity of applied agents. It

can be surmised from the findings that microbial agents exhibit a reliable level of mortality

against R. ferrugineus. Hence it would be fruitful to replace the conventional reliance on

chemical approaches.

Keywords: Rhynchophorus ferrugineus, Beauveria bassiana, Bacillus thuringiensis,

development, diet consumption, frass production

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6.1 Introduction


Curculionidae) is considered one of the most destructive pests of variety of palm species,

including date palms (Giblin-Davis, 2001). The weevil lives and breed into the tree trunk and

devastate its vascular system which lead to the tree collapse and death of host plant. Until 1917,

it was considered that RPW is the only pest of coconut palm but latter on it was found

devastating date palms in Pakistan, India and Iraq (Mohan, 1917; Milne, 1918; Buxton, 1920). In

Pakistan infestation of the RPW was observed during 1913 when researcher found the beetle

attack on the date palm trees imported from Middle East and later on Milne an Economist of

Punjab Agriculture College, Lyallpur (Presently University of Agriculture, Faisalabad, Pakistan)

observed RPW infestation in date palms and collected insect specimens from Multan,

Muzaffargarh and Dera Ghazi Khan Districts of Punjab (Milne, 1918). Since last 30 years RPW

has caused huge economic losses and no effective control measure has been invented so for

(Murphy and Briscoe, 1999; Faleiro, 2006).

For the successful control of RPW trapping, deployment of chemical insecticides and

fumigants has been considered a core component since long time. The unwise use of these

chemical insecticides and fumigants lead the resistance against this voracious pest which lead the

researchers to search for the alternative control strategies which are safer for human beings and

compatible to environment (Abraham et al., 1998). Moreover, RPW live in the self-made tunnels

which make them less vulnerable to Chemical and mechanical control. Thus, self-pathogens have

been proposed for the successful control of RPW (Dangar, 1997). Entomopathogens are the

safer, environment friendly and economic alternative to the chemical insecticides and getting

serious attention against RPW control. Fungal entomopathogens play a key role in managing

plant pathogens and herbivorous insects by improving the plant host defense mechanism or by

directly affecting plant pathogens (Sivasithamparam, 1998; Arnold et al., 2003).

Entomopathogenic fungi are preferred to the other entomopathogens due to its unique mode of

action by infecting their host through contact action and penetrating into insect hemocoel by

breaching the host cuticle. Fungal infection can be transformed by direct contact of infected

individuals to the healthy ones or subsequent development via new generation of spores (Lacey

et al., 1999; Quesada-Moraga et al., 2004).

On the other hand, an entomopathogenic bacterium from the genus Bacillus is also an

economical and potential alternative to the chemical insecticides. They are key antagonists of

insect pests of economic importance, their products and by products (Salama et al., 2004). It is

often an integral part of products used in biological control strategies worldwide, about 95%

microbial pesticides being used globally are bacterial in origin with annual sale of about $100

million (Federici et al., 2006). A number of species of this genus particularly Bacillus

thuringiensis (Berliner) are frequently used against vast array of insect pests from the order

Coleoptera, Lepidoptera and Diptera etc, which exhibit specificity towards the host and specific

stage of the host (Salama et al., 2004). It is a gram-positive soil bacterium, spore forming

mesophile having ability to produce proteinaceous parasporal inclusions during sporulation. It

produces δ-endotoxins in the form parasporal crystals which may vary from one to several types

and formed from different δ-endotoxins that are related to each other (Aronson et al., 1986).

So for more than 350 genes from B. thuringiensis has been discovered which are encoded for

specific toxic proteins which are specific to the larvae of various orders (Schnepf et al., 1998).

Both pathogens are widely distributed in the environment and found from the soil of

different origin and insect cadavers (Martin and Travers, 1989). The intervention of more than

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one biocontrol agent can enhance the effectiveness of the other partner, many studies have been

conducted in this regard. The combined effect of B. bassiana and B. thuringiensis working

synergistically delivers more harm to insect pests (Wraight and Ramos, 2005) and hence can be a

hint for those willing to manage it. Combine effect of B. thuringiensis and Entomopathogenic

fungi has synergistic effect performed by several researchers (Navon, 2000). Wraight and Ramos

(2005) observed that combinations of B. thuringiensis and B. bassiana have been successful in

increasing mortality in some insects. Sandner and Cichy (1967) applied a mixture of B.

thuringiensis var. kurstaki (Bt-k) and B. bassiana against larvae of the Mediterranean flour moth.

Results revealed that the two agents acted independently (mean mortalities from B. bassiana, B.

thuringiensis and the mixture were 57, 44, and 71% respectively).

The present study aiming at the endophytically colonizing B. bassiana isolates in date

palm and their evaluation against R. ferrugineus alone and in integrated manners with B.

thuringiensis



Different developmental stages (larvae, pupae and adult) of RPW were collected from

fallen and infested date palm trees with the permission of farmers (owners) from date palm

growing areas of west Punjab, Pakistan. During collections, adult, larvae and pupae were kept in

separate plastic jars until brought to the laboratory Microbial Control Laboratory, Department of

Entomology, University of Agriculture, Faisalabad, Pakistan. After arriving to the laboratory

larvae were provided with sugarcane (Saccharum officinarum L.; Poales: Poaceae) stems for

feeding and pupation, while adults were offered shredded sugarcane pieces for feeding and

oviposition substrates. On pupation, pupal cocoons were kept in separate boxes for adult

emergence in incubator at 25±2 oC, 65±5% RH and 12:12 (D: L) hours photoperiod. As adults

emerged, they were transferred to the adult’s jars for feeding and mating with shredded sugracne

pieces. Colony was developed in plastic boxes (15×30×30 cm) having a lid covered with mesh

wire gauze (60 mesh size) in the middle (7 cm diameter) for aeration. Rearing was carried out in

Microbial Control Laboratory. The rearing conditions were maintained as mentioned above.

Adult’s diet was changed after every three days and replaced sugarcane pieces were kept in

separate jars (8×8×12 cm) for egg hatching. On egg hatching, neonate larvae were allowed to

feed for 3 days in the same sugarcane set and then were transferred to the sugarcane sets for

feeding and pupation. Larvae were shifted to the new sugarcane sets biweekly until pupation.

6.2.2 Fungal isolates

Five isolates of B. bassiana used in the study belonging to the culture collection of

Microbial Control Laboratory, originally isolated from soil samples from crop fields (WG-11),

recovered endophytically from tomato plants (WG-40) and R. ferrugineus cadavers (WG-41,

WG-42 and WG-43). The cultures were maintained continuously on slants and sub-cultured for

14 days at 25±2 °C under a 12:12 (D: L) hours, tubes were tightly sealed and the culture was

stored at 4 °C. Mass culturing was done by inoculating Petri plates containing Sabourad

Dextrose Agar (SDA, from BD, Becton, Dickinson and Company Spark, MD 21152 USA)

media. Spore concentration of 1×106 spore ml-1 was determined with a Neubauer

haemocytometer.

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6.2.3 Preparation of Bacillus thuringiensis spore-crystal mixtures

The commercial formulation of B. thuringiensis var. kurstaki (Bt-k) was obtained from

Microbial Control Laboratory, originally obtained from National Center for Genetic Engineering

and Biotechnology (BIOTEC) in Thailand. This strain was then subjected to sporulation by

culturing in 50 ml nutrient broth media. Harvesting of culture was carried out by centrifugation

at 6000 rpm for 15 min (Crecchio and Stotzky, 2001; Hernández et al., 2005). The pellets formed

resultantly were washed twice in cold 1M NaCl and thrice in sterile distilled water (SDW), re-

suspended in distilled water (5 ml). From the suspension formed, 1 ml was centrifuged for 5 min

at 10,000 rpm, dried for 4 hours at 37 °C and weighed (Wakil et al., 2013).

6.2.4 Screening of fugal isolates

Date palm trees less than one year old were selected in the date palm plantations located

at Faisalabad during 2014 and 2015. Inoculation of B. bassiana isolates were carried out

following the method of Gómez-Vidal et al., (2006). From each palm, three petioles were rubbed

with 70% ethanol and 30 µl of conidial suspension was injected using insulin syringe. To avoid

sun drying and external contamination the inoculated area were wrapped with Parafilm. For re-

isolation, after 15 and 30 days post inoculation, petioles were sampled from inoculation site, 2, 4,

6, 8 and 10 cm above or below the inoculation site with the help of sterilize cork borer (0.6 cm

diameter). The samples were surface-sterilized with 1% sodium hypochlorite for 1 min, followed

by rinsing three times with sterile distilled water (Gómez-Vidal et al., 2006). Samples were then

placed on PDA for 8 days at 25±2 ͦ C for spore germination. Different fungi were recovered from

petiole pieces that were further purified by sub-culturing on SDA medium, and fungal isolates

were identified following the keys developed by Barnett and Hunter (1999).

6.2.5 Bioassay procedure

To evaluate the effect of endophytically colonized B. bassiana WG-41, a piece of

inoculated palm petiole (2×2 cm2), 2 cm away from the inoculated site was offered to the 2nd, 4th

and 6th instar larvae of R. ferrugineus separately in 150 ml plastic cups measuring (6×6 cm)

individually. While in control treatment untreated palm petiole was offered to the respective

larval stages. The larvae were allowed to feed on palm piece for 48 hours and then shifted to

artificial diet (Martín and Cabello, 2006) for rest of the period and provided with fresh diet every

day. For Bt-k treatments, three concentrations (30, 40 and 50 µg ml-1) were prepared. An

untreated piece of date palm petiole (2×2 cm2) immersed in respective doses of Bt-k suspension

for 90s and offered to the respective larval stages individually. The larvae were allowed to feed

on treated palm piece for 48 hours and shifted to the artificial diet for rest of the period

individually. In combined treatments WG-41 inoculated date palm petiole piece was immersed in

respective doses of Bt-k suspensions for 90s and then offered to the larvae. After last instar dry

coir was provided for pupation to the last instar larvae. Larval mortality was counted daily until

larvae pupated or died. Larvae that failed to respond on slight prodding by blunt needle were

considered as dead. From the surviving individual percent pupation, adult emergence and egg

eclosion were also recorded. Three replicates of ten larvae were used for each treatment and

same count of larvae fed on normal petiole served as untreated check. All the treatments were

incubated at 27±2 °C and 65±5 % RH and a 12: 12 (D: L) hours photoperiod in an incubator

(Sanyo, Japan). Entire experiment was repeated thrice.

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6.2.6 Bioassay on development of R. ferrugineus

To determine the effect of sub-lethal doses of WG-41 and Bt-k on developmental

parameters viz. larval duration, larval weight, pre-pupal duration, pre-pupal weight, adult

longevity (male and female) and adult weight (male and female) was recorded on 4th instar larvae

of R. ferrugineus. The piece of endophytically colonized WG-41 (6 cm away from inoculation

site) and Bt-k (10, 15 and 20 µg ml-1) were applied alone and in combination against 4 th instar

larvae and maintained at above motioned conditions. The larvae were allowed to feed on treated

petiole piece for 48 hours and then shifted to the artificial diet for rest of the period. After last

instar larvae was provided with dry coir (coconut coir) for pupal development. All the above

mentioned parameters were observed hereafter.

6.2.7 Bioassay on larval development

A new batch of 10th instar larvae of R. ferrugineus (L10) were encountered with sub-lethal

dose of WG-41 and Bt-k (10 µg ml-1). A piece of endophytically colonized palm petiole (6 cm

away from inoculated area) alone and in combination with Bt-k was offered to the larvae. The

larvae were allowed to feed for the whole 10th instar on the treated diet. Before exposure to the

palm petiole each larval instar were weighed and transferred to rearing vial with palm piece.

Every day until the larvae pupated or died; larvae were changed to new cups individually and

provided with fresh diet every day. Frass produced during this period was separated from vials

using tip of fine camel hair brush and weighed. Diet left unused in each vial was recovered, dried

in drying oven at 80 °C. Prior to assay, diet in 30 cups was dried to obtain an estimate of the dry

weight. Diet consumption of each larva was determined by subtracting the after feeding mass of

diet from before feeding mass. Moreover, frass production and weight gains during this period

were also determined.



formula and subjected to one way analysis of variance (ANOVA) in Minitab (Minitab, 2002) and

means were separated using Tukey’s Kramer test (HSD) (Sokal and Rohlf, 1995) at 5%

significance level. Type of interaction among combined treatments of Bt-k and WG-41 was

determined by equation; CTF = (Oc-Oe)/ Oe×100, where CTF is the co-toxicity factor, Oc is the

observed mortality (%) in combined application, and Oe the expected mortality (%), that is the

sum of individual mortality (%) encountered in each of the treatments used in the combination

(Mansour et al., 1966). The interactions were categorized as additive, synergistic or antagonistic:

CTF≥20 meaning synergism, CTF>20 - –20 meaning additive, and CTF<-20 meaning

antagonism (Mansour et al., 1966; Wakil et al., 2012). To inspect the impact of microbial agents

on the diet consumption, weight gain and frass production were analyzed by ANCOVA using

initial larval weight and diet consumption as covariates (Janmaat et al., 2014).

6.3 Results

6.3.1 Fungal colonization of date palm petioles

All the fungal isolates successfully colonized endophytically in date palm petioles more

or less from the inoculation point during both years. At the injection site hydrophobic and

necrotic lesions appeared above and below the injection site. Different entomopathogenic fungi

were recovered from the site of injection up to 10 cm above and below. At the injection site all

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the inoculated fungi were recovered (90-100%) from all the plants after 15 days of inoculation

during 2014 and (80-100%) following year (Table 6.1). B. bassiana isolate WG-41 was

recovered 10 cm above and below the injection site, while no entomopathogenic fungi were

recovered from non-inoculating petioles. Non-entomopathogenic fungi were less near the

inoculation site and gradually increased towards the ends in both years.

6.3.2 Toxicity of microbial agents

Toxicity assay was conducted on 2nd, 4th and 6th instar larvae of R. ferrugineus by

deploying B. bassiana inoculated date palm piece and Bt-k alone and in integrated manners.

Significant differences (Table 6.2) were recorded for mortality among different treatments and

larval instars (treatment: F7, 172 =201.87, P≤0.05; instar: F2, 161=63.34, P≤0.05) but non-

significant interaction was recorded for (instar × treatment: F14, 161=1.56, P=0.093). Synergistic

effect (CTF≥20) on the mortality was observed when larvae were exposed to simultaneous

application of endophytic B. bassiana and 30 µg of Bt-k in case of all the three instars tested.

Second instar larvae of R. ferrugineus were more susceptible to both pathogenic agents followed

by 4th and 6th instar larvae in all the treatments tested. Highest level of larval mortality

(83.17±2.28%) was observed in second instar larvae with the simultaneous application of B.

bassiana and Bt-k (50 µg ml-1) followed by 71.01±2.39% when treated with B. bassiana and Bt-k

(40 µg ml-1) and 54.63±2.26% when treated with B. bassiana and Bt-k (30 µg ml-1) (Table 6.3).

Additive effect (CTF≤20) was recorded when tested instar were treated with low and high dose

of Bt-k in integration with endophytic B. bassiana. A similar trend in mortality was recorded for

4th and 6th instar larvae of R. ferrugineus while mortality was found increasing with increase of

concentration of Bt-k. Combined application of endophytic B. bassiana and Bt-k proved more

fatal to all the instar larvae as compared to their sole application. Both microbial agents were

found working additively and synergistically. Percent pupation, adult emergence and egg

eclosion from surviving individuals was found inversely correlated to toxic level of microbial

agents in all instar larvae. Increase in mortality, while decrease in pupation, adult emergence and

egg eclosion was found in concentration dependent manner.

6.3.3. Development of R. ferrugineus

Development of 4th instar larvae was adversely affected by the toxic effect of microbial

agents. When larvae were exposed to different concentrations of Bt-mixed diets and B. bassiana,

significant variations were recorded for larval duration, larval weight, pre-pupal duration, pre-

pupal weight, pupal duration, pupal weight, adult longevity and adult and adult weight (larval

duration: F7, 71 =72.73, P≤0.05; larval weight F7, 71 =47.54, P≤0.05; pre-pupal duration: F7, 71

=53.12, P≤0.05; pre-pupal weight: F7, 71 =33.80, P≤0.05; pupal duration F7, 71 =43.95, P≤0.05;

pupal weight F7, 71 =26.61, P≤0.05; adult longevity (female F7, 71 =58.20, P≤0.05 and male F7, 71

=65.03, P≤0.05); adult weight (male F7, 71 =12.50, P≤0.05 and female F7, 71 =7.76, P≤0.05).

Increase in larval and pupal duration while decrease in pupal weight and adult duration was

recorded depending upon the lethal action of the applied agent. When compared to control,

significantly increased larval and pupal duration was recorded for combined application of Bb

and highest concentration of Bt-k followed by Bb and middle concentration of Bt-k. Similarly

extended pre-pupal and pupal period and reduced adult life span was recorded in concentration

dependent manner. While decrease in pre-pupal and adult weight was also recorded in

concentration dependent manners (Table 6.5).

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6.3.4 Effect on larval development

Diet consumption by 10th instar larvae was significantly influenced by the treatments

applied diet consumption was low in combined treatments as compared to sole applications.

Least diet consumption was recorded for combined treatments followed by B. bassiana and Bt-k.

While, highest diet consumption was recorded for the control treatment (Fig 6.1a). Frass

production was influenced by treatments applied with lowest frass production for combined

treatments of Bt-k and B. bassiana from (0.61±0.05 to 0.00±0.00g) during experimental period.

More frass production was recorded during the initial days of treatments which gradually

decreased to zero before pupation. On the other hand, highest level of frass production was found

in untreated larvae for all the period of last instar larvae till pupation (Fig 6.1b). Larvae treated

with sub-lethal concentrations of B. bassiana and Bt-k gained more weight as compared to their

combined application (Single agent versus combined treatment). Initial weight of larvae (10th

instar: 4.35±0.12g) exerted its impact on the weight gain and among treatments, there was a

trend of weight gain linked to pathogenicity. Combined application of B. bassiana and Bt-k had

an adverse impact on the weight gain and lowest gain (-0.54±.04g) was recorded for the

combined treatment while highest gain (-0.11±.02g) was recorded in untreated larvae (Fig 6.1c).

6.4 Discussion

The endophytic colonization by B. bassiana is commonly practiced to combat voracious

pests of different field crops. Endophytic fungi are also capable of colonizing date palm tissues

after petiole wounding. The B. bassiana isolate WG-41 was recovered from up to 10 cm away

from the inoculation site during both years. The results confirmed that entomopathogenic fungi

survived endophytically and colonized petiole tissue of date palms. Gómez-Vidal et al., (2006)

also confirmed the endophytic colonization of entomopathogenic fungi in live and detached palm

petioles of date palm. They confirmed the movement in the parenchyma and sparsely within

vascular tissue using microscopy techniques.

An effective remedy to combat voracious insect pests is influenced by a number of

factors which includes the toxic nature, compatibility to other control agents, speed of kill,

feeding deterrence to insect, effect on development of insect, acceptance to insect and

environmental persistence. Integration of two or more entomopathogens to fight against insect

pests may brighten the chances of targeting multiple hosts (Pingel and Lewis, 1999). Marzban et

al. (2009) reported that the integration of two or more myco-pathogens interact positively than

their individual effect. In most combinations the virulence of an agent is rectified by the action of

the other agent which resultantly increases the speed of kill, retard growth, feeding sesation,

improve virulence and broaden host range.

The findings of our study revealed higher mortality levels of RPW in combined

treatments of B. bassiana inoculated date palm piece and Bt-k as compared to their sole

application. Moreover, these deterrent effects increased with the increase of number of Bt spores.

Similar findings were revealed by Khalique and Ahmad (2002), who reported the extended larval

duration with the increase in Bt-k concentration. While, larvae in check treatments consumed

more food which are in accordance with the findings of Marzban et al. (2009). The studies of Ma

et al. (2008) and Marzban et al. (2009) also support our findings who reported the growth

retardation of Ostrinia furnacalis and H. armigera when challenged with Cry1Ac from Bt-

treated diets and combined action of Bt (Cry1Ac) and HaCPV respectively.

The mode of action of entomopathogens is the key to success factor which make them fit

for the use in IPM program to combat any insect pest. For infection to occur Bt toxins attach to

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the specific bindings sites of the insect’s midgut which then leads to cell lysis. This lysis may

result in the insect to stop feeding, become lethargic and ultimately die (Marzban et al., 2009).

While B. bassiana exhibit novel mode of action by adhering to the insect cuticle; later on

breaching the insect cuticle with the aid of enzyme complexes (beauvericin, bassianolide and

oosporein) followed by infection to the hemocoel where fungal conidia germinate and proliferate

in the insect body resultanting in insect death (Vey and Fargues, 1977). In combined treatments

of Bt and B. bassiana both agents work synergistically weakening the insect and affecting the

insect immune response to allow entomopathogens to infect the host more efficiently. Moreover,

conidial concentrations of both agents are the key factor in the degree of disease severity.

B. thuringiensis is rather quick in action which gets amplified when a larger number of

fungal spores are available to target the insect host. When B. bassiana gains access to the insect

gut, it boosts the infection of Bt toxins. In this way both agents help each other in retardation of

normal physiological functions of an insect host. These findings are further supported by Allee et

al. (1990) who found B. bassiana germinating and invading the insect favors the Bt toxins to

increase severity in grubs of Colorado potato beetle. Synergistic interaction was reported by

Wraight and Ramos (2005) when B. bassiana (GHA) and Bt-k were sprayed on potatoes to

protect against potato beetle (Leptinotarsa decemlineata). Similar findings were also reported by

Furlong and Groden (2003) who reported synergistic interaction between B. bassiana and B.

thuringiensis. Other researchers have also observed the similar results when combined B.

bassiana and B. thuringiensis withsynthetic insecticides (Fargues, 1975; Lewis and Bing, 1991;

Sander and Chichy, 1967). Contrarily, no synergistic interaction was observed between B.

bassiana and B. thuringiensis against 4th stage larvae of L. decemlineata (Costa et al., 2001).

Here the method of fungal spore application may retard the synergistic effect and respond

differently in terms of time and level of mortality. Different response of Chilo partellus was

reported when fungal spores were applied on leaf disk (Tefera and Pringle, 2003a).

In our present study synergistic interaction may be due to the treatment of RPW larvae

with fungal spores by larval dip method in which dorso-ventral infection of fungal spores may

weaken the larvae and render it more susceptible to Bt-k spores. Tefera and Pringle (2003b)

observed that loss of feeding H. armigera after exposure to Bt mixed diet make it easier for B.

bassiana to grab the physiology and hence proliferate rather easily. Similar loss of feeding was

observed by Tefera and Pringle (2003b) when second and third instar C. partellus were exposed

to simultaneous action of Bt (Cry1Ac) and B. bassiana as behavioral response elucidated reduced

diet uptake. Jadhav et al. (2012) found similar retardation in growth and survival of H. armigera

and Spodoptera litura after treatment with three flavonoids namely chlorogenic acid, quercetin

and rutin. The feeding experiments on RPW exhibited that B. thuringiensis invades the

hemolymph and the total circulating hemocytes decreased, mainly the plasmatocytes, after 19 h,

and for the first time, many Bt vegetative forms were recorded in the hemolymph of RPW after

Bt commercial product ingestion (Manachini et al., 2011).

Integrated action of Bt and fungi may also hypothesized by delayed larval molts with Bt

treatments which enhance the inter-molt period, thereby providing fungus extended period of

time for infection before next molt. The reduced food consumption with Bt treatments (Nathan et

al., 2005; Ramalho et al., 2011), food utilization (Prutz and Dettner, 2005; Ramalho et al., 2011)

and reduced larval weight has been observed. On the other hand in healthy larvae loss of conidia

may happen during molting, hence conferring interruption in fungal colonization (Wraight and

Ramos, 2005). Contrarily, Slansky (1993) reported surprising results by observing the reduction

in larval weight, food consumption and frass production in fungal treated larvae as compared to

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the untreated control. Our findings suggest that assimilation activities were found related to toxic

effect of pathogen as indicated by decreased quantity of frass produced. Our results are in

accordance with the study of Janmaat et al. (2014) who reported reduced frass production in

Trichoplusia ni with the increase of concentrations. This may be attributed to the fact that

enhanced Bt concentrations may alter the protein to carbohydrate ratio of the diet as required

optimally which resultantly disturb the growth response (Simpson and Raubenheimer, 1995).

Moreover, the toxicant larvae try to repair its midgut lining and the lysis effect is directly

proportional to the concentration of pathogen (Tanaka et al., 2012). Muñoz et al. (2014) reported

reduced food intake, growth and weight gain in H. armigera exposed to the sublethal doses of Bt

which in response did not allowed most of them to get the critical weight and pupate in time.

This type of feeding behavior may be the result of metabolic interference of the

entomopathogens with larva’s growth.

A greater knowledge of RPW biology and, in particular, of the interaction between

potential pathogens and immunocytes would be useful to improve RPW-IPM programs, which

should focus on the identification of more virulent natural pathogen strains and on improving the

virulence capacity of Bt (Manachini et al., 2011). While choosing microbial control agents to

fight insect pests, several uncontrolled factors must be considered as the comprehensive nature of

entomopathogens requires multidisciplinary targeted efforts. This study may help to apply

laboratory study to field conditions covering almost all the questions required to be answered.

However field study regarding the persistence of entomopathogens is required to have a detailed

knowledge about some of the factors that cannot be imagined under laboratory or green house

condition. This study could be a foundation and direction for the future researches.

Conclusions The present study showed that B. bassiana can colonize endophytically in date palm

petiole even after 30 days post inoculation. WG-41 isolate was recovered up to 10 cm from the

site of inoculation even after 30 days. Endophytic B. bassiana in integration with Bt-k can be

effectively used against 2nd, 4th and 6th instar larvae of this pest under laboratory conditions.

Moreover they also exert detrimental effect on their growth and development parameters such as

diet consumption, frass production and weight gain.

Acknowledgements



Fellowship Program.

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124

Table 6.1 Percentage of petiole fragments colonized by entomopathogenic (E) and other (O)

fungi in live palm petioles experiments

Isolate

Site

2014 2015 15 days 30 days 15 days 30 days

E O E O E O E O

WG-11

A5 - 100 - 100 - 100 - 100 A4 - 100 - 100 - 100 - 100 A3 20 80 20 80 15 85 25 75 A2 60 40 40 60 45 55 30 70 A1 100 - 60 40 90 10 50 50 INS 90 10 60 40 80 20 45 55 B1 80 20 60 40 70 30 40 60 B2 40 60 20 80 33 66 15 85 B3 - 100 - 100 - 100 - 100 B4 - 100 - - - 100 - - B5 - 100 - - - 100 - -

WG-40

A5 - 100 - 100 - 100 - 100 A4 35 65 20 80 25 75 15 85 A3 66 33 40 60 50 50 30 70 A2 80 20 40 60 66 33 55 45 A1 100 - 60 40 90 10 60 40 INS 100 - 100 - 100 - 100 75 B1 100 - 80 20 95 05 70 30 B2 75 25 80 20 66 33 66 33 B3 50 50 40 60 40 60 30 70 B4 25 75 15 85 20 80 - 100 B5 - 100 - 100 - 100 - 100

WG-41

A5 50 50 25 75 40 60 20 80 A4 70 30 50 50 66 33 40 60 A3 75 25 50 50 70 30 40 60 A2 90 10 75 25 75 25 65 35 A1 100 - 90 10 90 10 80 20 INS 100 - 100 - 100 - 100 - B1 100 - 80 20 90 10 70 30 B2 95 5 75 25 80 20 60 40 B3 80 20 66 33 70 30 50 50 B4 65 35 40 60 50 50 30 70 B5 40 60 33 66 40 60 20 80

WG-42

A5 - 100 - 100 - 100 - 100 A4 33 66 - 100 25 75 - 100 A3 60 40 33 66 40 60 25 75 A2 85 15 55 45 70 30 40 60 A1 100 0 65 35 90 10 50 50 INS 100 0 85 15 100 0 85 15 B1 100 0 60 40 85 15 50 50 B2 75 25 50 50 66 33 40 60 B3 66 33 20 80 50 50 15 85 B4 35 65 10 90 25 75 - 100 B5 - 100 - 100 - 100 - 100

125

WG-43

A5 - 100 - 100 - 100 - 100 A4 33 66 - 100 25 75 - 100 A3 55 45 40 60 40 60 30 70 A2 67 33 55 45 55 45 40 60 A1 90 10 75 25 75 25 60 40 INS 100 - 90 10 100 - 90 10 B1 85 15 60 40 70 30 50 50 B2 67 33 45 55 50 50 35 65 B3 50 50 33 66 40 60 20 80 B4 15 85 - 100 - 100 - 100 B5 - - - 100 - - - 100

Control

A5 - 100 20 80 - 100 - 100 A4 - 100 - 100 - 100 - 100 A3 10 90 - 100 - 100 20 80 A2 - 100 - 100 - 100 - 100 A1 - 100 - 100 20 80 10 90 INS - 100 - 100 - 100 - 100 B1 - 100 15 85 - 100 - 100 B2 - 100 - 100 - 100 - 100 B3 - 100 - 100 - 100 - 100 B4 - 100 - 100 - 100 - 100

B5 - 100 - 100 - 100 - 100 INS: site of injection; A1-A5 and B1-B5: site 2-10 cm above or below site of injection.

Table 6.2 Factorial analysis of mortality, pupation, adult emergence and egg eclosion of

R. ferrugineus exposed to endophytically colonized B. bassiana and B.

thuringiensis

S.O.V.

df

Mortality Pupation Adult Emergence

Egg Eclosion

F P F P F P F P

Instar 2 63.34 ≤0.05 59.23 ≤0.05 51.45 ≤0.05 34.56 ≤0.05 Treatment 7 201.87 ≤0.05 229.10 ≤0.05 233.45 ≤0.05 249 ≤0.05 Instar × Treatment

14 1.56 0.093 0.68 0.625 0.61 0.635 0.78 0.515

Error 149 - - - - - - - - Total 172 - - - - - - - -

126

Table 6.3 Mean mortality (%±SE) of 2nd, 4th and 6th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 2 cm

away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination (means

followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)

Treatments Second Instar Fourth Instar Sixth Instar

Actual Mortality (%)

Expected Mortality

CTF Actual Mortality (%)

Expected Mortality

CTF Actual Mortality (%)

Expected Mortality

CTF Type of Interaction

Bb 29.90±1.27de - - 23.36±1.18cde - - 20.67±1.17cde - - - Bt1 17.65±1.16e - - 12.53±1.09e - - 8.27±0.89e - - - Bt2 25.89±1.61de - - 18.58±1.12de - - 14.19±1.22de - - - Bt3 41.20±1.01cd - - 33.01±1.45cd - - 25.02±1.38cd - - - Bb + Bt1 54.63±2.26bc 47.55 14.88 42.13±1.32bc 35.89 17.38 34.07±1.50bc 28.94 17.72 Add. Bb + Bt2 71.01±2.39ab 55.79 27.28 54.07±1.65ab 41.94 28.92 47.87±1.42ab 34.86 37.32 Syn. Bb + Bt3 83.17±2.28a 71.1 16.97 65.86±1.50a 55.42 18.83 52.68±1.57a 44.96 17.17 Add. Control 2.4 - - 1.5 - - 1.00 - - - df 6 - - 6 - - 6 - - - F 33.3 - - 18.7 - - 26.1 - - - P ≤0.05 - - ≤0.05 - - ≤0.05 - - -

127

Table 6.4 Pupation, adult emergence and egg eclosion (%±SE) of 2nd, 4th and 6th instar larvae of R. ferrugineus treated with

endophytic B. bassiana (Bb: 2 cm away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone

and in combination (means followed by the same letter within each treatment are not significantly different; HSD test

P≤0.05)

Treatments Second Instar Fourth Instar Sixth Instar

Pupation (%) Adult

Emergence (%)

Egg Eclosion

(%)

Pupation

(%)

Adult

Emergence (%)

Egg Eclosion

(%)

Pupation

(%)

Adult

Emergence (%)

Egg

Eclosion (%)

Bb 63.31±2.35c 57.72±2.77c 51.13±2.20c 71.16±2.60cd 64.46±2.93c 57.73±2.23b 76.64±3.33bc 70.06±2.88c 64.46±2.93b

Bt1 78.82±3.15b 70.00±2.92b 63.37±2.48b 84.44±3.36ab 76.62±2.35b 65.97±3.01b 87.73±2.94ab 84.42±2.93ab 75.51±2.42b

Bt2 66.67±2.87c 58.83±2.60bc 52.28±2.73bc 75.56±2.75bc 67.73±3.13bc 58.75±2.23b 80.10±2.88bc 73.36±3.12bc 66.65±2.88b

Bt3 49.02±2.21d 37.96±1.53d 30.02±1.88d 58.81±2.60de 50.07±2.88d 43.31±2.35c 68.85±3.21c 52.24±2.77d 51.16±2.60c Bb + Bt1 32.24±2.12e 25.53±1.42e 18.84±1.15de 46.64±1.72e 38.83±2.09d 32.24±1.85c 53.36±2.40d 45.54±2.42d 37.72±2.77d

Bb + Bt2 18.79±1.12f 11.10±1.02f 7.72±0.86ef 31.15±1.51f 17.96±1.11e 15.56±1.05d 44.42±2.93de 31.16±1.88e 24.46±2.42e

Bb + Bt3 13.31±1.26f 6.66±0.63f 3.38±0.45f 24.41±1.34f 13.30±1.09e 8.83±0.69d 35.54±1.75e 22.27±1.23e 15.53±1.15e

Control 94.48±1.75a 91.14±2.60a 85.52±2.93a 96.64±1.66a 92.18±2.24a 90.08±2.35a 95.49±1.91a 93.32±2.35a 87.72±2.22a df 7 7 7 7 7 7 7 7 7

F 119.0 117 122 83.1 103 90.1 46.0 73.6 100

P ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05

128

Table 6.5 Growth parameters e.g. larval duration (days), larval weight (grams), pre-pupal duration (days), pre-pupal weight

(grams), pupal duration (days), pupal weight (grams), adult longevity (days) and adult weight (grams) (%±SE) of 2nd

instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away from inoculation site) and Bt-k

(Bt1: 10 µg; Bt2: 15 µg; Bt3: 20 µg ml-1) alone and in combination (means followed by the same letter within each

treatment are not significantly different; HSD test P≤0.05)

Treatments

Larval Duration (days)

Larval Weight (g)

Pre-Pupal Duration

(days)

Pre-Pupal Weight

(g)

Pupal Duration

(days)

Pupal Weight (g)

Adult Longevity (days) Adult Weight (g)

Male Female Male Female

Bb 109.16±3.56c 3.34±0.08bc 17.60±1.13de 3.20±0.06cd 24.16±1.12de 3.27±0.07cd 38.27±1.31a 42.16±1.15a 1.39±0.09abc 1.19±0.07bc Bt1 99.72±2.25d 4.07±0.08a 15.83±0.76fg 3.82±0.11ab 21.72±0.92fg 3.93±0.10ab 36.94±1.17ab 40.27±1.07ab 1.58±0.10ab 1.35±0.06ab Bt2 105.27±3.07cd 3.66±0.11b 16.48±0.57ef 3.52±0.12bc 22.94±1.18ef 3.65±0.10bc 35.50±1.05abc 39.16±1.25ab 1.50±0.11ab 1.28±0.06abc Bt3 111.71±3.10c 3.21±0.12cd 18.76±0.65cd 3.03±0.10de 25.83±1.04cd 3.12±0.11de 33.16±1.21bcd 37.94±1.03abc 1.37±0.08abcd 1.17±0.07bcd Bb + Bt1 118.62±2.80b 3.08± 0.10cde 20.16±1.38bc 2.77±0.06def 26.94±1.35bc 2.93±0.08def 30.94±0.91cde 35.61±1.14bcd 1.21±0.07bcd 1.02±0.06cde

Bb + Bt2 121.27±2.56ab 2.96±0.09de 21.41±0.70ab 2.61±0.09ef 28.27±1.26ab 2.78±0.08ef 28.83±1.06de 33.00±0.98cd 1.07±0.05cd 0.91±0.04de Bb + Bt3 125.41±3.23a 2.74±0.07e 22.72±1.33a 2.38±0.04f 29.61±1.41a 2.54±0.09f 25.72±1.11e 30.38±1.03d 0.98±0.04d 0.88±0.03e Control 85.72±2.19e 4.42±0.15a 14.16±0.86g 4.02±0.11a 20.27±0.94g 4.14±0.11a 39.50±1.40a 43.72±1.40a 1.70±0.08a 1.51±0.06a Df 7 7 7 7 7 7 7 7 7 7 F 72.73 47.54 53.12 33.8 43.95 26.61 65.03 58.20 7.76 12.5 P ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05

129

Table 6.6 Analysis of Co-variance for 10th instar larvae of R. ferrugineus regarding weight

gain, frass production and diet consumption when treated with endophytic B.

bassiana (Bb: 6 cm away from inoculation site) and Bt-k (Bt: 10 µg ml-1). Initial

weight of larvae and diet consumption were taken as covariate

S.O.V. df F P Covariate: Diet Consumption 1 0.193 0.660 Covariate: Weight Gain 1 3.062 0.081 Frass Production × Diet Consumption 24 2.318 ≤0.05 Diet Consumption × Weight Gain 1 0.138 0.710 Frass Production × Weight Gain 27 2.733 ≤0.05 Frass Production × Diet Consumption × Weight Gain

24 2.704 ≤0.05

Error 687 - - Total 828 - -

130

Figure 6.1 Mean mycosis (%±SE) in cadavers of R. ferrugineus treated with endophytic B.

bassiana (Bb: 2 cm away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg;

Bt3: 50 µg ml-1) alone and in combination (means followed by the same letter

within each treatment are not significantly different; HSD test P≤0.05)

.

Figure 6.2 Sporulation (conidia ml-1) on R. ferrugineus cadavers treated with endophytic B.

bassiana (Bb: 2 cm away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg;

Bt3: 50 µg ml-1) alone and in combination (means followed by the same letter

within each treatment are not significantly different; HSD test P≤0.05)

131

Figure 6.3 Diet consumption (grams) in 10th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away

from inoculation site) and Bt-k (Bt: 10 µg ml-1)

132

Figure 6.4 Frass production (grams) in 10th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away

from inoculation site) and Bt-k (Bt: 10 µg ml-1)

133

Figure 6.5 Weight gain (grams) in 10th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away from

inoculation site) and Bt-k (Bt: 10 µg ml-1)

134

CHAPTER 7

Integrated Effect of Entomopathogenic fungi and Entomopathogenic Nematode against

Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

Abstract

Research study was carried out to investigate the insecticidal properties of entomopathogenic

fungi Beauveria bassiana s.l. (Ascomycota: Hypocreales) strain WG-11, Metarhizium anisopliae

s.l. (Ascomycota: Hypocreales) strain WG-02 and the entomopathogenic nematode,

Heterorhabditis bacteriophora Poinar (Heterorhabditidae) for their virulence against 2nd, 4th and

6th instar larva of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). Both agents

were either applied alone or in combination, with H. bacteriophora 1 and 2 weeks after fungi

application. Moreover, the complete spectrum of toxicity, development, diet consumption, frass

production and weight gain were observed at sub lethal doses of both agents. In combined

treatments, additive and synergistic interactions were observed for all the three instars and effects

were not significantly different among the treatments either applied simultaneously or in

sequential combinations with each other. Enhanced morality was recorded for the combined

treatments when delayed application of H. bacteriophora was made 1 or 2 weeks after fungus

treatment as compared to their sole application. Decrease in pupation, adult emergence and egg

hatching were also found related to the toxic effect of treatments. Duration of different

developmental stages was significantly affected by the treatments applied. Decreased larval

weight, increased larval duration, increased pre-pupal and pupal period and decreased weight,

decreased adult weight and life span were recorded and compared to the control. Larvae fed on

sub lethal amounts of both agents revealed reduced food ingestion, reduced growth and weight

gain, preventing most of them from achieving the critical weight. Initial weight of larvae exerted

its impact on weight gain and diet consumption, and the trend was found linked to pathogenicity

of applied agents. A result of the present study suggests that R. ferrugineus can be successfully

managed by applying entomopathogenic fungi and H. bacteriophora. Additionally, their

simultaneous and sequential application may offer enhanced mortality as compare to the

application of either of them alone.

Keywords: Rhynchophorus ferrugineus, Beauveria bassiana, Metarhizium anisopliae,

Heterorhabditis bacteriophora, diet consumption, frass production

135

7.1 Introduction

The coleopteran insects are ranked among the most voracious pests of economically

important crops. Among these the Red Palm weevil (RPW) Rhynchophorus ferrugineus (Olivier)

(Coleoptera: Curculionidae) is most destructive to 29 different palm species particularly date

palms of economic importance in the Middle East, Africa and South East Asia (Malumphy and

Moran, 2009). Synonymously it is known as Asiatic palm weevil, coconut weevil, red stripe

weevil, hidden enemy and also called AIDS palm because of the damage and the slow death of

the palm tree (Khamiss and Abdel-Badeea, 2013). The pest has cryptic nature and mostly

damages the palm trees younger than 20 years (Nirula, 1956; Abraham et al., 1998) in which

crown, trunk and bole are the natural sites of damage, while the crown is the site of infestation in

older plantations. The larval stages destroy the vascular system while boring into the heart of the

host leading to tree collapse (Ju et al., 2011).

Insecticides and fumigants remained the mainstay of date palm growers for decades but

the cryptic nature of RPW presented an access challenge to treatments (Hussain et al., 2013).

Moreover, insecticides exert negative effects on the environment and human health and more

importantly pests have developed resistance against these chemicals (Abraham et al., 1998).

Alternatively entomopathogens can be used for suppression of this notorious pest in a wide array

of management approaches in versatile manners. Among microbial control agents

entomopathogenic fungi (EPFs) particularly, Beauveria bassiana s.l. (Ascomycota: Hypocreales)

and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales) are considered promising

alternatives to conventional chemical insecticides. They pose negligible detrimental effects on

the environment and human health (Khan et al., 2012), and have been reported to be effective

against a number of arthropod pests (Charnley and Collins, 2007; de Faria and Wraight, 2007).

Several researchers have isolated and successfully deployed these two strains against

different developmental stages of RPW as bio-control agents both under laboratory and field

conditions (Deadman et al., 2001; Gindin et al., 2006; El-Sufty et al., 2007, 2009, 2011; Sewify

et al., 2009; Torta et al., 2009; Vitale et al., 2009; Güerri-Aguilló et al., 2010; Merghem, 2011;

Francardi et al., 2012; Ricaño et al., 2013; Cito et al., 2014). EPFs are preferred over the other

microorganisms due to their novel mode of action by direct contact to the host cuticle instead of

ingestion or engulfing and their ability to transfer inoculum from treated insects to untreated

insects or to subsequent developmental stages via the new generation of spores (Quesada-

Moraga et al. 2004). Similarly, entomopathogenic nematodes (EPNs) are also promising

microbial control agents and declared efficient control agents against vast array of insect pests

(Abbas et al., 2001; Llácer et al., 2009; Dembilio et al., 2010a). They are obligate parasites in

the families Steinernematidae and Heterorhabditidae which kill insects with the aid of

mutualistic bacterium, which is carried in their intestine (Xenorhabdus spp. and Photorhabdus

spp. are associated with Steinernema spp. and Heterorhabditis spp., respectively) (Poinar, 1990).

Both agents are considered safer to non-target organisms (vertebrates and invertebrates) and

compatible to environment, and they are successfully integrated with each other exhibiting

strong additive and synergistic interactions (Thurston et al., 1993, 1994; Koppenhöfer and Kaya,

1997; Koppenhöfer et al., 1999).

This study aimed at the integration of B. bassiana, M. anisopliae and H. bacteriophora to

examine the mortality; development and growth of R. ferrugineus under laboratory conditions to

select the suitable application times of both agents for future field trials to successfully manage

R. ferrugineus populations in Pakistan.

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Survey was conducted for collection of R. ferrugineus in date palm growing areas of west

Punjab, Pakistan. Different developmental stages (larvae, pupae and adults) were collected from

fallen and infested date palm trees with the permission of farmers (owners). All the stages

collected were kept separately in plastic jars until brought to the Microbial Control Laboratory,

Department of Entomology, University of Agriculture, Faisalabad (UAF), Pakistan. Larvae were

fed with sugarcane (Saccharum officinarum L.; Poales: Poaceae) sets and the same were used for

pupation after last instar, while shredded sugarcane pieces were offered to adults for feeding and

substrate for oviposition. After pupation pupal cocoon were kept in separate plastic jars for adult

emergence in incubator set at 25±2 oC, 65±5% RH and 12:12 (D: L) hours photoperiod. After

adult emergence beetles were shifted to the adult’s jar for feeding, mating and oviposition.

Colony was developed in plastic boxes (30×60×60 cm) having a lid covered with mesh wire

gauze (60 mesh size) in the middle (10 cm diameter) for aeration. Adult’s diet was changed after

every three days and replaced sugarcane pieces were kept in separate jars (8×8×12 cm) for egg

hatching. After egg hatching neonate larvae were allowed to feed for some time in the same set

after 3 days larvae were transferred to the same sugarcane sets for feeding and pupation. Larvae

were shifted to the new sugarcane sets after every week until pupation. The rearing conditions

were maintained at 25±2 oC, 65±5% RH and 12:12 (D: L) hours photoperiod.

7.2.2 Entomopathogenic Nematode

Infective juveniles (IJs) of H. bacteriophora culture was obtained from Microbial Control

Laboratory which was used for the bioassay against 2nd, 4th and 6th instar larvae of R.

ferrugineus. H. bacteriophora was maintained in 3rd instar Galleria mellonella L. (Lepidoptera:

Pyralidae) following the procedure of Kaya and Stock (1997).

7.2.3 Entomopathogenic fungi

Two isolates of entomopathogenic fungi B. bassiana (WG-11) and M. anisopliae (WG-

02) used in the study were taken from the culture collection of Microbial Control Laboratory,

originally isolated from soils of vegetables and crop fields respectively. Mass culturing was done

by inoculating Petri plates containing Potato Dextrose Agar (PDA) media (BD, France). Spore

concentration of 1×106 spore ml-1 was determined with a Neubauer haemocytometer.

7.2.4 Treatment with entomopathogenic fungi

A spore concentration of 1×106 spore ml-1 was prepared from conidial powder of B.

bassiana and M. anisopliae using haemocytometer. Second, 4th and 6th instar larvae were directly

immersed in 100 ml conidial suspension for 60s individually and control was treated in aqueous

solution of 0.01% Tween-80 (Merck, KGaA, Darmstadt, Germany) (Dembilio et al., 2010b). The

fungal isolate treated and control larvae were individually shifted to 150 ml cylindrical plastic

cups, each measuring 6 cm in height with 6 cm diameter. The top of the cups were covered with

a fine mesh in order to avoid the insects to escape. A piece of 2×2 cm2 artificial diet (Agar,

brewer’s yeast, wheat germ, corn flour, ascorbic acid, benzoic acid, amino acid-vitamin mix,

chloramphenicol and nipagin) (Martín and Cabello, 2006) were kept in the center of cups and

incubated at 27±2 °C, 65±5 % RH and 12:12 (D: L) hours photoperiod in an incubator (Sanyo,

Japan). Three replicates of 10 larvae were treated to the fungal suspension. Each cup was opened

137

daily and checked for mortality, and the old diet was replaced with fresh artificial diet until dying

or pupation. After last instar dry coir (coconut coir) was provided to the surviving larvae for

pupation. The bioassay was repeated thrice to avoid the pseudo-replication phenomenon.

7.2.5 Treatment with H. bacteriophora

Nematode suspension was prepared with a concentration of 100 IJs ml-1 in glass jars and

1 ml of suspension was poured into the cylindrical plastic cups lined with Whatman filter paper.

After pouring 30 minutes were given for evenly distribution of nematodes on filter paper. A

small piece of artificial diet 2×2 cm2 was placed in middle of the cups as a food source. Ten

larvae for each treatment were used separate in each cup and each treatment was replicated three

times, while control treatment received 1 ml of distilled water. The cups were maintained at

above mentioned conditions. Each cup was opened daily and checked for mortality, and the old

diet was replaced with fresh artificial diet until dying or pupation. After last instar dry coir was

provided to the surviving larvae for pupation. Whole bioassay was repeated thrice to avoid the

pseudo replication phenomenon.

7.2.6 Treatment with entomopathogenic fungi and nematode

In combined treatments both agents were applied simultaneously or at different time

intervals as follows:

B. bassiana, M. anisopliae and H. bacteriophora were applied simultaneously: larvae

were immersed in fungal suspensions and transferred to the cylindrical plastic cups lined

with moisten filter paper treated with H. bacteriophora IJs and maintained at 27±2 °C

and 65±5% RH at 12: 12 (D: L) hours photoperiod.

Insects were first inoculated with B. bassiana and M. anisopliae, maintained at 27±2 °C

and 65±5% RH for one week, transferred to cylindrical plastic cups lined with moisten

filter paper treated with H. bacteriophora IJs and maintained at above mentioned

conditions.

Insects were first inoculated with B. bassiana and M. anisopliae, maintained at 27±2 °C

and 65±5% RH for two weeks, transferred to cylindrical plastic cups lined with moisten

filter paper treated with H. bacteriophora IJs and maintained at above mentioned

conditions.

Control insects were immersed in aqueous solution with 0.01% Tween-80 and maintained

in cylindrical plastic cups lined with moistened filter paper using conditions stated above.

Larval mortality was recorded after one, two and three weeks post application. For all treatments,

artificial diet was offered to the larvae as food source. Larvae that failed to respond on slight

prodding by blunt needle were considered dead. After the last instar dry coir was provided for

pupation. Percent pupation, adult emergence and egg eclosion were also recorded.

7.2.7 Effects of entomopathogens on R. ferrugineus development

To check the effect of entomopathogens on development of RPW, 4th instar larvae were

exposed to the sub-lethal dose of fungal entomopathogens (1×104 spore ml-1) and H.

bacteriophora (50 IJs ml-1). The larvae were fed on artificial diet and transferred to the treatment

cups with 1 ml of. Dry coir was provided to the each larva before pupation for cocoon formation.

While adult on emergence were offered with shredded sugarcane pieces. Developmental

138

parameters of each stage (Larval duration, larval weight, pre-pupal duration, pre-pupal weight,

pupal duration, pupal weight, adult longevity (male and female) and adult weight (male and

female) was recorded.

7.2.8 Effects of entomopathogens on larval development

For the larval development last instar larvae of RPW was exposed to sub-lethal doses of

B. bassiana (1×104 spore ml-1) and H. bacteriophora (50 IJs ml-1). Before exposure in all the

treatments larvae were weighed first and transferred to the rearing cups with artificial diet.

Larvae continued to feed until pupated under experimental conditions maintained at 25±2 °C,

65±5% RH and L: D (12: 12) hours photoperiod. Every day until the larvae pupated, larvae were

changed to a new clean cup and new piece of artificial diet was offered. Frass produced during

this period was separated from vials using tip of fine camel hair brush and weighed. Diet left

unused in each vial was recovered, dried in drying oven at 80 °C. Prior to assay, diet in fifteen

vials was dried to obtain an estimate of the dry weight. Diet consumption of each larva was

determined by subtracting after feeding mass of diet from before feeding mass. Three replicates

of ten insects were used for each treatment and same count of larvae fed on normal diet served as

untreated check while entire experiment was repeated thrice.


The fungus nematode interactions (synergistic, additive or antagonistic) were calculated

using formula devised by Nishimatsu and Jackson (1998). The type of interaction was

determined through a comparison of expected and observed percentage mortality of RPW.

Expected mortality was calculated using formula PE = P0 + (1- P0) (P1) + (1- P0) (1- P1) (P2),

where PE is the expected mortality of the combination, P0 is the control mortality, P1 is the

mortality from one pathogen treatment applied alone, and P2 is the mortality from the

other pathogen applied alone. A X2 test was applied to the observed and expected results: X2 =

(L0 - LE)2 / LE + (D0 - DE)2 / DE, where L0 is the number of living larvae observed, LE the number

of living larvae expected, D0 the number of dead larvae observed, and DE the number

of dead larvae expected. Interactions were additive if X2 < 3.84, antagonistic if X2 > 3.84 and PC

< PE, and synergistic if X2 > 3.84 and PC > PE, where PC is the observed mortality from the

combination and PE is the expected mortality from the combination. Data for pupation, adult

emergence, egg eclosion and developmental parameters were subjected to one way analysis of

variance (ANOVA) in Minitab (Minitab, 2003) means were separated using Tukey’s Kramer test

(HSD) (Sokal and Rohlf, 1995) at 5% significance level. To inspect the impact of microbial

agents on the diet consumption, weight gain and frass production were analyzed by ANCOVA

using initial larval weight and diet consumption as covariates (Janmaat et al., 2014).

7.3 Results

7.3.1 Entomopathogenic fungi and nematode interaction

In integrated applications of H. bacteriophora with B. bassiana and M. anisopliae

additive to synergistic interaction were observed when both agents were applied simultaneously

or delayed nematode application for all the three instars tested (Table 7.1, 7.2 and 7.3). During

simultaneous application, B. bassiana and H. bacteriophora produced additive lethality to 2nd

instar larvae for first two weeks, while synergistic interactions were observed the third week

after application. The degree of synergism increased with the delayed application of H.

139

bacteriophora one or two weeks after initial B. bassiana treatments. For M. anisopliae additive

effects were recorded for simultaneous application, while interactions were shifted towards

synergism when delayed nematode application was made after one and two weeks of fungal

spore application (Table 7.1). Similar trends were recorded for 4th and 6th instar larvae but 2nd

instars were less susceptible to treatments. Percent pupation, adult emergence and egg eclosion

from surviving individuals was found inversely related to toxic level of microbial agents and

delayed application of H. bacteriophora for all instars tested (Table 7.5). In factorial analysis

main effects for pupation adult emergence and egg eclosion were significant while their

interaction effects were non-significant except pupation (Table 7.4)

7.3.2 Development of R. ferrugineus

Growth and development of 4th instar RPW larvae was adversely affected by the toxic

effect of the microbial agents. When larvae were exposed to the sub-lethal doses of H.

bacteriophora, B. bassiana and M. anisopliae, significant variations were recorded for larval

duration, larval weight, pre-pupal duration, pre-pupal weight, pupal duration, pupal weight, adult

longevity and adult weight (larval duration: F5, 53 =9.92, P≤0.05; larval weight F5, 53 =27.3,

P≤0.05; pre-pupal duration: F5, 53 =6.59, P≤0.05; pre-pupal weight: F5, 53 =6.94, P≤0.05; pupal

duration F5, 53 =5.15, P≤0.05; pupal weight F5, 53 =11.10, P≤0.05; adult longevity (female F5, 53

=3.93, P≤0.05 and male F5, 53 =5.58, P≤0.05 ); adult weight (female F5, 53 =4.26, P≤0.05 and

male F5, 53 =12.7, P≤0.05). Increase in larval, pre-pupal and pupal duration while decrease in

weight was recorded for all the treatments tested. On the other hand decrease in adult life span

and weight (male and female) was also recorded. Highest detrimental effect on growth was

recorded for combined application of B. bassiana and H. bacteriophora followed by M.

anisopliae and H. bacteriophora, H. bacteriophora alone, B. bassiana and M. anisopliae (Table

7.6).

7.3.3 Effect on larval development

Diet consumption by 10th instar larvae was significantly influenced by the treatments

applied; diet consumption was low in combined treatments of H. bacteriophora and B. bassiana

as compared to their individual applications (Fig 6.1). Similarly frass production was influenced

by treatments applied with lowest frass production for combined treatments of H. bacteriophora

and B. bassiana (0.57±0.04 to 0.00±0.00g) during the experimental period. After treatment frass

production gradually decreased to zero before pupation. On the other hand, the highest frass

production was found in untreated larvae during the last instar larvae until pupation (Fig 6.2).

Larvae treated with sub-lethal concentrations of either B. bassiana or H. bacteriophora alone

gained more weight compared to larval treated with a combined application. Initial weight of

larvae (10th instar: 4.31±0.14g) exerted its impact on the weight gain and among treatments,

there was a trend of weight gain was linked to pathogenicity. Combined application of B.

bassiana and H. bacteriophora had an adverse impact on the weight gain. The lowest weight

gain (-0.54±0.04g) was recorded for the combined treatment while the highest gain (-

0.12±0.02g) was recorded in untreated larvae (Fig 6.3).

7.4 Discussion

This is very first study to investigate the combined effect of fungal isolates and H.

bacteriophora against larvae of RPW. The results revealed that both agents can effectively

control the larval stages, either applied simultaneously or delayed application of H.

140

bacteriophora 1 or 2 weeks of fungal treatment. Additive to synergistic interactions were

recorded for combined applications of both agents. Greater additive or synergistic interactions

were observed when fungi were applied 1 or 2 weeks before H. bacteriophora treatment. Our

study corroborates the findings of Ansari et al. (2004, 2006) who reported similar results with

combined application of H. megidis or S. glaseri with M. anisopliae CLO 53 against 3rd instar H.

philanthus under laboratory and greenhouse conditions, and between H. bacteriophora and M.

anisopliae CLO 53 under field conditions respectively. Similarly additive and synergistic effects

were observed in combined treatments of H. bacteriophora and M. anisopliae isolate MM

against barley chafer grub, C. curtipennis (Anbesse et al., 2008). They have suggested exposing

grubs 3 or 4 weeks before addition of nematodes to get stronger synergistic interaction. However

in our study enhanced efficacy and stronger interactions were recorded 1 or 2 weeks delayed

application of H. bacteriophora.

It is tempting to speculate that longer grubs were exposed to the fungus, the more

debilitated they become and subsequently were more susceptible to the EPN. The debilitated

insects respired more, attracting the EPNs, which followed a CO2 gradient to their hosts (Ansari

et al., 2008). Steinhaus, (1958) also suggested that the stressed insects were more vulnerable to

pathogen infection, hence enhancing insect mortality or facilitating the speed of kill and

enhancing additive or synergistic effects in combined treatments. For example, Paenibacillus

popilliae (Dutky) against scarab larvae acted as a stressor to nematode infection that caused

elevated larval mortality (Thurston et al., 1993; Thurston et al., 1994). Other authors also have

reported similar results during their studies (Kermarrec and Mauleon, 1989; Barbercheck and

Kaya, 1990; Thurston et al., 1993, 1994; Koppenhöfer and Kaya, 1997; Koppenhöfer et al.,

1999). Contrarily Shapiro-Ilan et al. (2004) found antagonism between EPNs and P.

fumosoroseus. This antagonism may be due to pathogen interactions prior to or during infection.

In case of Sternima marcescens and P. fumosoroseus, it is possible that these organisms are

directly pathogenic to EPNs, therefore nematodes may have been killed or their fitness reduced

prior to infection. The negative interactions may also be due to antagonistic toxins produced by

the entomopathogens after infection was initiated. The synergy shown between fungi and H.

bacteriophora provided an opportunity to reduce the cost of RPW control while increasing the

overall efficacy of the control strategy.

For performing normal daily functions, an insect needs to have proper growth and

development and any delay may render the insect susceptible to biotic and abiotic factors such as

natural enemies, and environmental regimes that ultimately influence the growth, development,

diet consumption and frass production. In this regard larval stages are considered vulnerable

towards these agents (Marzban et al., 2009). The extended larval and pupal period reduces time

span left for adult stage that directly affects the insect’s fecundity and in other words threatens

the survival in next generation. Outcomes of our present study indicate that entomopathogenic

fungi and H. bacteriophora can be applied successfully against RPW but their efficacy varies

depending upon the interval of nematode application. Larval development is greatly influenced

by the lethal action of both entomopathogenic fungi and H. bacteriophora affecting the degree of

diet consumption that establishes the foundation for insect control. The alone and combined

concentrations of both agent offers a range of toxicity that exerts corresponding effect on the

development and survival of target host.

In present study detrimental effects imposed by EPFs and H. bacteriophora decreased

larval, pre-pupal, pupation rate, pupal weight and prolonged the developmental period. It is

generally accepted that major part of the insect energy consumed fighting pathogens lead to the

141

weakness and growth retardation in insects (Sikorowski and Thampson, 1979; Wiygul and

Sikorowski, 1991). Similar observations have been made during the current research and the

integrated application of H. bacteriophora and EPFs increased the larval mortality compared

with their respective individual treatments. Therefore, in an IPM program they can be

recommended for pest control where this entomopathogens are important natural enemies.

Conclusions The present study showed that B. bassiana and M. anisopliae isolates in integration with

H. bacteriophora under laboratory conditions caused high mortality against larvae of red palm

weevil. The pathogens exerted detrimental effects on growth and development of different

developmental stages of R. ferrugineus. Hence, integrated application of H. bacteriophora in

sequential manners with B. bassiana and M. anisopliae can be effectively used for the successful

control of red palm weevil.

Acknowledgements



Fellowship Program.

142

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146

Table 7.1 Mean mortality (%±SE) of 2nd instar larvae of R. ferrugineus treated with B.

bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M. anisopliae

were used each @ 1×106 spore ml-1 and H. Bacteriophora was applied @ 100 IJs

ml-1.

Treatments Intervalsa Weekb Observed mortality

Expected mortality

Chi Sq. Type of interaction

Bb

- 1 11.22 - - - - 2 14.28 - - - - 3 20.40 - - -

Ma

- 1 8.16 - - - - 2 12.24 - - - - 3 17.34 - - -

EPN

- 1 14.28 - - - - 2 21.42 - - - - 3 29.59 - - -

Bb+EPN

0 1 27.55 23.90 0.48 Additive 0 2 43.87 32.65 2.87 Additive 0 3 61.22 43.96 4.86 Synergistic

Ma+EPN

0 1 23.71 21.28 0.24 Additive 0 2 32.98 38.21 0.82 Additive 0 3 48.45 36.94 2.73 Additive

Bb+EPN

7 1 32.99 26.53 1.26 Additive 7 2 51.54 37.46 3.84 Synergistic 7 3 73.19 51.86 6.21 Synergistic

Ma+EPN


Bb+EPN

14 1 51.54 37.46 3.84 Synergistic 14 2 69.07 51.86 4.28 Synergistic 14 3 88.65 62.51 7.70 Synergistic

Ma+EPN


a Intervals between the application of EPFs and EPNs. b Week after fungal application.

147




ml-1.

Treatments Intervalsa Weekb Observed mortality

Expected mortality


Bb

- 1 9.18 - - - - 2 11.22 - - - - 3 16.32 - - -

Ma

- 1 6.12 - - - - 2 9.18 - - - - 3 14.28 - - -

EPN

- 1 12.24 - - - - 2 17.34 - - - - 3 23.46 - - -

Bb+EPN


Ma+EPN


Bb+EPN


Ma+EPN


Bb+EPN


Ma+EPN



148




ml-1.

Treatments

Intervalsa Weekb Observed mortality (%)

Expected mortality


Bb

- 1 7.14 - - - - 2 9.18 - - - - 3 13.26 - - -

Ma

- 1 4.081 - - - - 2 7.14 - - - - 3 11.22 - - -

EPN

- 1 9.18 - - - - 2 14.28 - - - - 3 18.36 - - -

Bb+EPN


Ma+EPN


Bb+EPN


Ma+EPN


Bb+EPN


Ma+EPN



Table 7.4 Factorial analysis for pupation, adult emergence and egg eclosion of R.

ferrugineus exposed to B. bassiana, M. anisopliae and H. Bacteriophora

S.O.V. df Pupation Adult emergence Egg eclosion

F P F P F P

Instar 2 18.78 ≤0.05 39.94 ≤0.05 35.0 ≤0.05 Treatment 9 114.28 ≤0.05 84.44 ≤0.05 90.89 ≤0.05 Instar × Treatment 18 10.49 ≤0.05 0.48 0.96 0.46 0.97 Error 232 - - - - - - Total 269 - - - - - -

149

Table 7.5 Pupation, adult emergence and egg eclosion (%±SE) of 2nd, 4th and 6th instar R. ferrugineus larvae treated with B.

bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M. anisopliae were used each @ 1×106 spore ml-1 and

H. Bacteriophora was applied @ 100 IJs ml-1. Mean sharing the same letters are not significantly different. Means

sharing the same letters within columns are not significantly different

Treatments

Interval

Second instar Fourth instar Sixth instar

Pupation

(%)

Adult

emergence (%)

Egg eclosion

(%)

Pupation

(%)

Adult

emergence (%)

Egg eclosion

(%)

Pupation

(%)

Adult

emergence (%)

Egg eclosion

(%)

Bb - 62.22±2.23bc 57.77±2.77bc 53.33±2.33bc 71.11± 3.54b 66.66±3.08b 59.55±2.92b 80.00±2.88bc 74.44±3.37b 66.33±3.10b

Ma - 67.77±2.64b 61.11±3.21b 58.88±2.23b 73.33±3.40b 68.88±3.51b 62.22±2.77b 83.33±2.35ab 78.88±3.88ab 72.22±3.22b

EPN - 56.66±2.33bcd 50.55±2.69bcd 45.55±1.67bcd 62.22±2.77bc 57.77±2.33bc 51.11±2.51bc 69.44±3.42cd 65.55±2.75bc 59.33±2.44bc

Bb+EPN 0 45.55±1.93def 40.33±2.35def 36.66±1.88def 48.88±2.09cd 43.33±3.08cd 38.88±1.60cd 54.44±2.76ef 49.44±2.57de 43.33±2.35de Ma+EPN 0 51.11±1.51cde 44.77±2.89cde 39.44±1.36cde 54.44±2.12cd 49.44±2.16cd 44.44±2.23cd 60.00±2.88de 56.66±2.72cd 51.11±2.51cd

Bb+EPN 7 39.44±1.69ef 36.66±1.33def 31.11±1.51def 45.55±2.42d 40.55±1.93d 35.55±1.93d 51.11±2.51ef 45.55±2.93de 40.55±1.42de

Ma+EPN 7 43.55±1.73def 38.88±1.51def 34.44±1.37def 51.11±2.88cd 46.11±2.32cd 41.11±1.51cd 57.77±2.77def 52.22±2.23cde 46.66±1.33cde

Bb+EPN 14 32.22±1.27f 27.77±1.77f 22.22±1.46f 39.44±2.42d 36.66±1.33d 31.11±1.60d 44.44±2.93f 40.22±2.79e 34.44±1.93e Ma+EPN 14 35.55±1.43f 30.55±3.37ef 26.66±1.68ef 47.77±2.23cd 42.22±2.12cd 37.77±1.22cd 54.44±2.76ef 47.77±2.64de 42.22±1.22de

Control 90.55±2.11a 86.66±2.88a 81.11± 2.60a 93.33±2.66a 90.55±2.11a 83.33±3.33a 95.55±1.75a 92.22±2.22a 85.55±2.93a

df 9 9 9 9 9 9 9 9 9

F 35.7 31.3 31.0 23.4 23.6 28.8 30.6 28.1 32.4 P ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05

150

Table 7.6 Effect of B. bassiana, M. anisopliae and H. Bacteriophora on the development of R. ferrugineus. B. bassiana and

M. anisopliae were used each @ 1×104 spore ml-1 and H. Bacteriophora was applied @ 50 IJs ml-1. Mean sharing the

same letters are not significantly different

Treatmen

ts

Larval duration

(days)

Larval

weight

(g)

Pre-pupal

duration

(days)

Pre-pupal

weight

(g)

Pupal duration

(days)

Pupal weight

(g)

Adult longevity (days) Adult weight (g)

Male

Female

Male

Female

Bb 98.16±3.21b 4.01±0.12bc 15.16±0.58bc 4.02±0.20ab 22.94±1.20bc 3.92±0.18abc 39.05±1.54ab 42.83±1.78ab 1.411±0.12ab 1.14±0.11abc

Ma 96.50±3.88bc 4.41±0.15ab 15.94±0.79bc 4.07±0.19ab 23.72±1.36abc 4.11±0.15ab 41.83±1.40a 43.61±1.67a 1.33±0.14ab 1.28±0.15ab

EPN 101.16±3.63ab 3.72±0.12cd 16.72±0.95abc 3.85±0.14ab 24.16±1.41abc 3.67±0.10bcd 37.16±1.23ab 40.38±1.45ab 1.18±0.1bc 1.05±0.12abc Bb+EPN 109.27±4.16a 3.08±0.12e 20.16±1.14a 3.07±0.11c 27.61±1.24a 3.21±0.11d 34.94±1.29b 36.50±1.78b 0.84±0.10d 0.78±0.11c

Ma+EPN 104.38±3.32ab 3.27±0.11de 18.50±0.98ab 3.48±0.12bc 25.50±1.41ab 3.43±0.13cd 36.16±1.17b 38.71±1.68ab 1.01±0.17cd 0.92±0.16bc

Control 87.05±1.08c 4.87±0.14a 14.38±0.74c 4.17±0.25a 21.27±1.36c 4.24±0.10a 42.83±1.61a 45.27±1.43a 1.56±0.13a 1.37±0.11a

df 5 5 5 5 5 5 5 5 5 5 F 9.92 27.3 6.59 6.94 5.15 11.1 5.58 3.93 12.7 4.26

P ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05

151

Table 7.7 Analysis of co-variance for 2nd, 4th and 6th instar larvae of R. ferrugineus

regarding weight gain and frass production at a given level of diet consumption

when treated with B. bassiana and H. Bacteriophora alone and in combination.

Initial weight of larvae and diet consumption were taken as covariate

S.O.V. df F P Covariate: Diet Consumption 1 0.191 0.62 Covariate: Weight Gain 1 3.060 0.078 Frass Production × Diet Consumption 24 2.312 ≤0.05 Diet Consumption × Weight Gain 1 0.132 0.73 Frass Production × Weight Gain 27 2.730 ≤0.05 Frass Production × Diet Consumption × Weight Gain 24 2.701 ≤0.05 Error 687 - - Total 828 - -

152

.

Figure 7.1 Diet consumption in last instar larvae of R. ferrugineus when treated with B. bassiana and H. bacteriophora

153

.

Figure 7.2 Frass production in last instar larvae of R. ferrugineus when treated with B. bassiana and H. bacteriophora

154

.

Figure 7.3 Weight gain in last instar larvae of R. ferrugineus when treated with B. bassiana and H. bacteriophora

CHAPTER 8

Combined toxicity of Beauveria bassiana, Bacillus thuringiensis and Heterorhabditis

bacteriophora against red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera:

Curculionidae)

Abstract

Laboratory studies were carried out to evaluate the insecticidal effect of Beauveria bassiana

(Bb), Bacillus thuringiensis var. kurstaki (Bt-k) and an entomopathogenic nematode (EPN)

Heterorhabditis bacteriophora against distinct populations of Red Palm Weevil (RPW)

Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). Four populations of RPW

were collected from different districts of Punjab, Pakistan including Layyah, Dera Ghazi Kahn,

Muzaffargarh and Rahim Yar Khan. All the three agents were used alone and in all possible

combinations (Bt-k+Bb, Bt-k+EPN, Bb+EPN and Bt-k+Bb+EPN) against 6th instar larvae and

adults of RPW. The experiments were carried out at 25±2 °C and 70±5% RH and 12:12 (D: L)

hours, mortality counts were taken after 7, 14 and 21 days post incubation. H. bacteriophora was

more effective followed by B. bassiana and Bt-k in alone treatments, while in combined

treatments increased mortality was recorded. Combined treatments of Bb+Bt-k exhibited lowest

mortality followed by Bt-k+ EPN, Bb+EPN and BB+Bt-k+EPN. The maximum rate of mycosis

and sporulation in the cadavers of RPW was observed where B. bassiana was applied alone and

similar trend was recorded for nematode production. The results of the present study indicate that

all three control measures may provide effective control against RPW. But need of the hour is to

evaluate these agents under field conditions.

Keywords: Beauveria bassiana, Heterorhabditis bacteriophora, Bacillus thuringiensis

Rhynchophorus ferrugineus

8.1 Introduction

The Red Palm Weevil (RPW) is an important invasive pest which has almost been

invaded and fully established in more than 50% of the date palm growing areas of the world

which attributes to the high fecundity than the normal species (Faleiro, 2006), capability to live

and interbreed in the same tree even for several generations (Rajamanickam et al., 1995; Avand-

Faghih, 1996), adult flight capacity to a longer distance (Wattanapongsiri, 1966) and pest

tolerance to a wide range of climatic conditions due to its hidden habit in palm tree. To combat

RPW different control practices have been deployed among date palm growing areas of the

world. Treatments revolve around the deployment of conventional chemical insecticides, sterile

insect techniques, use of semio-chemicals (Paoli et al., 2014) and bio-control agents

(Wattanapongsiri, 1966; Murphy and Briscoe, 1999; Faleiro, 2006). Most commonly used

control treatments are insecticides such as Diazinon, Imidacloprid, and Phosmet (Abbas, 2010).

However, heavy use of chemical treatments causes environmental damage and harms non-target

organisms, and also leads to the development of insecticide resistance against RPW (Jalinas et

al., 2015).

Very few studies have been conducted on the natural entomophagous enemies of R.

ferrugineus or other Rhynchophorus species (Murphy and Briscoe, 1999; Faleiro, 2006).

Entomopathogenic fungi (EPFs) are commonly found in the nature and cause epizootics in insect

populations, thus play significant role in regulating insect population. Mostly, the member of

Entomophthorales and Hyphomycetes attack on terrestrial insects. EPFs from various strains of

B. bassiana and M. anisopliae have been found in association with RPW and found among the

most relevant biological agents suggested to control RPW (Faleiro, 2006). Unlike the other

entomopathogens, entomopathogenic fungi infect the host by contact, then germinate and

penetrate the insect cuticle. The host can be infected both by direct treatment and by horizontal

transmission from infected insects or cadavers to healthy insects. Subsequently, infection can

occur via the new generation of spores (Lacey et al., 1999; Quesada-Moraga et al., 2004). These

unique characters make EPF especially important for the control of concealed insects such as

RPW.

Bacillus thuringiensis (Bt) is another important microbial control agent which holds a

prominent position among commercial chemical compounds important for agricultural insect

pests. Different researchers have evaluated the pathogenic potential of Bt against RPW and

revealed successful control (Banerjee and Dangar, 1995; Alfazariy, 2004; Bauce et al., 2002;

Sivasupramaniam et al., 2007; Birda and Akhursta, 2007; Manachini et al., 2008; Manachini et

al., 2009). Studies reveled that feeding seasation and midgut damage were observed amongst the

larvae survived after treatments. Entomopathogenic nematodes (EPNs) have been declared an

efficient entomopathogen against variety of insect in integrated pest management program

against RPW (Abbas et al., 2001; Llácer et al., 2009; Dembilio et al., 2010a). They are obligate

parasites in the families Steinernematidae and Heterorhabditidae which kill their host with the

aid of mutualistic bacterium present in their intestine (Poinar, 1990). As for as the life cycle is

concerned the nematodes complete 2-3 generations within the host, after which free-living

infective juveniles emerge to seek new hosts (Poinar, 1990). Several formulations have been

developed to improve the activity of nematodes against insect pests (Georgis, 1990; Georgis and

Kaya, 1998). In coleopteran pests larvae of several weevil species (Coleoptera: Curculionidae)

such as the black vine weevil, Otiorhynchus sulcatus (F.), and the Diaprepes root weevil,

Diaprepes abbreviatus (L.) was successfully controlled with EPNs (Shapiro-Ilan et al., 2002).

The intervention of more than one biocontrol agent can enhance the effectiveness of the

other partner; many studies have been conducted in this regard. The combined effect of B.

bassiana and B. thuringiensis working synergistically delivers more harm to insect pests

(Wraight and Ramos, 2005). Similarly combined application of EPNs and EPFs have been

evaluated against different insect pests (Thurston et al., 1993, 1994; Koppenhöfer and Kaya,

1997; Koppenhöfer et al., 1999; Yadav et al., 2004; Ansari et al., 2004, 2006, 2008). Hence

integrated practices can be a hint for those willing to manage RPW.



Four different populations of R. ferrugineus were collected from Layyah, Dera Ismail

Khan (D.I. Khan), Muzaffargarh and Rahim Yar Khan (R.Y. Khan) districts of Punjab

(Pakistan). Different developmental stages were collected from infested and fallen trees with the

permission of farmer (owner). From each area insects were collected and kept in different plastic

boxes assigned for a specific stage and brought to the laboratory until enough collection was

done. Further multiplication for one generation was carried out in Microbial Control Laboratory,

Department of Entomology, University of Agriculture, Faisalabad, Pakistan. Larvae were offered

with pieces of sugarcane (Saccharum officinarum L.; Poales: Poaceae) stem for feeding and

pupation, while shredded sugarcane pieces were offered to adults for feeding and substrate for

oviposition. After pupation, pupal cocoon were kept in separate plastic jars for adult emergence

in an incubator (Sanyo, Japan). After emergence beetles were shifted to the adult’s jar for

feeding, mating and oviposition. Colony was developed in plastic boxes (30×60×60 cm) having a

lid covered with mesh wire gauze (60 mesh size) in the middle (10 cm diameter) for aeration.

Adult’s diet was changed after every three days and replaced sugarcane pieces were kept in

separate jars for egg hatching. After egg hatching neonate larvae were allowed to feed for 3 days

in the same set and then shifted to the sugarcane sets for feeding and pupation. Larvae were

shifted to the new sugarcane sets after every week until pupation. The rearing conditions were

maintained at 25±2 oC, 65±5% RH and 12:12 (D: L) hours photoperiod.

8.2.2 Preparation of B. thuringiensis spore-crystal mixtures

The commercial formulation of B. thuringiensis var. kurstaki (Bt-k) was obtained from

Microbial Control Laboratory, originally obtained from National Center for Genetic Engineering

and Biotechnology (BIOTEC) in Thailand. This strain was then subjected to sporulation by

culturing in 50 ml nutrient broth media. Harvesting of culture was carried out by centrifugation

at 6000 rpm for 15 min (Crecchio and Stotzky, 2001; Hernández et al., 2005). The pellets formed

resultantly were washed twice in cold 1M NaCl and thrice in sterile distilled water (SDW), re-

suspended in distilled water (5 ml). From the suspension formed, 1 ml was centrifuged for 5 min

at 10,000 rpm, dried for 4 hours at 37 °C and weighed (Wakil et al., 2013).

8.2.3 Entomopathogenic nematode

The Infective Juveniles (IJs) of H. bacteriophora were obtained from the culture

collection of Microbial Control Laboratory. Second instar larvae and adult of R. ferrugineus

were encountered with 300 IJs under laboratory conditions. H. bacteriophora was maintained in

3rd instar Galleria mellonella L. (Lepidoptera: Pyralidae) following the procedure of Kaya and

Stock (1997).

8.2.4 Preparation of fungi

The fungal isolate of B. bassiana (WG-43) used in the study was taken from the culture

collection of Microbial Control Laboratory, originally isolated from dead cadaver of RPW. Fungi

were sub-cultured on Sabouraud Dextrose Agar (BD, Becton, Dickisonand Company sparks, MD

21152 USA). Conidial suspension was prepared with 0.01% Tween-80 (Merck, KGaA,

Darmstadt, Germany) in sterile distilled water and conidial concentration of 1×107 conidia ml-1

determined using a Neubauer haemocytometer.

8.2.5 Treatment with B. bassiana

Sixth instar larvae and adults of uniform age from each population were directly

immersed into the conidial suspensions for 60 and 90s respectively and control was treated in

aqueous solution with 0.01% Tween-80. Isolate-treated and control insects were individually

shifted to 150 ml cylindrical plastic cups, each measuring 6 cm in height with 6 cm diameter.

The top of cups were covered with fine mesh in order to avoid the insects to escape. A piece of

2×2 cm2 artificial diet (Agar, brewer’s yeast, wheat germ, corn flour, ascorbic acid, benzoic acid,

amino acid-vitamin mix, chloramphenicol and nipagin) (Martín and Cabello, 2006) was kept in

the center of the cups for larvae and a shredded sugarcane piece was offered to the adults. All the

treatments were incubated at 25±2 °C, 70±5 % RH and a 12:12 (D: L) hours photoperiod and

mortality counts were made after 7, 14 and 21 days post-incubation. The causal agent of dead

larvae or adults were confirmed by shifting the cadavers into a Petri dish lined with wet filter

paper and incubating them at 25±2 °C and 70±5 % RH for up to 15 days.

8.2.6 Treatment with B. thuringiensis var. kurstaki (Bt-k)

Sixth instar larvae form each population was individually offered with artificial diets

(Martín and Cabello, 2006), mixed with the diluted spore-crystal (70 µg g-1). To each larvae, Bt-k

treated diet piece of (2×2 cm2) was provided to feed. For adults shredded sugarcane pieces were

dipped in known concentration of Bt-k for 90s and offered to respective populations.

8.2.7 Treatment with H. bacteriophora

H. bacteriophora suspension was prepared with a concentration of 300 IJs in glass jars

and 1 ml of suspension was poured into the cylindrical plastic cups lined with damp Whatman

filter paper. The top of the cups were covered with a fine mesh in order to avoid the insects to

escape. After pouring nematodes 30 minutes time was given for their even distribution on filter

paper (Atwa et al., 2014). A small piece of artificial diet 2×2 cm2 was placed in middle of the

cups as a food source for larvae and provided with new food every day. In each cup one 6 th instar

larvae from each population was placed on top of the filter paper. Same procedure was repeated

for adult population and shredded sugarcane pieces were offered as food source. Each treatment

was replicated three times, while control treatment received 1 ml of distributed water. The cups

were placed in an incubator at 25±2 °C and 70±5% RH at 12: 12 (D: L) hours photoperiod.

Mortality data was recorded after 7, 14 and 21 days after treatment and whole bioassay was

repeated thrice to avoid the pseudo-replication phenomenon. Dead individuals were transferred

to the modified White traps (White, 1927) and left for 10 more days for IJs emergence. The

insects exhibiting typical odor and color (signs for nematode infestation) were considered to be

killed by nematodes (Woodring and Kaya, 1988).

8.2.8 Treatment with B. bassiana, Bt-k and H. bacteriophora

In combined treatments of B. bassiana, Bt-k and H. bacteriophora 6th instar larvae and

adults were directly immersed in 100 ml of conidial suspensions for 60 and 90s respectively and

control was treated in aqueous solution with 0.01% Tween-80 solution. After fungal treatments

larvae and adults were offered with Bt-k treated artificial diet and sugarcane pieces respectively

on H. bacteriophora treated plastic cups lined with damp Whatman filter paper. Experimental

conditions were maintained at 25±2 °C, 70±5% RH and 12:12 (L: D) hours photoperiod and

mortality data was recorded after 7, 14 and 21 days. Three replicates of ten insects were used for

each treatment and same count of larvae fed on normal diet served as untreated check while

entire experiment was repeated thrice. Larvae that exhibiting fungal infection symptoms

(hardening of the cadaver or emergence of conidiophores) were maintained as described above

and production of spores on the cadavers were evaluated following 14 day of incubation at 25±2

°C. Larvae demonstrating symptoms of EPN infection (changes in pigmentation) were

maintained in White traps for 10 days for production of IJs.

8.2.9 Sporulation and Nematode production

Mycosed larvae after 14 days of incubation were vortexed for 30 minutes in distilled

water containing 0.01% Tween-80 and number of spores was estimated in 1 ml from the

suspension using a haemocytometer. Concentration of IJs was measured by 1 ml sample from the

final solution and counting IJs with the help of a Peters’ slide and microscope.



formula and subjected to one way analysis of variance (ANOVA) in Minitab (Minitab, 2003)

means were separated using Tukey’s Kramer test (HSD) (Sokal and Rohlf, 1995) at 5%

significance level.

8.3 Results

8.3.1 Mortality of larvae and adult

The results of present study revealed that the larval and adult mortality was significantly

affected by the main effects and their associated interactions for all the population tested (Table

8.1). The mortality of both larvae and adult was non-significant (P≤0.05) among all the

population tested after each exposure interval, except the Bt-k+EPN for larvae after 7 days of

exposure and EPN, Bt-k+EPN and Bb+EPN for larvae and Bb+EPN for adults after 14 days of

exposure, after 21 days of exposure the treatments Bb and EPN for larvae and EPN, Bt-k+EPN

and Bb+EPN for adult were significantly different (P≤0.05). Overall the mortality was higher on

combined treatments as compared to individual applications of either B. bassiana, Bt-k or H.

bacteriophora for both larvae and adult, while the larval mortality was higher as compared to the

adult beetles in all the treatments applied at all the exposure intervals (Table 8.2, 8.3 and 8.4).

The laboratory population was more susceptible followed by R.Y. Khan, D.G. Khan and

Muzaffargarh at all the exposure intervals. After the last count Bb+EPN treatment exhibited

100% larval and adult mortality for all the populations tested, while Bt-k+EPN exhibited 100%

mortality for the laboratory population after 21 days post incubation.

8.3.2 Mycosis and sporulation

The maximum mycosed larvae (85.74%) and adults (69.07%), and sporulation in larvae

and (189.22 conidia ml-1) adults (164.56 conidia ml-1) was observed in treatments where B.

bassiana was applied alone against larvae and adults respectively in laboratory population

(Figure 8.1a, b and 8.2a, b), however low rate of mycosis and sporulation was observed in the

treatments where H. bacteriophora and B. bassiana were applied in combined manners. Similar

trend was recorded for the R.Y. Khan, D.G. Khan and Muzaffargarh populations.

8.3.3 Insects affected by EPF and EPN and their production

The maximum lethality in larvae affected by nematode was 92.40% and in adults

81.29%. The maximum number of nematode production on white trap was (178 IJs ml-1) adult

(153 IJs ml-1) was observed in treatments where H. bacteriophora was applied alone against

larvae and adult respectively in laboratory population (Figure 8.4a and 8.4b), however low rate

of nematode affected and production was recorded in the treatments where H. bacteriophora and

B. bassiana were applied in combined manners. Similar trend was recorded for R.Y. Khan, D.G.

Khan and Muzaffargarh population.

8.4 Discussion

Evidence suggests that entomopathogens play a key role in the host biology like

production of essential nutrients (amino acids and vitamins) and indispensable compounds which

influence some essential parameters such as growth, development, longevity, fertility, vector

capability, immunological competences and deliver protection against natural enemies (Valzano

et al., 2012). Numerous studies have documented sole and integrated applications of

entomopathogens against a number of insect pests. Generally the combined treatment of

entomopathogens exhibit enhanced mortality as compared to their individual application.

Therefore, simultaneous use of these agents did not cause any harmful effects on the efficiency

of the other agent. All agents have different modes of actions which enhance the disease severity

in a short period of time, hence reduce the time span to inflict damage to the host crop.

The entomopathogens that curtail RPW infestations are more effective in managing

weevil a population as compared to the plant protection (Salama et al., 2004; Dembilio et al.,

2010; El-Sufty et al., 2011). A number of entomopathogens are available worldwide that are

very effective against RPW including entomopathogenic fungi, bacteria and nematodes. Among

bacteria members of genus Bacillus like B. sphaericus, B. lentimorbus, B. popilliae are important

antagonists of RPW. These bacteria produce insecticidal proteins that target specific

developmental stages of RPW (Bulla et al., 1975; Salama et al., 2004). The entomopathogenic

fungi M. anisopliae and B. bassiana inflict mortality in different developmental stages of RPW

(Gindin et al., 2006; Dembilio et al., 2010). Moreover, the combined treatments of B. bassiana

and Bt exhibit enhanced larval mortality as compared to their individual applications. Similar

findings were observed by a number of researchers (Sander and Chichy, 1967; Kaliuga, 1968;

Fargues, 1973, 1975; Kalvish and Krivstova, 1978; Lewis and Bing, 1991).

Synergistic effects resulting from a combination of entomopathogenic nematodes with

other entomopathogens have been reported in a number of studies (Thurston et al., 1993, 1994;

Koppenhöfer and Kaya, 1997; Koppenhöfer et al., 1999). Contrarily Shapiro-Ilan et al. (2004)

found antagonism between entomopathogenic nematodes and P. fumosoroseus. Such

antagonisim may be due to pathogen interactions prior to or during infection. In case of Sternima

marcescens and P. fumosoroseus, it is possible that these organisms are directly pathogenic to

entomopathogenic nematodes, and therefore the nematodes may have been killed or their fitness

reduced prior to infection. These negative interactions may also be due to antagonistic toxins

produced by the entomopathogens after infection was initiated.

In our present study high mortality was recorded after different exposure intervals in

combined treatments as compared to their sole application. Higher mortalities were recorded for

(Bb+EPN) followed by (Bt-k+EPN) and (Bt-k+Bb) at all the exposure intervals. While in sole

treatments H. bacteriophora was found more effective followed by B. bassiana and Bt-k. Our

results are in accordance with the findings of Koppenhöfer and Kaya (1997) who reported

additive or synergistic interactions among entomopathogens when applied simultaneously.

Koppenhöfer and his collogues also recorded positive interaction between H. bacteriophora and

B. thuringiensis against Cyclocephala pasadenae, C. hirt and Anomala orientalis after different

exposure intervals (Koppenhöfer et al., 1999). Similar findings were reported by many other

scientists (Barbercheck and Kaya, 1990; Kermarrec and Mauleon, 1989).

Ansari et al. (2008) found synergistic interaction against black vine weevil larvae when

applied M. anisopliae and EPNs simultaneously. They also found similar results with H.

philanthus Füessly white grubs and revealed that type of interaction between EPN and fungal

entomopathogens depends on the time of application and specie of EPNs (Ansari et al., 2004,

2006). Similarly additive or slight synergistic interaction was recorded between M. anisopliae

and EPNs against Holotrichia consanguinea larvae (Yadav et al., 2004) and the larvae of pecan

weevil (Shapiro-Ilan et al., 2004).

A good knowledge of biological parameters of RPW and most importantly the interaction

among entomopathogens could play a key role to expand RPW-IPM programs. This, calls for the

isolation and identification of more virulent strains of entomopathogens (Manachini et al., 2011).

Moreover, the field evaluation of these substances in combined manners can provide substantial

information and help in developing new strategies by deploying IPM production systems (Neves

et al., 2001). In summary, the results of the present study indicate that the integration of

entomopathogens may be preferable to the use of a single agent. The integration takes advantage

of the positive characteristics of each agent. For example, the Bt treatments lead the gut to

septicemia causing the insect to stop feeding, and weakening the host immune system. This will

favor the B. bassiana to work efficiently with very low resistant of the host immune system

thereby increasing mortality.

Conclusions The present study showed that B. bassiana and Bt-k and H. bacteriophora can kill the

larvae and adult of R. ferrugineus from different populations collected across Punjab and Khyber

Pkhtunkhwa, Pakistan. They also exert the detrimental effect on their growth and development

which can be use effectively against this pest.

Acknowledgements



Fellowship Program.

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Table 8.1 ANOVA parameters for the main effects and associated interactions for mortality

levels of R. ferrugineus larvae and adults

S.O.V. Larvae Adult

df F P F P

Treatment 5 454.81 ≤0.05 437.56 ≤0.05

Interval 2 1546.71 ≤0.05 1099.79 ≤0.05

Location 4 25.54 ≤0.05 17.88 ≤0.05

Treatment × Interval 10 19.98 ≤0.05 26.11 ≤0.05

Treatment × Location 20 0.58 0.92 0.64 0.88

Interval × Location 8 0.93 0.49 1.24 0.27

Treatment × Interval ×

Location

40 0.73 0.89 0.41 0.99

Error 550 - - - -

Total 647 - - - -

Table 8.2 Mean mortality (%±SE) of R. ferrugineus populations collected from Layyah,

D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B.

bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in

combination after 7 days of exposure (means followed by the same letter within

each treatment and insect populations not significantly different; HSD test

P≤0.05)

Stage Treatments Insect Populations

Layyah D.G. Khan Muzaffargarh R.Y. Khan F P

Larvae

Bt-k 9.47±0.78e 6.47±0.55d 6.08±0.78d 7.93±0.71e 0.38 0.77 Bb 13.48±1.18de 10.47±1.12cd 8.39±1.01cd 11.68±1.16de 0.30 0.82

EPN 25.57±1.39cd 21.57±1.38bc 19.58±1.17bc 23.45±1.13cd 0.62 0.60

Bt-k + Bb 32.28±1.61bc 28.36±1.46ab 25.06±1.30b 30.17±1.65bc 1.28 0.29

Bt-k + EPN 45.37±2.15ab 33.73±1.50ab 28.08±1.57ab 42.11±2.36ab 6.46 ≤0.05

Bb + EPN 51.68±2.34a 42.19±2.51a 39.40±2.14a 46.82±2.54a 2.05 0.12

F 24.8 30.5 18.8 14.4 - -

P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -

Adult

Bt-k 6.54±0.70d 4.69±0.49d 4.02±0.53c 5.46±0.74d 0.24 0.87

Bb 10.46±1.12d 8.41±0.87cd 7.56±1.04bc 9.29±1.09cd 0.16 0.92

EPN 19.18±1.24cd 14.90±1.01bcd 13.22±1.35bc 16.51±1.21cd 0.72 0.54

Bt-k + Bb 24.70±1.65bc 21.24±1.96abc 19.69±1.56ab 22.06±1.56bc 0.31 0.81

Bt-k + EPN 35.41±2.35ab 28.84±2.11ab 27.09±1.75b 31.52±2.13ab 0.90 0.45

Bb + EPN 39.73±2.09a 32.37±2.03a 30.89±2.05a 36.08±2.45a 1.38 0.26

F 19.2 8.90 12.4 14.4 - -

P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -






P≤0.05)


Layyah D.G. Khan Muzaffargarh R.Y. Khan F P

Larvae

Btk 22.50±1.58c 20.67±1.39c 17.70±1.08c 19.50±1.23c 0.43 0.73

Bb 31.02±1.81c 27.76±1.85c 23.39±1.92c 25.64±1.08c 0.59 0.62

EPN 60.38±2.71b 55.66±2.44b 49.22±2.65b 51.57±2.18b 1.38 0.26

Bt-k + Bb 65.24±3.10b 59.10±2.18b 52.55±2.77b 55.09±3.07b 3.39 ≤0.05

Bt-k + EPN 89.66±3.26a 83.10±3.72a 74.57±3.14a 78.42±3.87a 4.07 ≤0.05

Bb + EPN 97.37±2.73a 91.55±3.27a 82.68±3.86a 86.17±3.03a 3.67 ≤0.05

F 86.2 73.1 46.3 60.3 - -

P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -

Adult

Btk 17.40±1.46c 15.78±1.04c 12.01±0.88c 13.76±1.07c 0.73 0.54

Bb 24.07±1.50c 21.85±1.28c 17.85±1.33cd 20.52±1.32c 0.37 0.77

EPN 45.06±2.48b 40.40±1.65b 34.54±2.03bc 37.62±1.99b 0.93 0.43

Bt-k + Bb 52.54±2.49b 48.01±2.14b 39.98±2.19b 43.27±2.75b 2.56 0.07

Bt-k + EPN 72.37±3.21a 67.77±3.10a 61.64±2.93a 65.80±2.57a 1.48 0.23

Bb + EPN 81.29±3.37a 75.74±3.34a 64.43±3.06a 70.18±2.28a 3.87 ≤0.05

F 50.5 42.1 27.9 41.8 - -

P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -






P≤0.05)


Layyah R.Y. Khan Muzaffargarh D.G. Khan F P

Larvae

Bt-k 58.36±4.55d 54.66±4.46c 46.86±4.53d 49.78±2.45c 1.55 0.22

Bb 72.63±3.88c 66.79±3.45c 57.27±2.75cd 55.55±2.92c 6.04 ≤0.05

EPN 84.54±2.71bc 81.06±3.14b 70.45±3.19bc 73.92±2.74b 4.75 ≤0.05

Bt-k + Bb 87.01±3.04b 82.17±2.90b 75.77±4.75b 78.24±2.39b 2.09 0.12

Bt-k + EPN 100.00±0.00a 98.41±1.58a 93.35±3.06a 95.57±2.85a 1.75 0.17

Bb + EPN 100.0±0.00a 100.0±0.00a 100.0±0.00a 100.0±0.00a - -

F 29.8 34.9 35.5 69.0 - -

P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -

Adult

Bt-k 39.04±2.41d 32.56±3.80d 26.79±3.68f 30.74±2.96f 2.45 0.08

Bb 47.55±2.60d 43.07±4.15d 38.13±4.09e 36.12±3.31e 2.03 0.12

EPN 61.13±3.81c 58.15±3.25c 52.37±2.20d 54.35±1.82d 1.83 0.16

Bt-k + Bb 80.53±2.66b 76.69±3.11b 67.96±3.92c 71.04±3.18c 2.99 ≤0.05

Bt-k + EPN 94.24±2.28a 90.03±2.82a 81.27±2.28b 86.48±1.46b 5.88 ≤0.05

Bb + EPN 100.0±0.00a 100.0±0.00a 100.0±0.00a 100.0±0.00a - -

F 95.3 70.9 80.5 130 - -

P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -

Figure 8.1a Mean mycosis (%±SE) in larvae of R. ferrugineus populations collected from

Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B.


combination (means followed by the same letter within each treatment are not

significantly different; HSD test P≤0.05)

Figure 8.1b Mean mycosis (%±SE) in adults of R. ferrugineus populations collected from





Figure 8.2a Sporulation (conidia ml-1) in larvae of R. ferrugineus populations collected from





Figure 8.2b Sporulation (conidia ml-1) in adult of R. ferrugineus populations collected from





Figure 8.3a R. ferrugineus larvae affected by H. bacteriophora (%±SE) from different

populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan

treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H.

bacteriophora (300 IJs) applied alone or in combination (means followed by the

same letter within each treatment are not significantly different; HSD test P≤0.05)

Figure 8.3b R. ferrugineus adult affected by H. bacteriophora (%±SE) from different

populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan

treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H.

bacteriophora (300 IJs) applied alone or in combination (means followed by the

same letter within each treatment are not significantly different; HSD test P≤0.05)

Figure 8.4a Nematode production (IJs ml-1) in larvae of R. ferrugineus affected by H.

bacteriophora from different populations collected from Layyah, D.G. Khan,

Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107

conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination

(means followed by the same letter within each treatment are not significantly

different; HSD test P≤0.05)

Figure 8.4b Nematode production (IJs ml-1) in adult of R. ferrugineus affected by H.

bacteriophora from different populations collected from Layyah, D.G. Khan,

Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107

conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination

(means followed by the same letter within each treatment are not significantly

different; HSD test P≤0.05)

Summary


Curculionidae) is one of the major and destructive insect pests of 29 different palm species all

around the world. It is an important invasive pest that has invaded and established in more than

50% of the date palm growing areas of the world attributed to the high fecundity of this species

(Faleiro, 2006), ability to live and interbreed in the same tree for several generations

(Rajamanickam et al., 1995; Avand-Faghih, 1996), adult flight capacity (Wattanapongsiri, 1966)

and pest tolerance to a wide range of climatic conditions due to its hidden habit in palm tree. To

combat RPW different control practices has been deployed among date palm growing areas of

the world. Treatments revolve around the deployment of conventional chemical insecticides,

sterile insect techniques, use of semio-chemicals (Paoli et al., 2014) and bio-control agents

(Wattanapongsiri, 1966; Murphy and Briscoe, 1999; Faleiro, 2006). Integration of RPW

associated microbial control agents with other control practices such as entomopathogens,

chemical insecticides and attract-and-kill techniques.

Various management strategies have been adopted for controlling this pest mostly relying

upon the use of broad-spectrum insecticides, but the injudicious use of such chemicals raises

various environmental and human health related issues that necessitates review of prevailing

control measures and evaluation of the new and alternative control methods. The utilization of

entomopathogenic microorganisms such as entomopathogenic fungi, entomopathogenic bacteria

and entomopathogenic nematodes are considered to be promising alternatives to conventional

insecticides in managing this voracious pest.

Prior to the application of any control strategy, sampling or monitoring of the pest

population and their genetic analysis can give a better idea of the exact status of the insect

populations and can facilitate the adaptation of appropriate curative measures. In order to have

base line data about the genetic diversity of R. ferrugineus from local populations and their

comparison with the rest of the world populations can give the idea of their native and invaded

range and their distribution pattern. Moreover, local populations of R. ferrugineus have gained

resistance to commonly used chemical insecticides and phosphine due to the excessive and

unwise use of these chemical insecticides. Resistance against seven different populations of R.

ferrugineus was determined from very low to low and moderate to high level against agents

commonly used insecticides. Phosphine, cypermethrin and deltamethrin exhibited highest

resistance against almost all populations of this insect pest.

Entomopathogenic fungi are potent alternatives to these chemical insecticides. Screening

of 19 different fungal isolates of B. bassiana and M. anisopliae exhibited variable ranges of

mortality against larvae and adults. Five best isolates that caused highest mortality against larvae

and adult after 5, 10 and 15 days of incubation were screened by virulence assays. WG-41 and

WG-42 were the best isolates that caused highest mortality and significantly reduced the

developmental parameters. B. bassiana are capable of colonizing endophytically in live date

plam petioles even after 30 days of inoculation and can significantly reduce the weevil

population when exposed to the endophytically colonized date palm pieces. Moreover Bt-k is

also an effective agent that can also cause detrimental effects of larval and adult survival alone

and in combination with endophytically colonized date palm pieces. Both agents also had great

influence on the developmental parameters such as larval duration, larval weight, prepupal

duration, prepupal weight, pupal duration, pupal weight, adult longevity and adult weight etc. the

agents also affect the developmental parameters like, diet consumption, frass production and

weight gain.

The alone and integrated use of entomopathogenic fungi, Bt-k and nematodes can also

cause to suppress the weevil population collected from 4 different areas of Punjab and Khyber

Pkhtunkhwa, Pakistan under laboratory conditions. Hence we can use microbial

entomopathogens against this voracious pest which are safer to environment and compatible to

environment.

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DEPARTMENT OF ENTOMOLOGY, FACULTY OF AGRICULTURE, …prr.hec.gov.pk/jspui/bitstream/123456789/7973/1/Muhammad_Yasin... · ENTOMOPATHOGENS AS POTENTIAL BIOCONTROL AGENTS AGAINST RED