Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/2739/1/3117S.pdf · Certificate This is to certify that the research work entitled “Isolation of Herbicidal Constituents

ISOLATION OF HERBICIDAL CONSTITUENTS

FROM CULTURE FILTRATES OF DRECHSLERA

SPP. FOR THE MANAGEMENT OF SOME

NOXIOUS WEEDS OF WHEAT

MUHAMMAD AKBAR

INSTITUTE OF AGRICULTURAL SCIENCES,

UNIVERSITY OF THE PUNJAB,

LAHORE, PAKISTAN.

2012

ISOLATION OF HERBICIDAL CONSTITUENTS

FROM CULTURE FILTRATES OF DRECHSLERA

SPP. FOR THE MANAGEMENT OF SOME

NOXIOUS WEEDS OF WHEAT

By

Muhammad Akbar

A THESIS SUBMITTED FOR THE FULFILLMENT OF DEGREE OF

Doctor of Philosophy

in

Mycology & Plant Pathology

Supervisor DR. ARSHAD JAVAID

Co-supervisor

Dr. EJAZ AHMED

INSTITUTE OF AGRICULTURAL SCIENCES UNIVERSITY

OF THE PUNJAB, LAHORE

PAKISTAN

Certificate

This is to certify that the research work entitled “Isolation of Herbicidal Constituents

from Culture Filtrates of Drechslera spp. for the Management of Some Noxious Weeds of

Wheat” described in this thesis by Mr. Muhammad Akbar, is an original work of the author

and has been carried out under our direct supervision. We have personally gone through all the

data, results, materials reported in the manuscript and certify their correctness and authenticity.

We further certify that the material included in this thesis has not been used in part or full in a

manuscript already submitted or in the process of submission in partial or complete fulfillment

of the award of any degree from any institution. We also certify that the thesis has been

prepared under our supervision according to the prescribed format and we endorse its

evaluation for the award of Ph. D degree through the official procedures of the University of

the Punjab, Lahore, Pakistan.

Supervisor Co-supervisor

DR. ARSHAD JAVAID Dr. EJAZ AHMED

Assistant Professor Assistant Professor

Institute of Agricultural Sciences, Institute of Chemistry,

University of the Punjab, Lahore, University of the Punjab, Lahore,

Pakistan. Pakistan.

Dated: Dated:

Declaration Certificate

I hereby certify that the research work reported in this thesis entitled “ISOLATION OF

HERBICIDAL CONSTITUENTS FROM CULTURE FILTRATES OF DRECHSLERA SPP.

FOR THE MANAGEMENT OF SOME NOXIOUS WEEDS OF WHEAT” is an original work

carried out under the supervision of Dr. Arshad Javaid and co-supervision of Dr. Ejaz Ahmed.

I further certify that I have written this thesis independently and used no other aids and

resources than those indicated.

Muhammad Akbar Ph. D Scholar

Institute of Agricultural Sciences,

University of the Punjab,

Lahore, Pakistan.

Dated:

DEDICATIONS

To Allah Almighty for giving me the perseverance to carry this work to the end in spite of all

the hurdles.

To my beloved mother and father, I am highly indebted to you for your prayers, you both

could not live to see me prosperous. Allah may rest their souls in Heaven, Aameen.

ACKNOWLEDGMENTS All praises and thanks to the grace of Allah Almighty Who is the ultimate source of all

knowledge to mankind. He bestowed man with intellectual power and understanding and gave

him spiritual insight enabling him to discover his “Self” know his Creator through His wonders

and conquer nature. Bow in obeisance, I before my Lord, WHO bestows me to fortitude and

impetus to accomplish this task and elucidate a drop of already existing ocean of knowledge.

WHO made me reach at present pedestal of knowledge with quality of doing something

adventurous, novel, thrilling, sensational, and path bearing.

Next to all His Messenger Hazrat Muhammad (Peace Be upon Him) Who is an

eternal torch of guidance and fountain of knowledge for humanity. Who made mankind to get

out of depths of evil & darkness.

I give a sincere gratitude to my Ph. D supervisor, Dr. Arshad Javaid, Assistant

Professor, Institute of Agricultural Sciences and co-supervisor, Dr. Ejaz Ahmed, Assistant

Professor, Institute of Chemistry, University of the Punjab, Lahore, Pakistan for their personal

supervision, cordial co-operation and ever contribution, inspiring guidance, valuable

suggestions and sympathetic attitude in the preparation of this manuscript.

My thanks are due to Prof. Dr. M. Saleem Haider, Director, Institute of Agricultural

Sciences, University of the Punjab, Lahore, Pakistan for his valuable help towards the

completion of this research work.

I express my humblest thanks to compassionate dignified Prof. Dr. Rukhsana Bajwa,

ex-director Institute of Mycology and Plant Pathology, University of the Punjab, Lahore,

Pakistan for her motivating behavior and personal interest in the accomplishment of my Ph. D

degree.

My special and sincere thanks are due to Dr. Shakil Ahmed for his friendly co-

operation, sound advices, encouragement, motivation and valuable suggestions throughout

the course of this study.

It gives me immense pleasure to express my deep sense of gratitude to Prof. Dr. Phillip

Crews, Department of Chemistry & Biochemistry, University of California, Santa Cruz, USA

for providing lab. facilities to accomplish analyses of natural compounds.

Thanks are due to Haji Muhammad Ramzan Mayo and Mr. Akbar Ali Mayo, Dhing

Shah, District Qasur, for providing all facilities to carry out field experiment. Their unselfish

and honest passion is memorable. Thanks also to Dr. Javed Saleem and Dr. Muhammad Islam

for their guidance related to field experiment.

Higher Education Commission (HEC), Government of Pakistan needs a separate

mention as all this would have been impossible with out fellowships granted by HEC.

I am thankful to Federal Seed Certification Department, Lahore for providing certified

seeds of wheat varieties.

I am highly indebted to all my family members for their prayers, encouragements, deep

affections and patience. I deprived them of the love I owe them because of my studies.

Thanks are due to First Culture Bank of Pakistan for providing necessary fungal

cultures and Professor Dr. J. H. Mirza (Late), Dr. Uzma Bashir, Dr. Noureen Akhtar, Dr.

Irum Mukhtar and Miss Sobia, for helping me in the identification of fungal cultures.

Dr. Salik Nawaz Khan, I would say thanks for your guidance in research work and friendly

behavior.

Dr. Ghazala Nasim, I will always remember your appreciation and your efforts to aim for

excellence.

Thanks to Dr. Aamir Ali , Dr. Abdul Majid Khan, Dr. Akram Tariq Sial, Dr. Tariq

Riaz, Mr. Javaid Akram and Mr. Hassan Siddqi for helping me during all stages of my

thesis.

I will always remember honest contributions of Dr. Ahmed Ali Shahid, Dr. Safdar A.

Anwar, Dr. Tehmina Anjum, Miss Shabnam Javed, Mrs. Ruqia Suleman, Mr. Noor

Zaman, Dr. Abdul Hanan, Dr. Asad Shabbir, Mrs. Saira Sroya, and Mr. Muhammad

Khalil Ahmed Khan.

Mr. Waheed Anwar, Mr. Aqeel Ahmed, my dear friends and colleagues, you both were

my computer mentors, I cannot pay your honesty and passion towards me.

Mr. Muhammad Aslam, Mr. Taufiq Asghar, Mr. Ehsan Zaidi, Mr. Khurram, Mr.

Amad, Mr. Irfan Ali, Mr. Irfan Mahmood, Mr. Ishtiaq Ahmed, Mrs. Aliya, Mr.

Muhammad Iqbal Shad, Mr. Sarfraz Nawaz, Mr. Ishfaq, Mr. Muhammad Akram, Mr.

Abid, Mr. Abdul Raffay, Mr. Amjad, Mrs. Shazia, Miss Faiza, Mr. Manzoor Ilahi, Mr.

Niaz, Chacha Sadiq, Mr. Abbas, Mr. Imran, Mr. Muhammad Nasir Shah, Mr. Asif and

Mr. Samsoon Masih, I say a big thank to all of you.

In addition, special thanks to all my friends, lab members and hostel fellows for their

kind cooperation, constructive criticism, and valuable suggestions during the progress of my

studies and research and in preparation of this manuscript. Thanks are also to the members of

the IAGS for their time devotion, synergistic help, cooperation and valuable input during my

studies and research.

Muhammad Akbar

http://pu.edu.pk/faculty/index/454

Contents

Title Page #

Certificate

Acknowledgments

List of Abbreviations i

Summary iii

Chapter 1: Introduction 1.1. Importance of Wheat 1

1.2. Importance of Weeds 2

1.3. Management of Weeds 4

1.3.1. Mechanical Control 4

1.3.1.1. Tillage 4

1.3.1.2. Flooding 5

1.3.1.3. Fire 5

1.3.1.4. Hand Hoeing 6

1.3.1.5. Mulching 6

1.3.2. Cultural Methods 6

1.3.2.1. Competitive Crops and Cultivars 6

1.3.2.2. Crop Rotation 7

1.3.2.3. Increased Crop Density 7

1.3.2.4. Intercropping 8

1.3.2.5. Companion Cropping 8

1.3.3. Biological Weed Control 8

1.3.4. Chemical Method 11

1.3.5. Natural Products as Herbicides 14

1.3.5.1. Herbicides from Plants 14

1.3.5.2. Herbicides from Fungi 16

1.4. Genus Drechslera 19

1.5. Objectives 22

Chapter 2: Materials and Methods 23 2.1. Selection of Test Fungal Isolates 23

2.2. Single Spore Isolation 23

2.3. Selection of Weeds of Wheat 23

2.4. Selection of Test Wheat Varieties 24

2.5. Preparation of Culture Filtrates of the Test Fungi 24

2.6. Laboratory Screening Bioassays 24

2.7. Foliar Spray Bioassays 25

2.8. Field Trials 26

2.8.1. Field Preparation 26

2.8.2. Sowing of Seeds 26

2.8.3. Treatments and Experimental Layout 27

2.8.4. Schedule of Foliar Sprays 27

2.8.5. Harvesting and Data Collection 28

2.9. Statistical Analysis 28

2.10. Organic Solvent Extraction 28

2.11. Leaf Discs Bioassays with Crude Organic Fractions 29

2.12. Isolation of Compounds through Chromatographic Techniques 31

2.12.1. Thin Layer Chromatography 31

2.12.2. Preparative Thin Layer Chromatography 32

2.12.3. Reversed Phase High Performance Liquid Chromatography 32

2.13. Spectroscopic Analyses 32

2.14. Leaf Discs Bioassays with Purified Chromatographic Fractions 33

Chapter 3: Results 34 3.1. Laboratory Bioassays 34

3.1.1. Effect of Fungal Culture Filtrates on Germination 34

and Growth of C. album

3.1.1.1. Effect on Germination 34

3.1.1.2. Effect on Shoot Growth 34

3.1.1.3. Effect on Root Growth 35


and Growth of R. dentatus





and Growth of P. minor





and Growth of A. fatua





and Growth of Wheat

3.1.5.1. Wheat var. Inqlab 91 38

3.1.5.1.1. Effect on Germination 38

3.1.5.1.2. Effect on Shoot Growth 38

3.1.5.1.3. Effect on Root Growth 39

3.1.5.2. Wheat var. Sehar 2006 39




3.1.5.3. Wheat var. Uqab 2000 40




3.2. Foliar Spray Bioassays 52

3.2.1. Effect of Fungal Culture Filtrates on Growth of C. album 52



3.2.2. Effect of Fungal Culture Filtrates on Growth of R. dentatus 52



3.2.3. Effect of Fungal Culture Filtrates on Growth of P. minor 53



3.2.4. Effect of Fungal Culture Filtrates on Growth of A. fatua 54



3.2.5. Effect of Fungal Culture Filtrates on Growth of Wheat 54



3.3. Field Experiment 70

3.3.1. Effect of Fungal Culture Filtrates on Weed Biomass 70

3.3.2. Effect of Fungal Culture Filtrates on Wheat Growth and Yield 70

3.4. Leaf Discs Bioassays Using Crude Organic Fractions 76

3.5. Leaf Discs Bioassays Using Purified Chromatographic Fractions 76

3.6. Spectroscopic Data of Isolated Compounds 84

3.6.1. Compound 1, (Holadysenterine) 84

3.6.2. Compound 2, (Z)- docos-5-en-1-oic acid 85

Chapter 4: Discussion 86

Conclusion 94

Future Prospects 94

References 95

Appendices

List of Tables Title Page #

Chapters 3: Results Table 1 42

Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera

species on germination and growth of Chenopodium album in laboratory bioassays.

Table 2 42


species on germination and growth of Rumex dentatus in laboratory bioassays.

Table 3 45


species on germination and growth of Phalaris minor in laboratory bioassays.

Table 4 45


species on germination and growth of Avena fatua in laboratory bioassays.

Table 5 48


species on germination and growth of wheat var. Inqlab 91 in laboratory bioassays.

Table 6 48


species on germination and growth of wheat var. Sehar 2006 in laboratory

bioassays.

Table 7 48


species on germination and growth of wheat var. Uqab 2000 in laboratory

bioassays.

Table 8 72

Effect of foliar spray of herbicide bromoxynil+MCPA and culture filtrates of four

Drechslera spp. on biomass of Rumex dentatus.

Table 9 78

Leaf discs bioassays using crude organic fractions on punctured leaf surface.

Table 10 79

Leaf discs bioassays using crude organic fractions on unpunctured leaf surface.

Table 11 81

Leaf discs bioassays using purified chromatographic fractions from chloroform

fraction of D. australiensis on punctured leaf surface.

Table 12 82

Leaf discs bioassays using purified chromatographic fractions from ethyl acetate

fraction of D. australiensis on punctured leaf surface.

List of Figures Title Page #

Chapters 3: Results Fig. 1 56

Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-week

and 2-week old Chenopodium album plants.

Fig. 2 58


and 2-week old Rumex dentatus plants.

Fig. 3 60

Effect of foliar spray of culture filtrates of Drechslera spp. on growth of 1-week and 2-

week old Phalaris minor plants.

Fig. 4 62


and 2-week old Avena fatua plants.

Fig. 5 64


and 2-week old plants of wheat var. Inqlab 91.

Fig. 6 66


and 2-week old plants of wheat var. Sehar 2006.

Fig. 7 68


and 2-week old plants of wheat var. Uqab 2000.

Fig. 8 73

Effect of foliar spray of full (FD) and half dose (HD) of Bromoxynil+MCPA and

culture filtrates of four Drechslera spp. on different growth parameters of field grown

wheat.

Fig. 9 74

Effect of foliar spray of full (FD) and half dose (HD) of Bromoxynil+MCPA and

culture filtrates of four Drechslera spp. on grain yield and 100 grains weight of field

grown wheat.

Fig. 10 84

Chemical structure of holadysenterine

Fig. 11 85

Chemical structure of (Z)- docos-5-en-1-oic acid

List of Plates Title Page #

Chapters 2: Materials and Methods Plate 1 30

Scheme for leaf discs bioassays using crude organic fractions.

Chapters 3: Results Plate 2 43

Effect of culture filtrates of four Drechslera species on germination and growth of C.

album in laboratory bioassays.

Plate 3 44

Effect of culture filtrates of four Drechslera species on germination and growth of R.

dentatus in laboratory bioassays.

Plate 4 46

Effect of culture filtrates of four Drechslera species on germination and growth of P.

minor in laboratory bioassays.

Plate 5 47

Effect of culture filtrates of four Drechslera species on germination and growth of A.

fatua in laboratory bioassays.

Plate 6 49

Effect of culture filtrates of four Drechslera species on germination and growth of

Inqlab 91 in laboratory bioassays.

Plate 7 50


Sehar 2006 in laboratory bioassays.

Plate 8 51


Uqab 2000 in laboratory bioassays.

Plate 9 57


and 2-week old Chenopodium album plants

Plate 10 59


and 2-week old Rumex dentatus plants.

Plate 11 61


and 2-week old Phalaris minor plants.

Plate 12 63


and 2-week old Avena fatua plants.

Plate 13 65


and 2-week old wheat var. Inqlab 91 plants.

Plate 14 67


and 2-week old wheat var. Sehar 2006 plants.

Plate 15 69


and 2-week old wheat var. Uqab 2000 plants.

Plate 16 75

Effect of foliar spray of recommended (RD) and half dose (HD) of

Bromoxynil+MCPA and culture filtrates of four Drechslera spp. on field grown weed

and wheat.

Plate 17 80

Effect of crude chloroform (A) and ethyl acetate (B) fraction of culture filtrate of

Drechslera australiensis on punctured leaf discs of Rumex dentatus.

Plate 18 83

Effect of 2,4-D and chromatographic fractions(A), (C), (D), (F)and (H) of culture

filtrate of Drechslera australiensis on punctured leaf discs of Rumex dentatus.

i

List of Abbreviations

kg ha-1

Kilogram per hectare

GR Glyphosate-resistant

µg-1

Per microgram

AAL Alternaria alternata

ppm Parts per million

2,4-D 2, 4- dichlorophenoxyacetic acid

mM Milli molar

µM Micro molar

M Molar

mL Milliliter

Monocot. Monocotyledon

Dicot. Dicotyledon

P cm-2

Pascal per square centimeter

rpm Revolutions per minute

cm Centi meter

CRD Completely randomized design

g Gram

mg kg-1

Milligram per kilogram

NPK Nitrogen phosphorous potassium

RCBD Randomized complete block design

cm Centi meter

mg Milli gram

µg µl-1

Micro gram per microliter

µl Microliter

TLC Thin Layer chromatography

PTLC Preparative Thin Layer Chromatography

RPHPLC Reversed Phase High Performance Liquid Chromatography

UV Ultra violet

Rf Retention factor

DI Deionized

MS Mass Spectrometry

EIMS Electron Impact Mass Spectroscopy

HREIMS High Resolution Electron Impact Mass Spectroscopy

NMR Nuclear Magnetic Resonance

MHz Mega Hertz

1D One Dimensional

2D Two Dimensional 1NMR Hydrogen NMR

13NMR Carbon NMR

COSY Correlation Spectroscopy

HMBC Heteronuclear Multiple Bond Correlation

Conc. Concentration

mm Millimeter

ii

wt. Weight

FCF Fungal Culture Filtrates

var. Variety

CF Culture filtrate

FD Full dose

HD Half dose

g plot-1

Gram per plot

RD Recommended dose

m.p. Melting point

cm-1

per centimeter

Calcd. calculated

iii

Summary

Wheat (Triticum aestivum L.) is a major crop of Pakistan and is regarded as the

staple food of people of the country. The average grain yield in Pakistan is very low as

compared to yield potential possessed by most of the wheat cultivars. One of the most

important reasons for the low yield of the wheat in the country is the infestation of weeds.

Up to 45 weed species have been reported from different wheat growing areas of

Pakistan. Use of synthetic herbicides is believed to be the most effective strategy for the

management of weeds in wheat fields. However, indiscriminate use of these agro-

chemicals leads to environmental and health problems on one hand and evolution of

herbicide resistance in weeds on the other hand. The alternative to these synthetic agro-

chemicals is the use of natural products isolated from plants and fungi or their synthetic

analogues. The present study was, therefore, designed to evaluate the herbicidal potential

of culture filtrates of four Drechslera species viz. D. australiensis (Bugnicourt)

Subramanian & Jain., D. biseptata (Sacc. & Roum.) Richardson & Fraser, D. hawaiiensis

M.B. Ellis, and D. holmii (Luttr.) Subramanian & Jain, against four problematic weeds of

wheat namely Chenopodium album L., Rumex dentatus L. (dicotyledonous), Avena fatua

L. and Phalaris minor Retz. (monocotyledonous), and identification of active herbicidal

constituents through various chromatographic and spectroscopic techniques.

Culture filtrates of the four test Drechslera species were prepared by incubating

these fungi in M-1-D broth. In laboratory bioassays, seeds of the four selected weed

species and the three wheat varieties were exposed to original (100%) and diluted (50%)

fungal culture filtrates in Petri plates of 9-cm diameter. Culture filtrates of all the four

Drechslera spp. exhibited herbicidal activity against all the four test weeds. However, the

activity varied with the fungal species, concentration of the culture filtrates and the target

weed species. R. dentatus was found to be the most susceptible weed species. Original

culture filtrates of various Drechslera spp. significantly reduced germination, shoot

length and biomass as well as root length and biomass of R. dentatus by 12–56%, 67–

85%, 68–88%, 69–94% and 63–88% respectively. In general, culture filtrates of D.

australiensis exhibited the best herbicidal activity followed by culture filtrates of D.

hawaiiensis. Germination as well as root and shoot growth of the three test wheat

varieties was also adversely affected by culture filtrates of the various Drechslera

iv

species, however, wheat showed less susceptibility to the application of fungal culture

filtrates as compared to the target weed species especially R. dentatus.

In foliar spray bioassays, pot grown 1-week and 2-week old plants of the test

weed species and the three wheat varieties were sprayed with original Drechslera culture

filtrates four times with intervals of 4 days. Growth response of various weeds species to

fungal culture filtrates was highly variable in these bioassays. R. dentatus was found to be

the most susceptible weed species. Culture filtrates of all the four Drechslera species

significantly reduced shoot length and shoot biomass of 1-week old R. dentatus by 23–

42% and 54–60%, respectively, over control. The effect of foliar spray was more

pronounced in case of 1-week than in 2-week old plants. Culture filtrates of D.

australiensis exhibited the highest herbicidal activity against this weed species followed

by culture filtrates of D. hawaiiensis. The effect of foliar spray of fungal culture filtrates

was generally nonsignificant on the growth of other three target weed species and wheat

varieties.

The most susceptible weed species R. dentatus was selected for field trials. R.

dentatus was grown in field plots in 1:1 ratio with a wheat variety Sehar 2006. Over all

twelve treatments were made to assess the effect of culture filtrates of four Drechslera

spp. and a commercial synthetic herbicide on different growth parameters of the weed

and wheat. Culture filtrates of D. australiensis proved to be highly effective causing 58%

reduction in weed biomass over weedy check with subsequent increase of 27% in grain

yield of wheat.

In laboratory, pot and field studies, metabolites of D. australiensis exhibited the

best herbicidal activity against R. dentatus. This fungal species was thus selected for

isolation of active herbicidal ingredients. Culture filtrates of this fungal species were

successively extracted with n-hexane, chloroform, ethyl acetate and n-butanol followed

by evaporation in a rotary evaporator under reduced pressure. Solutions of different

concentrations of these crude extracts were prepared and were applied to wounded and

non-wounded leaf discs of R. dentatus. A positive reaction was indicated by the

appearance of a necrotic spot. Chloroform fraction exhibited the best herbicidal activity.

Six chemical constituents from this fraction were separated through Thin Layer

Chromatography (TLC), and the compounds were purified by Preparative Thin Layer

v

Chromatography (PTLC), followed by Reversed Phase High Performance Liquid

Chromatography (RPHPLC). Herbicidal activities of these isolated constituents were

evaluated by leaf discs bioassays. Two of the six isolated compounds exhibited the best

herbicidal activity. These compounds were identified as holadysenterine and (Z)-docos-5-

en-1-oic acid. through various spectroscopic techniques viz. Electron Impact Mass

Spectroscopy (EIMS), High Resolution Electron Impact Mass Spectroscopy (HREIMS)

and One Dimensional and Two Dimensional Nuclear Magnetic Resonance Spectroscopy

(1D and 2D NMR).

Results of the present study suggest that the metabolites of the test Drechslera

species possess herbicidal activity. The herbicidal activity of these metabolites varies

with test Drechslera species as well as the target weed species. Metabolites of D.

australiensis were found the most effective natural herbicides against broad-leaf weed R.

dentatus. All the test wheat varieties were found resistant to these metabolites. Further

studies are required to use structures of the two isolated herbicidal constituents as

analogues for the preparation of eco-friendly herbicides for the management of R.

dentatus.

1

Chapter 1

Introduction

1.2. Importance of Wheat

Wheat (Triticum aestivum L.), family Poaceae, is a globally pivotal cereal crop

with respect to area and production (Ashrafi et al. 2009). It is grown under irrigated as

well as rain-fed conditions worldwide (Zhao et al. 2009). It is regarded as the staple food

of Pakistan. Due to the presence of characteristic protein called gluten, wheat is widely

used as the principal cereal for the making of bread (Van Der Borght et al. 2005). Wheat

grains are rich in bioactive compounds which provide nutritional benefits to humans

(Loladze 2002). It contains carbohydrates as maltose, fructose, glucose, raffinose,

sucrose, starch and fructan (Högy et al. 2011). In addition to protein gluten, wheat also

contains minerals including macro-elements as K, Ca, Mg, P, S and Na; micro-elements

as Fe, Co, Se, Zn,, Cu, Mn, Cr, Mo and Ni and trace elements as Al, B, Cd, Pb and Si.

In Pakistan, wheat is cultivated as a winter crop on a huge area. It occupied an

area of 8666 thousands hectares during the year 2011–2012 having an average grain yield

equals to 2714 kg ha-1

and total production 23515 thousands tonnes (Anonymous 2012),

which is too less when compared to per hectare yield of advanced countries of the world,

inspite of the fact that most of its cultivars possess much higher yield potential. One of

the major reason for this low yield is weed infestation. In some studies, these weeds have

been investigated to incur yield losses from 10–83%, depending upon type of weed as

well as wheat cultivar, when grown in 1:1 ratio under experimental conditions (Siddiqui

et al. 2010; Anjum and Bajwa 2010).

1.2. Importance of Weeds

Weeds are plants which grow out of their proper places and whose virtues have

not yet been discovered (Kazi et al. 2007). These are regarded as the most undesirable,

aggressive and noxious element of world's vegetation. Weeds are unwanted plants, which

wrought noteworthy reduction in the yield of crop plants to variable extent depending

2

upon type and severity of infestation of weed species, type and density of crop plants,

environmental factors and soil fertility level etc. (Ahmadvand et al. 2009; Armin and

Asghripour 2011; Chauhan et al. 2012). Weeds compete with the crop plants by

occupying the space, which would otherwise be available to the crop plants (Wright et al.

2001). Weeds are also extremely likely to be competing for other sources such as water,

light and nutrients (Rajcan et al. 2001; Blackshaw et al. 2005; Erbs et al. 2009).

Water requirement for the growth of weeds is primarily of interest from the stand-

point of competition with the crop plant for the available moisture. It has been reported

that black mustard (Brassica nigra L.) transpires about four times more water than a crop

plant (Thakur 1984). In areas of low rainfall, enormous cover of weeds prevents a large

proportion of the rain falling in moderate shower from reaching the ground at all.

Moreover, weeds exert effect on nearby crop plants through uptake of water and the

intensity of this influence depends on relative rooting depths of the weed and the crop

plant (Soffe 2011).

Nutrition is another important factor that promotes plant growth. However, in

weed-crop competition, generally application of nutrients benefits weeds more than the

crop plants because of greater ability of weeds to accumulate mineral elements.

Tollennaar et al. (1997) found that under reduced nitrogen conditions, maize (Zea mays

L.) yield was reduced to 47% due to weeds infestation. Holm (1971) reported that weeds

contained approximately twice the nitrogen, 1.6 times phosphorus, 3.5 times potassium,

7.6 times calcium and 3.3 times magnesium as compared to that of corn. In other studies,

application of nitrogen fertilizers favored wild oat (Avena fatua L.) and green foxtail

[Setaria viridis (L.) Beauv.] over wheat, indicating that the addition of nitrogen profits

weeds more than crop plants (Peterson and Nalewaja 1992). It has been discovered that

there exists a correlation between density of weeds and protein content of wheat grains.

About one and half wild oat plants per meter square reduce protein content of wheat grain

by 1% (Khan 2008). Iqbal and Wright (1999) investigated the competitive ability of

lamb's quarters (Chenopodium album L.), wild mustard (Sinapis arvensis L.) and

littleseed canarygrass (Phalaris minor Retz.), in relation to wheat crop. Although there

was no effect of weed density on wheat plant height but there was significant decrease in

uptake of total nitrogen by wheat plants, dry biomass and grain yield. More over, weed

3

competition in wheat significantly effect number of tillers per square meter as well as

number of grains per spike (Chaudhary et al. 2008; Siddiqui et al. 2010). Growth habit of

some weeds, for example, orange eye butterflybush (Buddleja davidii Franch.) profits

them in behaving as strong competitors for light allowing lesser and lesser light to reach

crop plant resulting in frailty of crop plant (Richardson et al. 1996). Some weeds

intercept light due to their greater height than crop plants. e.g. wild oat reduces light

penetration and ultimately growth of wheat by being taller than the wheat plants (Cudeny

et al. 1991). Similarly, velvetleaf (Abutilon theophrasti Medik.) intercepts light due to its

greater height than soybean [Glycine max (L.) Merril.] (Akey et al.1990). Studies have

shown that weed canopy architecture perspectives especially plant height, location of

branches and leaf area determine the impact of interspecific light competition resulting in

low yield of crop plants (Naseri et al. 2012).

Inhibitory effects of weeds on crop plants through the release of phytochemicals

are also well documented. Reduction of 14–19% in the yield of soybean by the extracts of

dried residues of several weed species including C. album, red-root amaranth

(Amaranthus retroflexus L.) and A. theophrasti has been reported (Bhowmik and Doll

1992). Similar cases of reduction in crop yield by allelopathic weeds include: Purple

nutsedge (Cyperus rotundus L.) and Indian shot (Canna indica L.) in rice (Oryza sativa

L.), C. album and A. retroflexus in safflower (Carthamus tinctorius L.), Dogbane (Rhazya

stricta Decne.) on maize and crabgrass (Digitaria horizontalis Willd.) on dry bean

(Phaseolus vulgaris L.), and turnip (Brassica rapa L.) and soybean (Javaid et al. 2007;

Lin et al. 2009; Rezaie and Yarnia 2009; Khan et al. 2011; Teixeira et al. 2011).

Infestation of weeds is among the major causes of low yield of wheat that

harnesses most of the moisture and nutrients. Wheat crop faces both monocot. and dicot.

weeds infestation. Siddiqui and Bajwa (2001) and Qureshi and Bhatti (2001) have

reported 45 types of weeds from wheat growing regions of Pakistan. In these studies, P.

minor, wild oat, burclover (Medicago polymorpha L.), lesser swinecress [Coronopus

didymus (L.) Sm.], small meliot (Melilotus parviflora Desf.), toothed dock (Rumex

dentatus L.) and C. album appeared to be the most frequently occurring and densely

populated weeds. Yield losses due to these weeds in different wheat cultivars were

estimated as 20-60% (Siddiqui et al. 2010). The wheat yield losses by these weeds has

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4

been recorded up to 80% taking into account various environmental factors, weed type

and its density, and also wheat density and cultivar (Khera et al. 1995; García-Martín et

al. 2007; Qasem 2007). Thus weed management has become a serious peril to wheat

yield as weeds not only reduce yield of the crop but also deteriorate quality of the

produce in many cases (Memon et al. 2003).

1.3. Management of Weeds

At present weeds are being considered to be the major cause of suboptimal crop

yield throughout the world despite centuries of efforts in their management (Oerke 2006).

Weed management in organic agriculture uses preventive methods that include different

strategies like intercropping, cover crops, green manure and mulches. Roots of

allelopathic plants release compounds in the soil that are toxic to weeds (Campiglia et al.

2009; Isik et al. 2009; Flower et al. 2012). However suppressive effect on weeds is

influenced by type of species, seeding rate and method, planting date, decomposition

time of plant residues and weather (Yalcin and Cakir 2006; Ortiz-Monasterio and Lobell

2007; Kalinova 2010; Chauhan et al. 2011). Several methods of the weed management

are in vogue such as cultural and mechanical, biological, chemical and management

through natural products.

1.3.1. Mechanical Control

It involves the removal of weeds by various tools/implements including tillage,

flooding, fire, hand hoeing, pulling and mulching.

1.3.1.1. Tillage

The principle of this method is simply to turn the weeds under or bring them to

the soil surface where they die on account of desiccation (Swanton et al. 1999). This

method also affects the nature and extent of weed populations (Blackshaw et al. 1994).

This practice can be employed before and after planting. Tillage before planting results in

germination of weed seeds and subsequent destruction of seedlings during soil

preparation (Dirk 2007). However the behavior of weeds and their interaction with crops

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5

is a complex phenomenon which is still under investigation. Weed species having

photoblastic germination tend to be more problematic in conservation agriculture. Also,

in the absence of tillage, perennial weeds may also become more problematic (Chauhan

et al. 2012).

1.3.1.2. Flooding

The principle of this technique lies in promoting weed seed decay and

germination. For this tactic to be useful, seeds of weeds should be submerged for an

extended period of time (Rao 2000). For example, weedy rice (Oryza nivara S.D. Sharma

& Shastry) is a noxious weed of cultivated rice. Infestations of weedy rice have been

recorded to have spread to 40-75% of the total of rice cultivation region in European

countries (Ferrero 2003). In a field experiment, weedy rice plant density declined

significantly by the application of winter flooding. Here flooding caused more than 95%

decrease in the number of viable weed seeds when compared to fields which were left dry

between rice crops (Fogliatto 2010). Winter flooding is a common management practice

in America, where rice fields are flooded in autumn following rice harvest until the

spring before tillage operations (Van Groenigen et al. 2003).

1.3.1.3. Fire

Use of conventional fire has long been witnessed to control unwanted vegetation.

Nowadays a modified technique of fire known as flame cultivation is used on very small

scale. In this method, fire is used for selective control of weeds in crop rows. But it

requires great care as it can damage the crop as well as having negative effects on soil

(Tu et al. 2001). Gleadow and Narayan (2007) carried out a study in Australia where a

weed sweet pittosporum (Pittosporum undulatum Vent.) has colonized into many

habitats, causing a serious damage to structural diversity and floristic composition due to

its competitive ability that created an environment conducive to its own progeny and

deleterious for other plant species. They concluded that high temperatures associated with

wildfires are enough to disrupt the invasion cycle of test plant species.

6

1.3.1.4. Hand Hoeing

This method has proven to be very effective to eradicate annual and biennial

weeds as it not only eradicates weeds but also improves soil aeration due to stirring.

However, this method is less effective in case of perennial weeds. In spite of all its

advantages, it is not considered to be cost effective (Tu et al. 2001). Besides, there are

reports that hand hoeing is less effective when compared with chemical control (Subhan

et al. 2004).

1.3.1.5. Mulching

Organic mulching is a strategy in which at least 30% of the surface of soil is

covered by plant material. It conserves the soil, improves the soil ecology, stabilizes and

enhances crop yield and improves various environmental factors. Although mulching

practices are no solutions but they represent technological options that integrate

conservation and productivity considerations (Erenstein 2003). Mulching has smothering

effect on weeds by casting shadow on weed plants, which results in little photosynthesis

resulting in frail weed plants offering lesser competition to main crop. Hiltbrunner et al.

(2007) in a field experiment concluded that legumes can be used to suppress weeds in

wheat field. Due to high cost, it is considered cost-effective only in case of high value

crops like tea and coffee. For example, guatemala grass (Tripsacum laxum Nash.) is used

as mulching in tea fields (Rao 2000). However, mechanical methods are not feasible

where weeds resemble morphologically to crop. e.g. wild oat (Avena ludoviciana

Durieu.) and P. minor mimic wheat before flowering. Also, mechanical weed control is

not cost-effective and becomes difficult in broadcast sown wheat.

1.3.2. Cultural Methods

1.3.2.1. Competitive Crops and Cultivars

Under field conditions, both weeds and crop compete with each other for same

resources (Turk and Tawaha 2003). So if those cultivars of crops are planted that have

7

high vigor and able to grow more rapidly as compared to weeds, significant losses due to

these weeds can be prevented (Corre-Hellou et al. 2011). In this regard, competitiveness

of the crop seems to be important in weed behaviour. For example, pea (Pisum sativum

L.), a weak competitor, had much higher biomass at harvest compared with oats and

winter wheat (Lundkvist et al. 2008). More rapidly growing crops cause stifling effect on

the growth of weeds by casting its shadow as well as excluding weeds out of competition

by utilizing resources like nutrients, sunlight, moisture and carbon dioxide (Aldrich and

Kremer 1997; Radicetti et al. 2012).

1.3.2.2. Crop Rotation

Monocrop culture practiced for an extended period of time in a particular area

helps to establish associated weeds of that particular crop in that area. Management of

weeds through crop rotation is effective because changing patterns of disturbance

diversifies selection pressure that prevents the proliferation of weed species well suited to

the practices associated with a monoculture crop. For example, wild mustard populations

can be reduced by selective treatment of small grain grown in rotation with row crops

(Turk and Tawaha 2003). Crop rotations have also been reported to break disease cycle,

improve nitrogen fixation and water-use efficiency (Ryan et al. 2008). Thus wheat-

legume rotation system has proven to be the best in certain regions as it results in the

highest yield and protein content of wheat crop. Also this treatment does not need

fertilizers to achieve better crop yield and is considered to be more sustainable system for

low rainfall zones (Galantini et al. 2000).

1.3.2.3. Increased Crop Density

The principle of this practice lies in the fact that greater number of crop plants in

the field offers more competition to weeds on account of their smothering effects as well

as competition for limited resources. This technique uses enhanced seed rate and narrow

inter and intra row spacing. Also there exists optimum seed rate for obtaining higher

grain yield. However, such practices need to be optimized in a particular area

(Ahmadvand et al. 2009; Marwat et al. 2011).

8

1.3.2.4. Intercropping

Intercropping is considered to be an efficient tool for better land use efficiency

and weed suppression. As an example, wheat and bean (Vicia faba L.) were grown as

intercrops. Regarding weed suppression, intercrops were more effective than wheat sole

crops (Eskandari 2011). In another study, pea-barley intercrops have shown to lessen the

weed biomass when compared with the pea and barley sole crops (Corre-Hellou et al.

2011).

1.3.2.5. Companion Cropping

Judicious use of living mulches is very important factor as it requires adapted

seeding rate and technique as well as type of main and cover crop. In a study, living

mulches belonging to four different genotypes of legumes were used in order to tap its

full potential in winter wheat crop. It was found that legumes producing more dry matter

namely birdsfoot trefoil (Lotus corniculatus L.) and white clover (Trifolium repens L.)

and controlled weeds better than species producing less dry matter such as subclover

(Trifolium subterraneum L.) and strong-spined medick (Medicago truncatula Gaertner)

(Hiltbrunner et al. 2007).

Although cultural and mechanical practices are equally effective yet large

numbers of farmers are not very well trained in this regard (Thomas et al. 1999). Also

these methods become more effective when used in an integrated form (O’Donovan et al.

2001; Derksen et al. 2002). Under such circumstances weed control through chemical

herbicides has become the most popular method among the farmers.

1.3.3. Biological Weed Control

Biological control strategies are utilized in a classical (Kurose et al. 2012),

augmentative (Vorsino et al. 2012) or inundative mode (Gerber et al. 2011). Classical

biological control by means of plant pathogens has been used in many agro climatic areas

to control exotic weeds. The concept of this approach is simple to use: Discover effective

and highly host-specific agents from the weed's native geographic range, confirm their

biosafety and effectiveness, and introduce them into regions where the weed has been

9

newly invaded and needs control (Charudattan and Dinoor 2000; Cripps et al. 2011).

Augmentative approaches on the other hand depend on the release of additional numbers

of a natural enemy when too few are present to control a pest effectively (Lv et al. 2011).

While inundative approach depends on the release of large numbers of biological control

agents to control a pest when there is very low risk of the biological control agent to

spread or establish it permanently. In this approach, application may be made to induce

disease epidemics or to act as microbial pesticides (Williams et al. 2003; Fernando et al.

2010). Biological control of weeds using phyto-pathogens started in the 1970s when a

small number of noxious weeds were controlled by different strategies. Since then,

scientists are evaluating different strategies in a hope to solve some of the most

intractable weed problems (Charudattan and Dinoor 2000). In selecting classical

biological control agents for landscape-level suppression of weeds, prior evaluation of

their biosafety together with their effectiveness is of utmost importance.

A bioherbicide is defined as a plant pathogen used as a weed-control agent

through inundative and repeated applications of its inoculum. Bioherbicides provide

envovironment friendly, non-chemical method to control a number of weeds (Saxena and

Pandey 2002). Inspite of huge research work done on microbial herbicides, only few

bioherbicides have been registered so far. As for example, DeVine composed of isolate of

Phytophthora palmivora Butl.; Collego and BioMal, both based on Colletotrichum

gloeosporioides (Penz.) Penz. & Sacc.; Dr. BioSedge based on the Puccinia canaliculata

Arthur; CAMPERICO based on bacterium Xanthomonas; and Stumpout based on a

basidiomycete Cylindrobasidium. These have been used to control stranglervine

[Morrenia odorata (Hooker & Arnott) lindley], northern jointvetch, [Morrenia odorata

(Hooker & Arnott) lindley], round-leaved mallow (Malva pusilla Smith) and bluegrass

(Poa annua L.) (Yongqang 1998; Charudattan 2000; Charudattan and Dinoor 2000).

However, mass production of bioherbicides is difficult, and because of their specific

requirement for action conditions, these products are not world-famous and did not bring

significant economic benefits (TeBeest et al. 1992; Makowski 1993). There are many

other examples of bioherbicides from microbes. For example, mixture of Drechslera

gigantea Heald & Wolf, Exserohilum longirostratum (Subramanian) Sivanesan, and

Exserohilum rostratum (Drechsler) Leonard et Suggs, have also been described to control

10

many grassy weeds with 100% disease incidence having no injurious effects on crops

(Chandramohan and Charudattan 2001). C. rotundus and yellow nutsedge (Cyperus

esculentus L.) are serious weeds in Florida and in many other parts of the world.

Dactylaria higginsii (Luttr.) M.B. Ellis is a promising fungal bioherbicide candidate for

these weeds (Shabana et al. 2010a). The fungal pathogen Microsphaeropsis amaranthi

(Schwein.) Kuntze has been considered as a promising bioherbicide for the control of

waterhemp (Amaranthus tuberculatus Sauer) (Shabana et al. 2010b). The bacterium

Pseudomonas fluorescens (Flügge) Migula, has potential for biocontrol of S. viridis and

a large number of grassy weed species (Pedras et al. 2003; Banowetz et al. 2009).

Deleterious rhizobacteria have been shown to affect the invasive species cheatgrass

(Bromus tectorum L.) and can likely serve as biological control agents (Dooley and

Beckstead 2010). However, limited commercial interest, complexities in production,

assurance of efficacy and shelf-life of inoculum are serious limitations that have led to

the abandonment of several promising plant pathogens for the biocontrol of weeds

(Charudattan and Dinoor 2000). Lack of host specificity of potential biocontrol agent is a

major criterion that leads to ultimate rejection of proposed biocontrol agent (Yobo et al.

2009).

Biological control of weeds using insects as enemies has also been widely studied

successfully in many parts of the world (Manrique et al. 2008; Hough-Goldstein et al.

2009; Myint et al. 2012). For example, some floating invasive plants of major importance

have been controlled by biological agents such as American weevil (Stenopelmus

rufinasus Gyllenhal) controlled mosquito fern (Azolla filiculoides Lam.). Similarly,

species of Neochetina weevils controlled water hyacinth [Eichhornia crassipes (Mart).

Solms] and Salvinia weevil (Cyrtobagous salviniae Calder & Sands) controlled giant

salvinia (Salvinia molesta Mitchell) (McConnachie et al. 2004; Hill and McConnachie

2009; Coetzee et al. 2009; Julien et al. 2009). Deploying multiple biological control

agents where they can partition the target resources in space and/or time has been proven

to be more effective, e.g. in Australia, individual and combined effects of the rust fungus

Puccinia myrsiphylli (Thuem.) Wint. and a leafhopper (Zygina sp.) for biocontrol of a

weed, bridal creeper [Asparagus asparagoides (L.) Druce] have been investigated. In this

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11

study, synergistic effect of both biological control agents was evident (Turner et al.

2010).

Classical biocontrol has not been a world-famous approach of weed management

in intensively managed crops due to the slowness of the classical biocontrol process

relative to the short duration of the cropping season. The frequent disruptions associated

with cropping practices can also have adverse effects on classical biocontrol agents

(Charudattan and Dinoor 2000). Also a biological control agent for a weed developed for

one country has proven to be ineffective against the same weed in another country

(Balciunas 2007). Depending on one’s point of view, this biological control approach has

been quite successful or wrought with limitations (Charudattan and Dinoor 2000). Recent

investigations point out certain unavoidable non target effects of biological control agents

due to complexities involved in the interactions of bio-control agents and target species

(Pearson and Callaway 2005). Also all introduced bio-control agents incur effects on

nontarget species (Delfosse 2005). So, there has been considerable debate on risks

associated with biological control, e.g. non-target impacts of bio-control agents (Barratt

et al. 2010).

1.3.4. Chemical Method

To combat weeds, chemical herbicides have been a major breakthrough. At

present, control of weeds by the application of herbicides is considered to be the most

effective method of controlling weeds. So herbicides have become the basis for weed

control in intensive agriculture (Rüegg et al. 2007). Chemical weed control is preferred

because it is cost-effective as well as it offers no physical damage to the crop that

happens during hand hoeing and tillage. Moreover, the control is more effective as the

weeds even within the rows are killed which otherwise escape invariably during

mechanical control, on account of morphological mimicry to wheat. Different

acetamides, aliphatics, arsenicals, benzamides and sulfonylureas are being used as

herbicides (Verstraeten et al. 2002).

There are many successful attempts of weed management through chemical

herbicides (Jordan et al. 2009; Krutz et al. 2009; Hulting et al. 2012). Sometimes

herbicidal mixtures are used because none of the herbicides alone controls a wide enough

12

range of weeds to be suitable in all circumstances. It is, therefore, desirable to mix

herbicides to get the best fit for the weeds in the crop. Various chemical herbicides such

as Topic (clodinafop-propargyl), Puma Super (fenoxaprop-p-ethyl), Affinity

(carnfentrazone ethyl + isoproturon), Buctril Super (bromoxynil octanovate +

heptanovate ester), imidazolinone herbicides, imazapyr, imazapyr plus imazapic, and

imazapyr plus imazamox etc. are very effective in controlling weeds of wheat fields in

various parts of the world (Bibi et al. 2005, 2008; Cheema et al. 2006; Usman et al. 2010;

Kleemann et al. 2009). However efficacy of different herbicides to control weeds in

wheat varies and in some cases it is site dependant (Zand et al. 2010).

Although synthetic herbicides are every effective in the management of weeds,

however, indiscriminate use of these agrochemicals have created a number of problems.

Due to frequent use of herbicides, there have been dramatic increase in the frequency and

diversity of weed biotypes that are herbicide-resistant, that poses a threat to the

sustainability of agriculture worldwide (Yuan et al. 2007; Llewellyn et al. 2009). For

example, overwhelming evolution of resistance of a number of weeds including wild oats

to various chemical herbicides have been reported (Vila-Aiub et al. 2005; Singh et al.

2012). More than 200 distinct weed biotypes that are resistant to various herbicides, have

evolved world over (Devine and Shukla 2000). Furthermore, many herbicides are very

toxic to some sensitive crops and may cause severe crop injury in some cases as in

cotton, corn, many vegetable crops and wheat (Kadir and Charudattan, 2000; Usman et

al. 2010; Sikkema et al. 2007). Since herbicides have also shown to affect crops so in this

scenario herbicide resistant crops are being developed. e.g. glyphosate-resistant crops like

soybean, cotton (Gossypium hirsutum L.) and maize have been widely adopted in USA

(Sankula 2006). Due to the development of glyphosate-resistant crops, many farmers

depend solely on glyphosate for weed control and its recurrent use is the main cause for

the development of herbicide resistant weeds (Holt 1992). Also, in recent years, the use

of chemicals is becoming more restrictive due to public awareness regarding ill effects of

all the chemical herbicides (Marin et al. 2003; Rial-Otero et al. 2005). The herbicides

used to boost agricultural food production may not only combat pests and weeds but also

present toxic properties and cause genetic aberrations into exposed fauna and flora (Losi-

Guembarovski et al. 2004). The herbicides have a long residence time in the atmosphere

13

and come to earth in the form of wet and dry depositions (Waite et al. 2005). There are

evidences that herbicides present in the atmosphere induce intracellular overproduction of

reactive oxygen species and herbicide induced oxidative stress disturbing the

photosynthesis of target plants e.g. in wheat and tilapia (Oreochromis niloticus L.), thus

damaging plant cells (Peixoto et al. 2006; Song et al. 2006, 2007; Wang and Zhou 2006).

Phenylurea herbicides are used world over, that often pollute surface and groundwater in

concentrations exceeding the limiting value of 0.1 µg-l for drinking water (Badawi et al.

2009). Herbicides are also known to affect aquatic life. For example, in green alga

Raphidocelis subcapitata (Korsh) Nygaard et al., a reduction in photosynthesis has been

reported. The most toxic herbicides documented in this regard include, atrazine,

ametryme, chlorotoluron, cyanazine, isoproturon and diuron (Ma et al. 2006). Natural

waters have been investigated to contain complex mixtures of herbicides as well as

herbicide breakdown products as contaminants potentially posing a threat to marine

communities through chemical interactions (Magnusson et al. 2010). In some

experiments, application of herbicides have shown to effect aquatic plants (Huiyun et al.

2009; Vervliet-Scheebaum et al. 2010). Frequent use of herbicides has led to weed

adaptation via the selection of resistance mechanisms enabling weed plants to withstand

herbicide application in at least 194 weed species worldwide (Heap 2008; Heap 2010;

Johnson et al. 2009; Knezevic et al. 2010). For example, Wimmera ryegrass (Lolium

rigidum Gaudin) is the most prevalent and noxious grassy weed of winter cereals in

Spain. Due to frequent use of herbicides to control this weed, its populations are evolving

herbicide resistance (Loureiro et al. 2010). This herbicide resistance has posed serious

concerns for agriculture because it disrupts herbicide-based weed eradication and also

because alternative strategies of controlling weeds have proven to be less effective (Bond

and Grundy 2001; Bastiaans et al. 2008). Moreover, due to greater water solubility, high

polarity and heat stability, it is difficult to fade away them from the atmosphere (Bonnet

et al. 2008; Rashid et al. 2010).

In addition to their harmful effects on environment, high cost associated with the

use of herbicides is a limiting factor in the profitability of crop production (Partridge et

al. 2006). Agricultural producers cannot give up their chemical tools for weed control

until research provides them with workable alternatives (Quimby et al. 2002). Moreover,

14

at many places, the use of chemical herbicides is severely restricted or mostly banned

(Vurro et al. 2012). Hence, there is dire need of alternate eco-friendly, cost effective and

bioefficaceous methods of weed control. In this scenario, there have been numerous

efforts to control invasive weeds by natural products from plants and microbes (Van

Driesche et al. 2010; Javaid 2010; Aliferis and Jabaji 2011; Berner and Bruckart 2012).

1.3.5. Natural Products as Herbicides

1.3.5.1. Herbicides from Plants

Chemical interactions between and among both plants and microorganisms

through release of biologically active chemical compounds into the environment is

known as allelopathy. Allelopathic potential of certain weeds and crop species that exists

in nature can influence the growth and distribution of associated weed species and the

yield of desired crops. By virtue of this, allelopathy has been harnessed successfully in

biocontrol programs to restrain noxious weeds. Allelopathy thus plays an important role

in an agroecosystem and a better understanding of this phenomenon would help in crop

improvement through sustainable agriculture (Farooq et al. 2011).

Herbicides derived from naturally occurring materials are gaining fame as these

are environmentally safe. Numerous allelopathic plants have been exploited for their use

as bioherbicides as these plants contain natural growth inhibitors (Xuan et al. 2005). For

example, sunflower (Helianthus annuus L.) cv. Suncross-42 extracts exhibited herbicidal

activity against R. dentatus and C. album (Anjum and Bajwa 2007a,b). A bioactive

annuionone H was isolated from leaves of sunflower. The isolated compound showed

pronounced herbicidal activity against P. minor, C. album, R. dentatus, C. didymus and

M. polymorpha (Anjum and Bajwa 2005). Céspedes (2006) studied some phytochemicals

of plant origin and found that some of them possessed potent herbicidal activities.

Similarly, rice allelopathy has been exploited against world’s noxious weed of rice,

barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] (Mennan et al. 2011). Ergosterol

peroxide and 7-oxo-stigmasterol proved to be the most active herbicidal compounds

isolated from rice extracts. In case of ergosterol peroxide the herbicidal activity was

higher than the commercial chemical herbicide Logran (Trisulfuron) (Macías et al. 2006).

15

Likewise, catmint [Anisomeles indica (L.) Kuntze] have shown potential herbicidal

activity like that of chemical herbicide against P. minor and other weeds of the wheat

under natural field conditions (Batish et al. 2007). Salamci et al. (2007) found that

essential oils isolated from Turkish Tanacetum aucheranum and Tanacetum

chiliophyllum var. Chiliophyllum exhibited herbicidal effects. Herbicidal effects of the

oils were evaluated on seed germination and seedling growth of test weed species viz. C.

album, curly dock (Rumex crispus L.) and A. retroflexus. Similarly, natural plant product,

benzoxazolin-2(3H)-one has been investigated to be having herbicidal activity against

lettuce (Lactuca sativa L.) (Sánchez-Moreiras et al. 2008). Likewise, essential oil of

letswaart [Origanum acutidens (Hand.-Mazz.)] has shown phytotoxicity against C.

album, R. crispus and A. retroflexus. The oil, thymol and carvacrol completely arrested

the seed germination and seedling growth of all the test plant species (Kordali et al.

2008). Glucosinolates form a group of allelochemicals produced by many species of

plants like Brassica, Sinapis, Lepidium, Nasturtium and Limnanthes spp. Glucosinolate

degradation products have shown herbicidal activity on B. tectorum coleoptile emergence

(Stevens et al. 2009). Similarly, herbicidal potential of sorghum (Sorghum bicolor L.)

water extract alone and in combination with water extracts of other allelopathic plants:

sesame, sunflower, tobacco, eucalyptus and brassica, against two problematic weeds of

wheat, P. minor and A. fatua have been demonstrated. In this study application of

sorghum and sunflower extracts each were found more effective than rest of the test plant

species (Jamil et al. 2009). In another study, wollemi pine (Wollemia nobilis Jones, Hill

& Allen) plant material exhibited herbicidal effects against L. rigidum and wild radish

(Raphanus raphanistrum L.) (Seal et al. 2010). In the same way, Aswagandha [Withania

somnifera (L.) Dunal] has been exploited as natural herbicide against Parthenium weed

(Parthenium hysterophorus L.) and P. minor under laboratory conditions, foliar spray

bioassays and soil amendment bioassay (Javaid et al. 2010a, 2011a). Similar herbicidal

activity of extracts of Datura (Datura metel L.) has also been reported against P. minor

and P. hysterophorus (Javaid et al. 2008, 2010b).

16

1.3.5.2. Herbicides from Fungi

The literature abounds with examples of herbicidal compounds (phytotoxins)

isolated from fungal world. Phytotoxins are largely low molecular weight secondary

metabolites capable of deranging the vital activity of plant cells viz. enzyme inhibition,

interference with the properties of membranes and interference with defense responses.

Phytopathogenic fungi are best known as phytotoxin producers. Phytotoxins are usually

isolated from in vitro cultures of the pathogen grown either on solid or liquid media and

have the ability to damage plants. Tal et al. (1985) isolated novel compounds, named

radianthin and radicinin having phytotoxic activity from liquid culture broth of Alternaria

helianthi (Hansf.) Tubaki & Nishih. Similarly, AAL-toxin TA isolated from Alternaria

alternata (Fr.) Keissl. and Fumonisin B1 from Fusarium moniliforme J. Sheld. proved

potent phytotoxins against duckweed (Lemna pausicostata L.) (Abbas et al. 1998).

Ascaulitoxin characterized as N2-(2,4,7-triamino-5-hydroxy)-octanedioyl-β-D-gluco-

pyranoside was isolated from the culture filtrate of Ascochyta caulina (P. Karst.) Aa &

Kesteren found a promising natural herbicide for the control of many noxious weeds.

Ascaulitoxin, when assayed in the leaf-puncture assay on weeds including C. album,

common sowthistle (Sonchus oleraceus L.), noogoora burr (Xanthium occidentale

Bertol.), annual fleabane [Erigeron annuus (L.) Pers.] and Tree of Heaven (Ailanthus

glandulosa Desf.), ascaulitoxin caused the appearance of necrotic spots (Evidente et al.

1998). Latter on, a phytotoxic metabolite trans-4 aminoproline was isolated from culture

filtrate of the same fungus and also found very effective in controlling C. album

(Evidente et al. 2000). In addition, Vurro et al. (2001) also identified another herbicidal

constituent aglycone of ascaulitoxin from culture filtrates of A. caulina that was found

very effective in controlling C. album. Latter on Vurro et al. (2012) showed that

metabolites with herbicidal properties from A. caulina can be produced on pre-industrial

level. Fukushima et al. (1998) reported that culture filtrate of Nigrospora sacchari

(Speg.) E.W. Mason showed strong herbicidal activity against weeds such as E. crus-

galli, S. viridis, A. theophrasti and slender amaranth (Amaranthus viridis L.) They

isolated three lactones from the culture broth of test fungus. The major component was

identified as (+)-phomalactone, 6-(1-propenyl)-5,6-dihydro-5-hydroxy-2H-pyran-2-one.

http://en.wikipedia.org/wiki/L.

http://en.wikipedia.org/wiki/Pers.

17

Others were 5-[1-(1-hydroxybut-2-enyl)]-furan-2-one and 5-[1-(1-hydroxybut-2-enyl)]-

dihydrofuran-2-one. The herbicidal activity of these fungal metabolites was caused by

cellular disruption when applied at concentrations higher than 50 ppm. Similarly, two

new phytotoxic nonenolides viz. herbarumins I [(7S,8S,9R)-7,8-dihydroxy-9-propyl-5-

nonen-9-olide] and herbarumins II [(2R,7S,8S,9R)-2,7,8-trihydroxy-9-propyl-5-nonen-9-

olide], were identified from Phoma herbarum Westend. Their phytotoxicity was

evaluated as pre-emergent herbicides against amaranth (Amaranthus hypochondriacus

L.). Herbarumins I appeared to be more potent herbicide than the positive control 2,4-D,

while herbarumins II exhibited a herbicidal potency similar to that of 2,4-D (Fausto

Rivero-Cruz et al. 2000). Macías et al. (2001) reported two phytotoxic naphthopyranone

derivatives by investigating fermentation broth and mycelium of the coprophilous fungus

Guanomyces polythrix M.C. González, Hanlin & Ulloa. They named these isolated

compounds as (2S, 3R)-5- hydroxy-6,8-dimethoxy-2,3-dimethyl-2,3-dihydro-4H-

naphtho[2,3-b]-pyran-4-one and (2S, 3R)-5-hydroxy-6,8,10-trimethoxy-2,3- dimethyl-

2,3-dihydro-4H-naphtho[2,3-b]-pyran-4-one. The isolated compounds significantly

arrested radicle growth of two weed seedlings, E. crus-galli and A. hypochondriacus.

Likewise, Macrocyclic trichothecene toxins produced by phytopathogen Myrothecium

verrucaria (Alb. & Schwein.) Ditmar and the non-trichothecene toxin atranone B from

Stachybotrys atra Corda have shown their phytotoxicity against kudzu (Pueraria lobata

L.) and duckweed plantlets (Abbas et al. 2002).

Destruxins are secondary metabolites isolated from entomopathogenic fungus,

Oospora destructor (Metschn.) Delacr. Destruxins exhibited a wide variety of biological

activities, but are well known for their phytotoxic activities. Phytotoxic activity of

destruxins has been demonstrated against many herbs including oat (Avena sativa L.) and

quinoa (Chenopodium quinoa Willd.) (Pedras et al. 2002). Phytotoxicity tests of the

metabolite solutions or crude toxins of A. alternata on aquatic weeds especially E.

crassipes, developed phytotoxic symptoms (Babu et al. 2003a). Moreover, an herbicidal

glycoprotein, produced by Phoma eupyrena Sacc. brought about blighting and necrosis of

leaf tissues when 1-5 µg was introduced into the mesophyll tissue of water lettuce [Pistia

stratiotes (L.) Fam.] (Babu et al. 2003b). Phytotoxic fungal metabolites namely

leptosphaerodione, elsinochrome A and cercosporin have been isolated from different

18

isolates of phytopathogenic fungal species Stagonospora convolvuli Dearn. & House.

These metabolites have been shown to be toxic to field bindweed (Convolvulus arvensis

L.) and hedge bindweed [Calystegia sepium (L.) R. Br.] (Ahonsi et al. 2005). Luis et al.

(2005) isolated two phytotoxic compounds namely 1-hydroxy-2-oxoeremophil-

1(10),7(11),8(9)-trien-12(8)-olide and penicillic acid from fungus Malbranchea

aurantiaca Sigler & Carmichael. These metabolites caused significant inhibition of

radicle growth of A. hypochondriacus. Similarly, two compounds, macrocidin A and B

have been isolated from the liquid culture filtrate of fungus Phoma macrostoma Mont.

They were the first representatives of a new family of cyclic tetramic acids. These

phytotoxic metabolites caused bleaching and chlorosis to several broadleaf weed species

(Graupner et al. 2006). Zonno et al. (2008) envisaged the use of Phyllostictine A

produced by a pathogen Phyllosticta cirsii Desm. as a natural herbicide against

Californian thistle [Cirsium arvense (L.) Scop.]. Phyllostictine A was proved to be a

promising natural herbicide against host and non host plant species. In another

investigation on the same fungus, four oxazatricycloalkenones, named phyllostictines A-

D, have been isolated, characterized and tested for herbicidal activity. Phyllostictine A

was proved highly phytotoxic against the weed C. arvense (Evidente et al. 2008).

Some plant pathogens have been found virulent enough to control weed species

and to compete commercially available synthetic chemical herbicides but most pathogens

are not sufficiently virulent to control weeds. However, this hindrance can be overcome.

As an example, there are certain amino acids that exhibit inhibitory effects on the growth

and development of certain plants. Pathogens or their mutants that can overproduce such

inhibitory amino acids can be selected. Such augmentation of biocontrol efficiency in

three separate pathogen-host systems, one with Pseudomonas and two with Fusarium has

already been reported (Sands and Pilgeram 2009). They outlined a stepwise approach that

can be followed to obtain enhanced weed control agents that would be capable of

producing inhibitory levels of selected amino acids in situ. Zhang et al. (2010) isolated

and identified the structure of herbicidal component, dimethyl o-phthalate from

phytopathogenic fungus Pythium aphanidermatum (Edson) Fitzp. When assayed on

weeds including hairy crabgrass [Digitaria sanguinalis (L.) Scop.] and A. retroflexus, the

ethyl acetate extract exhibited strongest herbicidal activity in terms of inhibition of seed

19

germination as well as seedling growth. Many species of Trichoderma are also known to

exhibit herbicidal activity. For example, Trichoderma harzianum Rifai, Trichoderma

pseudokoningii Rifai, Trichoderma reesei Simmons and Trichoderma viride Pers. have

been described to have herbicidal activity against P. minor and R. dentatus (Javaid and

Ali 2011). In order to harness maximum benefits of natural resources, total synthesis of a

number of phytotoxins have been accomplished (Nanda 2005; Leyva et al. 2008; Tanaka

et al. 2009; Selvam et al. 2009; Kamal et al. 2009).

1.4. Genus Drechslera

This Genus Drechslera is known mainly because it has a number of plant

pathogenic species and has done serious damages to crops in past. e.g. the Bengal

epiphytotic of 1942 was the most devastating plant disease in plant pathological history

(Padamadhan 1973). This epidemic was caused by Drechslera oryzae (Breda de Haan)

Subram. & B.L. Jain (Yun et al. 1988). Also in 1970, the corn crops of the Canada and

United States were severely destroyed by a corn-blight epidemic caused by the fungus

Drechslera maydis (Y. Nisik. & C. Miyake) Subram. & B.L. Jain. This epidemic incurred

the greatest crop loss in the shortest time span of any plant disease ever reported.

The genus Drechslera is well known for the production of secondary herbicidal

metabolites. Extensive research regarding isolation and purification of a number of novel

compounds having phytotoxic properties on host and non host species have been done in

past so far. A phytotoxic metabolite (─)-Dihydropyrenophorin was isolated from

Drechslera avenae (Eidam) Shoem. This toxin was found active against a number of

weed species including . A. fatua, Johnsongrass, [Sorghum halepense (L.) Pers] bermuda

grass (Cynodon dactylon Pers.) goosegrass (Eleucine indica Gaertn.), yellow foxtail

[Setaria glauca (L.), Beauv. ], and S. viridis (Sugawara and Strobel 1986). Similarly,

macrodiolide pyrenophorol (5,13-dihydroxy-8,16-dimethyl-1,9-dioxa-cyclohexadeca-

3,11-diene-2,10-dione), a metabolite isolated from D. avenae was found toxic to sterile

oat (Avena sterilis L.) and A. fatua when used at a concentration of 320 mM. Although

seed germination of A. sterilis was not affected but seedling cuttings which were partially

immersed in pyrenophorol solution showed leaf necrosis (Kastanias and Chrysayi-

Tokousbalides 2000). A similar herbicidal compound macrodiolide (8R,16R)-(-)-

20

pyrenophorin (8,16-dimethyl-1,9-dioxa-cyclohexadeca-3,11-diene2,5,10,13-tetraone) was

isolated by Kastanias and Chrysayi-Tokousbalides (2005) from culture of D. avenae. The

isolated compound inhibited seed germination of A. sativa, A. fatua and A. sterilis at a

concentration of 60 µM. The metabolite caused abnormal chlorophyll retention in leaf

sections of all the plant species tested. Also a phytotoxin named Tryptophol has been

identified from culture medium of Drechslera nodulosum Berk and curt. When tested,

this compound produced necrotic spots on leaves of goosegrass at a concentration of 6.2

x l0-4

M (Sugawara and Strobel 1987). Sugawara et al. (1987) isolated a series of

phytotoxic sesterterpenoids belonging to the ophiobolin family from culture filtrates of D.

maydis and Drechslera sorghicola (Lefebvre & Sherwin) M.J. Richardson & E.M.

Fraser. These ophiobolins were named as Ophiobolin I, Ophiobolin A, Ophiobolin C, 25-

Hydroxyophiobolin 1, 6-Epianhydroophiobolin A, 6-Epiophiobolin A. These ophiobolins

produced characteristic lesions on host plants. Culture filtrate and mycelia of D. maydis is

also known to produce phytotoxins named drechslerol-A [(cis) hentetracont-10-ene-12-

hydroxymethyl-4-ol], drechslerol-B [3-hydroxy-eicos-11(Z)-enyl eicos-4(Z)-enoate] and

Drechslerol-C [3-hydroxy-eicos-11(Z)-enylheptacos-11(Z)-enoate]. Drechslerol-A caused

necrotic lesion on the leaves of Wild ginger [Costus speciosus (Koenig) Smith] at 1.6 ×

10−4

M concentration. Drechslerol-C when applied with concentrations from 2.85 × 10−5

to 2.28 × 10−4

M produced characteristic necrotic and chlorotic lesions on the leaves of C.

speciosus (Shukla et al. 1987; 1989; 1990). Phytotoxic ophiobolins 6-Epiophiobolin A

and 3-anhydro-6-epiophiobolin A have also been isolated from D. maydis race T

(Canales and Gary 1988). A number of other phytotoxins from D. oryzae have also been

isolated and characterized as 6-epiophiobolin I, ophiobolin J and 8-deoxyophiobolin J

with the help of spectroscopic analyses and comparisons with already identified

ophiobolin I (Sugawara et al. 1988). Culture of Drechslera siccans (Drechsler)

Shoemaker is also reported to yield a phytotoxin named as 6,8-dihydroxy-3-(2’-

hydroxypropyl) isocoumarin (de-o-methyldiaporthin). Phyto-toxicity of this compound

has been estimated in terms of necrotic spot area when tested on A. sativa, Smooth

crabgrass [Digitaria ischaemum (Schreb.) Schreb. ex Muhl.], E. crus-galli, spiny

amaranth (Amaranthus spinosus L.), maize and soybean (Hallock et al. 1988). Triticone

A is reported to be synthesized by several fungi including Drechslera tritici- repentis

http://en.wikipedia.org/wiki/Johann_Christian_Daniel_von_Schreber

http://en.wikipedia.org/wiki/Gotthilf_Heinrich_Ernst_Muhlenberg

21

(Died.) Shoemaker. By undergoing racemization it forms triticone B and when tested, the

enantiomeric mixture evoke chlorosis and necrosis on a variety of plants including wild

oat (Kenfield et al. 1988). Same Drechslera species also synthesizes Triticones B, C, D,

E, and F along with Triticone A Amongst these Triticones, mixture of Triticone A and

Triticone B was found the most phytotoxic of the Triticones. Its biological activity was

assessed by observing yellowish-brown lesions by the leaf puncture method on weeds

including C. album and A. retroflexus (Hallock et al. 1993). Phytotoxic compounds

curvulin and O-methylcurvulinic acid were isolated from Drechslera indica (J.N. Rai,

Wadhwani & J.P. Tewari) Mouch. These toxins caused necroses on purslane (Portulaca

oleracea L.) and spiny amaranth (Kenfield et al. 1989). Bunkers and Strobel (1991)

proposed the mode of action of numerous phytotoxins belonging to the eremophilane

family from D. gigantea. They concluded that green island formation by these

eremophilanes proceeds through the inhibition of protein synthesis in detached oat leaves.

Although metabolites of a number of Drechslera species have been tested against

some problematic weed species, however, studies regarding the herbicidal activity of

Drechslera spp. from Pakistan are scarce. The present study was therefore, carried out to

investigate the herbicidal potential of culture filtrates of four Drechslera species from

Pakistan, namely, D. hawaiiensis, D. holmii, D. biseptata and D. australiensis against

some problematic weeds of wheat.

22

Objectives

The present research work was undertaken to seek nature friendly alternatives to

synthetic herbicides from culture filtrates of Drechslera spp. for the management of some

noxious weeds of wheat. To achieve this goal the present study was aimed:

To evaluate the in vitro and in vivo herbicidal activity of culture filtrates of four

Drechslera spp. against four noxious weeds of wheat and different wheat

varieties.

To investigate the herbicidal activity of culture filtrates Drechslera spp. against

the most susceptible weed species under field conditions.

To isolate the herbicidal compounds from the most active Drechslera species

through bioactivity guided bioassays using various chromatographic techniques.

To elucidate the structures of active herbicidal compounds through various

spectroscopic techniques.

23

Chapter 2

Materials and Methods

2.1. Selection of Test Fungal Isolates

Four species of Drechslera viz. D. australiensis, D. biseptata, D. hawaiiensis and

D. holmii, were procured from Fungal Culture Bank of Pakistan, Institute of Agricultural

Sciences, University of the Punjab, Lahore, Pakistan.

2.2. Single Spore Isolation

For the purpose of single spore isolation of the test fungi, 1 mL of autoclaved

water was poured into a vial containing the fungal culture. The vial containing fungal

culture was shaken vigorously for 1 minute and water containing the spores was poured

into 9 mL of autoclaved water to give 10-1

dilution. This procedure was repeated twice

get a 10-3

dilution. One milliliter suspension was taken from this dilution and poured on

autoclaved malt extract agar medium in 9-cm diameter Petri dish. There were three

replicates of this treatment. Sterilized glass spreader was used to spread the spores evenly

on the surface of medium in Petri plate. These plates were incubated for three days at

28±2 °C in an incubator to allow the spores to germinate until the colonies were visible.

Each colony free from contamination was removed with the help of sterilized fine needle

and transferred to another Petri plate containing malt extract agar medium. These plates

incubated at 28±2 °C for 15 days in an incubator for an appreciable conidial and mycelial

formation. After this, pure fungal cultures were confirmed and stored at 4 C.

2.3. Selection of Weeds of Wheat

Four frequently occurring and problematic weeds of wheat viz. Avena fatua L.,

Phalaris minor Retz. (Monocot.), Chenopodium album L., Rumex dentatus L. (Dicot.)

were chosen for this study. Seeds of the test weed species were collected from wheat

24

fields of University of the Punjab Lahore, Pakistan, at the end of growing season of

wheat in May 2009. These seeds were sun dried, cleaned and stored at room temperature.

2.4. Selection of Test Wheat Varieties

Three commonly cultivated wheat varieties in Punjab, Pakistan namely Inqlab 91,

Sehar 2006 and Uqab 2000 were selected to evaluate their germination and growth

response to culture filtrates of the test Drechslera species. Certified seeds of these

varieties were procured from Federal Seed Certification Department, Lahore, Pakistan.

2.5. Preparation of Culture Filtrates of the Test Fungi

Minimal medium (M-1-D) was prepared in distilled water as described by

Evidente et al. (2006b). This medium consisted of 1.2 mM Ca(NO3)2, 0.79 mM KNO3,

0.87 mM KCI, 3.0 mM MgSO4, 0.14 mM NaH2PO4, 87.6 mM sucrose, 27.1 mM

ammonium tartrate, 7.4 µM FeC13, 30 µM MnSO4, 8.7 µM ZnSO4, 22 µM H3BO3 and

4.5 µM KI. The pH was adjusted to 5.5 with 0.1 M HCl. Medium was poured into 500

mL conical flasks at 200 mL medium in each flask. Flasks were autoclaved at 121°C and

103425 P cm-2

pressure for 20 minutes and cooled to room temperature. Flasks were

individually inoculated with 5 mm agar discs of each of the four test fungal species from

the margins of actively growing fungal colonies. Inoculated flasks were incubated at

252 ºC in an incubator for 28 days. Cultures were filtered through four layers of muslin

cloth, centrifuged at 4000 rpm for ten minutes followed by filtration through sterilized

Whatman filter paper No. 1. These filtrates were stored at 4 ºC in a refrigerator. Sterilized

distilled water was added to the original filtrates (100%) to prepare dilution of 50%

(Javaid and Adrees 2009). Filtrates were generally used within a week to avoid any

contamination or chemical alteration.

2.6. Laboratory Screening Bioassays

The effect of original and diluted culture filtrates of the four selected fungal

species was evaluated on germination and early seedling growth of the test weed species

as well as against three selected wheat varieties. Seeds of weeds and wheat varieties were

25

surface sterilized with 1% sodium hypochlorite for 10 minutes. Twenty seeds of each of

the test weed species and three wheat variety were placed at equal distance in sterilized 9

cm diameter Petri dishes lined with sterilized filter papers, moistened with 3 mL of the

two concentrations of fungal culture filtrates. Treatments in a similar manner with M-1-D

medium (Original and 50% diluted) served as positive control, whereas treatment with

distilled water served as negative control. All tests were performed in quadruplicate. Petri

dishes were arranged in a completely randomized design (CRD) in a growth room

maintained at 16 C with 10 h light period daily. Data regarding germination of seeds

were recorded after 15 days. Plants were thinned and 10 uniform seedlings were selected

for measurement of different root and shoot growth parameters (Fig. 1). Materials were

dried at 60 °C in an electrical oven till constant weight (Javaid and Ali, 2011).

2.7. Foliar Spray Bioassays

Pot experiments were conducted during November-December 2009 in University

of the Punjab, Lahore, Pakistan, located on latitude 31.57 N and longitude 74.31 E.

Plastic pots of 8-cm diameter and 12-cm deep were filled with 450 g sandy loam soil

having organic matter 0.69%, pH 7.8, available phosphorus and potassium 6.3 mg kg-1

and 100 mg kg-1

respectively, with nitrogen content 350 mg kg-1

. The micronutrient Zn,

Mn, Cu, B and Fe were 1.3, 22.8, 1.9, 1.06 and 10.8 mg kg-1

respectively. NPK fertilizers

were used in each pot. Ten seeds of each weed species as well as each wheat variety were

sown in each pot. After germination, pots were arranged in two sets to perform the foliar

spray on 1-week and 2-week old seedlings. Each treatment was replicated four times. All

the pots were arranged in a completely randomized design in open under natural

environmental conditions.

Original culture filtrates of the four selected Drechslera species were sprayed on

1-week and 2-week old test weeds and wheat seedlings. Both of the sets were sprayed 4

times with an interval of four days. Treatment in a similar manner with distilled water

spray served as negative control whereas M-1-D medium without fungal inoculation was

used as positive control. All the sprays were carried out during evening hours. Plants

were harvested after 50-days growth. Plants were carefully uprooted and washed

thoroughly under tap water to remove soil. Moisture from plant surface was evaporated

26

under fan at room temperature. Parameters regarding length and fresh and dry biomass of

root and shoot were recorded (Javaid et al. 2011b).

2.8. Field Trials

2.8.1. Field Preparation

In laboratory bioassays and pot trials, R. dentatus was found the most susceptible

to various fungal culture filtrates. To evaluate the effect of culture filtrates of the four

Drechslera spp. against R. dentatus under field conditions, field trial was carried out

during the wheat growing season of 2009–2010 at Dhing Shah District Qasur, (30° 56' N

and 74° 13' E), 80 kilometers from Lahore, Pakistan.

All the recommended agronomic practices right from preparation of field till

harvesting were employed. A composite soil sample of the experimental field was taken

before launching of the experiment. Soil was got analyzed from soil and water testing

laboratory for research, Lahore, Pakistan. Soil was sandy loam in texture having available

potassium (43 mg kg-1

), ECmScm-1

(2.4), pH (7.7), organic matter (0.84%) and available

phosphorous (4.5 mg kg-1

). Experiment was laid out in randomized complete block

design (RCBD) with three replications. Each plot measured 1.54×1.54 m2. Fertilizers

were applied as recommended by the Punjab Agriculture Department, Pakistan, for

wheat. Nitrogen (N) was applied at 160 kg ha-1

as urea, P2O5 at 110 kg ha-1

as single

super phosphate and K2O at 60 kg ha-1

as sulphate of potash. Full doses of P2O5 and K2O,

and a half-dose of N were applied as basal, while half the N was top-dressed at flowering

stage.

2.8.2. Sowing of Seeds

Since all the three wheat varieties showed resistance to various fungal culture

filtrates, therefore, only one wheat variety Sehar 2006 was selected for field trials. Three

wheat seeds per hill were sown at 22 cm inter and intra row spacing accommodating

seven rows in each plot with 7 plants per row. After germination, thinning was carried

out at the stage of full emergence of first leaf to maintain only one wheat seedling at one

place. First irrigation was carried out twenty days after sowing and subsequent irrigations

27

were carried out according to the requirement of the crop. Over all six irrigations with

tube well water were carried out as dry spell was observed through out the course of

present study. Seeds of R. dentatus were sown in the field at the time first water. Weed

species belonging to different plant genera along with R. dentatus emerged in the field

after first irrigation. All weed species except R. dentatus were removed manually and 1:1

ratio of R. dentatus and wheat plants was maintained.

2.8.3. Treatments and Experimental Layout

The following twelve treatments were tried in the field trial:

T1 Weed free control.

T2 Weedy check.

T3 Application of culture filtrate of D. hawaiiensis.

T4 Application of culture filtrate of D. holmii.

T5 Application of culture filtrate of D. biseptata.

T6 Application of culture filtrate of D. australiensis.

T7 Bromoxynil + MCPA (recommended dose).

T8 Bromoxynil + MCPA (half dose).

T9 Culture filtrate of D. hawaiiensis + half dose of Bromoxynil + MCPA.

T10 Culture filtrate of D. holmii + half dose of Bromoxynil + MCPA.

T11 Culture filtrate of D. biseptata + half dose of Bromoxynil + MCPA.

T12 Culture filtrate of D. australiensis + half dose of Bromoxynil + MCPA.

2.8.4. Schedule of Foliar Sprays

A total of three sprays were carried out with fungal culture filtrates. First spray was

carried out when R. dentatus was at three to four leaves stage. Two successive sprays

with fungal culture filtrates were carried out with intervals of 7 days. Culture filtrates

were sprayed at 100 L ha-1

. Only one spray of synthetic herbicide Bromoxynil + MCPA

200/200EC, either alone or mixed with fungal culture filtrates was carried out with

Knapsack hand sprayer with 4T-jet nozzle. Both the recommended and half dose of the

28

herbicide was used where it was used alone. However, in combination with fungal culture

filtrates, only half dose of the herbicide was used.

2.8.5. Harvesting and Data Collection

The crop was harvested at maturity i.e. 150 days after sowing. Data for plant

height, weight, number of fertile tillers per plant, total seed weight, hundred seeds weight,

and biomass of weed plants were recorded. Wheat and weed biomass were measured

after placing plant materials in an electric oven at 60 °C to constant weight.

2.9. Statistical Analysis

All the data from laboratory screening and foliar spray bioassays as well as field

trials were subjected to analysis of variance (ANOVA) followed by Duncan’s Multiple

Range Test to delineate the treatment means using computer software COSTAT.

2.10. Organic Solvent Extraction

In the previous laboratory, pot and field trials, culture filtrate of D. australiensis

were found to be the most efficient in controlling growth of R. dentatus. This species was

thus selected for isolation and identification of active herbicidal constituents from its

culture filtrate. This fungus was grown in M-1-D medium in 500 mL conical flasks as

described in section 2.5. A total of 4 L of crude fungal culture filtrate of this were

collected and evaporated to yield 1.5 L concentrated filtrate. Four organic solvents viz. n-

hexane (C6H14), chloroform (CHCl3), ethyl acetate (C4H8O2) and n-butanol (C4H10O)

were successively used for extraction. These organic solvents were used in order of their

increasing polarity. First a volume of 300 mL of n-hexane was added to 300 mL crude

concentrated fungal culture filtrate in a separating funnel, shaken well and kept stationery

until the two phases got separated. The upper n-hexane layer was separated and the

process was repeated until all n-hexane compounds were separated from the aqueous

filtrates. Similarly, the rest of 1200 mL culture filtrates were treated with n-hexane. This

n-hexane phase was concentrated under vacuum in a rotary evaporator to yield crude n-

29

hexane fraction. The aqueous phase was then extracted similarly with chloroform, ethyl

acetate and n-butanol, yielding crude fractions.

2.11. Leaf Discs Bioassays with Crude Organic Fractions

Bioassays with crude organic fractions were carried out following procedure

described by Mahoney et al. (2003), with some modifications. R. dentatus seeds were

grown in plastic pots under natural environmental conditions. Young leaves from 30 days

old plants were detached and discs of 1-cm diameter were cut with the help of a cork

borer. The leaf discs were placed on glass slide and punctured with the help of a fine

needle. The glass slides were placed on a filter paper wetted with 2 mL of sterilized

distilled water in a Petri plate. Four milligrams of each crude fraction viz. n-hexane,

chloroform, ethyl acetate and n-butanol were dissolved in 100 µL of dimethylsulfoxide

(DMSO). Final volume of each fraction was raised to 1.0 mL with distilled water to

prepare a stock solution of 4 mg mL-1

concentration. The stock solution was serially

double diluted by adding distilled water to prepare lower concentrations of 2, 1, …,

0.0625 mg mL-1

. Droplets of 15 µL of each of the seven concentrations were applied on

the punctured leaf surface. Ten leaf discs of the test weed species were used for each

concentration. Positive control received DMSO at 100 µL mL-1

of distilled water at

highest concentration and subsequent lower concentrations were made by double diluting

it with distilled water. Treatment with distilled water alone served as negative control.

Treatments in a similar manner but with un-punctured leaf surface were also made. These

Petri plates were incubated at 25 °C under continuous fluorescent light in growth room.

Symptoms regarding appearance of necrotic spot and discolouration of leaf discs were

observed after 72 hours. Colour scale 0-3 and necrotic spot scale 4-10 was used for

comparisons (Plate 1).

30

Plate 1: Scheme for leaf discs bioassays using crude organic fractions.

water

Concentration of

Crude fraction

(mg mL-1

) Concentration of

DMSO (µl mL-1

)

0.0625

0.1250

0.2500

0.5000

4.0000

2.0000

1.0000

3.125

6.25

12

25

50

100

1.562

0.781

31

2.12. Isolation of Compounds through Chromatographic Techniques

Crude chloroform and ethyl acetate fractions of culture filtrate of D. australiensis

showing pronounced herbicidal activity were selected for Thin Layer chromatography

(TLC) analysis followed by separation through Preparative Thin Layer Chromatography

(PTLC) and Reversed Phase High Performance Liquid Chromatography (RPHPLC).

2.12.1. Thin Layer Chromatography

A thin strip of aluminum foil backed TLC (6.5 cm long and 1.5 cm wide) was cut

with a scissor. A base line was drawn near the bottom of the plate with lead pencil to

show the original position of the compound. Five milligrams of crude chloroform fraction

was taken in an eppendorf tube and was dissolved in 1 mL of methanol. A small drop of

solution was placed on the center of baseline with the help of capillary jet and allowed to

dry for few minutes. Solvent system or mobile phase was prepared in a glass jar. Ten

milliliter of solvent (chloroform, ethyl acetate and n-hexane) in 10:6:84 ratio was poured

into a glass jar to a depth of 0.9 cm. TLC strip was placed in the solvent so that its bottom

touched the solvent and solvent level remained below the baseline with the spot on it. The

container was closed with a lid and was left for a few minutes to let the solvent elute the

mixture of compounds spotted on chromatogram strip. When the solvent front moved to

about 1 cm below the upper end of the strip, the plate was removed and dried. Spot was

located under UV transilluminator, both at short and long wavelength as well as

visualized by spraying ceric sulphate solution accompanied by heating with heat gun. The

Retention factor (Rf) value for each spot was calculated using the formula:

Distance traveled by component

Rf =

Distance traveled by the solvent

Six fractions namely A (Rf 0.096), B (Rf 0.130), C (Rf 0.170), D (Rf 0.269), E (Rf

0.480) and F (Rf 0.576) were isolated from chloroform fraction of culture filtrate of D.

australiensis. Similarly, three fractions namely G (Rf 0.054), H (Rf 0.345) and I (Rf

0.618) were separated from ethyl acetate fraction of culture filtrate of the same fungal

32

species using solvent system n-hexane and ethyl acetate in a ratio of 3:7. The isolated

fractions were further purified by Preparative Thin Layer Chromatography (PTLC).

2.12.2. Preparative Thin Layer Chromatography

For preparative thick layer chromatography pre-coated silica gel GF-254

preparative plates (20 × 20 cm, 0.5 mm thick, E-Merck) were used. Solvent system was

same as in TLC. When developed with the solvent, the compounds separated in

horizontal bands. These bands were scraped from the plates and eluted with methanol.

Soluble compounds were carefully collected separately in another vial through filtration,

and were evaporated at 40 C to dryness and weighed.

2.12.3. Reversed Phase High Performance Liquid Chromatography

Compounds separated through Preparative Thin Layer Chromatography were

further subjected to Reversed Phase High Performance Liquid Chromatography. For

elution Acetonitrile (HPLC Grade) with 0.1% Formic acid added and Deionized Water

(DI water) with 0.1% Formic acid was used as solvent system. Gradient elution was

employed with initial ratio of Acetonitrile and Deionized (DI) water as 10:90 with an

increasing ratio of Acetonitrile to water as 100:0. Fractions containing purified

compounds were collected in glass vials and solvent was evaporated under continuous

currents of clean air at room temperature. The work was done at Department of

Chemistry & Biochemistry, University of California, Santa Cruz, USA.

2.13. Spectroscopic Analyses

Structural elucidation by 1D & 2D NMR techniques of only the most active

compounds was carried out. Less bioactive compounds were not subjected to mass

spectroscopy because their bioactivity was far low as compared to synthetic herbicide in

use.

Proton nuclear magnetic resonance (1H-NMR) spectra were recorded in CD3OD

using TMS as internal standard at 600 MHz and 500 MHz on Bruker AM-300, AM-600

33

and AM-500 nuclear magnetic resonance spectrometers. The 13

C-NMR spectra were

recorded in CD3OD at or 125 MHz on the same instruments.

EIMS spectra were recorded on a Finnigan MAT 311 with MASSPEC data

system.

2.14. Leaf Discs Bioassays with Purified Chromatographic Fractions

Bioassays with purified chromatographic fractions were generally carried out

using the same method as was adopted in case of crude organic fractions section 2.11,

except that these bioassays were only performed with punctured leaf disks. Stock

solutions of 2 mg mL-1

of various purified organic constituents were prepared by

dissolving 2 mg of the compound in 50 µL of DMSO and raised the volume to 1.0 mL by

adding distilled water. Lower concentrations of 1, 0.5, …, 0.03125 mg mL-1

were

prepared by serially double diluting the stock solutions. In total seven concentrations

were made with distilled water viz. 2, 1, 0.5, 0.25, 0.125, 0.0625 and 0.03125 mg mL-1

.

For positive control 50 µL DMSO was dissolved in distilled water to make the final

volume 1.0 mL. Lower concentrations were made by double diluting this mixture with

distilled water to prepare corresponding positive control treatments of DMSO for various

concentrations of the purified compounds. A positive control using 2,4-D (2, 4-

dichlorophenoxyacetic acid) was also included in these bioassays to compare the efficacy

of isolated compounds. Treatment with distilled water alone served as negative control.

Symptoms regarding appearance of necrotic spot and discoloration of leaf discs were

observed after 72 hours.

34

Chapter 3

Results

3.1. Laboratory Bioassays

3.1.1. Effect of Fungal Culture Filtrates on Germination and Growth of C. album

3.1.1.1. Effect on Germination

Original (100%) growth medium significantly reduced germination by 12% over

control. However, the effect of diluted (50%) growth medium was nonsignificant. The

effect of various fungal culture filtrates on germination was variable. In general, all the

culture filtrates significantly suppressed the germination by 28–50% over control. The

effect of filtrates of D. australiensis and D. hawaiiensis was more pronounced as

compared to filtrates of other two fungal species (Table 1, Plate 2).

3.1.1.2. Effect on Shoot Growth

Highest length, fresh weight and dry weight of shoot was recorded in control.

Original and diluted growth medium significantly reduced shoot length by 17% and 13%,

shoot fresh weight by 31% and 25%, and shoot dry weight by 21% and 16%, over

control, respectively. In general, culture filtrates of all the four Drechslera spp.

significantly suppressed plant growth. However, a marked variation in herbicidal activity

of culture filtrates of different fungal species was evident. Culture filtrates of D.

hawaiiensis were found the most effective followed by those of D. australiensis in

reducing shoot length and biomass of C. album. There was 91% and 84% reduction in

shoot length, and 84% and 68% reduction in shoot dry weight due to original culture

filtrates of D. hawaiiensis and D. australiensis, respectively, as compared to control.

Culture filtrates of the other two fungal species exhibited comparatively less herbicidal

activity against shoot growth of the test weed species. There was 55% and 54%

reduction in shoot length and 58% decline in shoot dry weight due to original culture

filtrates of D. biseptata and D. holmii as compared to control (Table 1, Plate 2).

35

3.1.1.3. Effect on Root Growth

Growth medium exhibited variable effect on length and weight of root. Root

length was significantly reduced by 16% and 12% due to original and diluted culture

medium. Similarly, the adverse effect of original filtrates was significant on root fresh

weight. By contrast, none of the two concentrations of growth medium had significant

effect on root dry weight. Root growth exhibited high susceptibility to the application of

culture filtrates of the four Drechslera species. Root length and dry biomass were

significantly reduced by 66–88% and 56–65% due to original culture filtrates of different

fungal species as compared to control. Original filtrates of D. hawaiiensis and D.

australiensis were found the most effective in reducing length and biomass of C. album

roots (Table 1, Plate 2).

3.1.2. Effect of Fungal Culture Filtrates on Germination and Growth of R. dentatus


The effect of M-1-D broth was nonsignificant on germination of test weed

species. Culture filtrates of all the four significantly reduced germination to variable

extents. The highest herbicidal activity was shown by filtrate of D. biseptata (up to 56%

reduction) followed by those of D. australiensis, D. hawaiiensis and D. holmii,

respectively. The original culture filtrates of other Drechslera spp. significantly reduced

germination by 12–49% (Table 2, Plate 3).


The effect of original growth medium was found significant on shoot growth

resulting in 15% in each of shoot length and shoot dry weight, respectively. The effect of

all the culture filtrate treatments except 50% D. holmii was significant as compared to

control. Original filtrates of D. australiensis were found to be the most effective in

suppressing shoot length and shoot dry biomass of R. dentatus by 85% and 88%,

respectively. Similarly, D. biseptata resulted in 81% and 83% decline in shoot length

and dry biomass of R. dentatus, Generally, culture filtrates of D. hawaiiensis and D.

36

holmii proved less toxic to R. dentatus growth as these two species caused 67% and 68%,

and 73% and 72% reduction in shoot length and shoot dry weight over control,

respectively (Table 2, Plate 3).


In general, root growth exhibited slightly more susceptibility to the application of

culture filtrates of various Drechslera species as root length and biomass were

significantly reduced by 69–94% and 63–88%, respectively. Filtrates of D. australiensis

were found the most effective in inhibiting various root growth parameters of R. dentatus.

This species incurred 94% and 88% reduction in root length and root dry weight


3.1.3. Effect of Fungal Culture Filtrates on Germination and Growth of P. minor


The effect of both original as well as diluted growth medium was nonsignificant

on seed germination. Both original and diluted culture filtrates of of all the four test

fungal species significantly reduced germination by 35–93%. The adverse effect of

original culture filtrates on germination was more pronounced as compared to diluted

ones. Original culture filtrates of D. australiensis incurred drastic effect inhibiting

germination of P. minor seeds by 93%. Culture filtrates of other three fungal species

proved less toxic to seed germination as compared to filtrates of D. australiensis (Table

3, Plate 4).


The effect of the original growth medium was significant on length as well as

biomass of shoot of P. minor seedlings as there was 19% and 23% decline in shoot length

and shoot dry weight, respectively. However, the adverse effect of the fungal culture

filtrates was much higher than the effect of growth medium. All the culture filtrates

either used in original or diluted form significantly reduced various shoot growth

37

parameters as compared to control as well as growth medium treatments. Filtrates of D.

hawaiiensis and D. australiensis incurred 65% and 60% decline in shoot length, and 64%

and 45% reduction in shoot dry weight of P. minor seedlings over control, respectively

(Table 3, Plate 4).


The effect of the original growth medium was significant on length as well as

biomass of root. There was 36% and 32% reduction in root length and root dry weight

due to original M-1-D broth. Root length as well as root weight were significantly

suppressed by culture filtrates of all the four Drechslera species. There was 81–90%

reduction in root length and 59–81% reduction in root dry weight of P. minor due to

different concentrations of the various culture filtrates as compared to control. Culture

filtrate of D. australiensis exhibited the highest phytotoxic activity inhibiting root length

and root dry weight of P. minor up to 90% and 81% respectively. The difference in root

fresh and dry weight was nonsignificant among the original concentrations of various

fungal culture filtrate treatments (Table 3, Plate 4).

3.1.4. Effect of Fungal Culture Filtrates on Germination and Growth of A. fatua


The effect of growth medium on germination was significant. There was 14% and

12% reduction in germination due to 100% and 50% M-1-D broth. Different culture

filtrate treatments significantly reduced the germination of A. fatua seeds by 28–54%.

Inhibitory effect of original culture filtrates of D. hawaiiensis and D. australiensis was

highest and comparable to each other (Table 4, Plate 5).


The effect of growth medium on shoot length of A. fatua was found significant as

reduction of 16% was observed in shoot length. However the effect of growth medium

was nonsignificant on shoot dry weight of A. fatua. Although all the culture filtrate

38

treatments except 50% D. biseptata significantly reduced shoot growth of A. fatua in

terms of length and biomass but adverse effects were more pronounced in case of D.

holmii where culture filtrate of this fungus consistently reduced shoot length as well as

shoot fresh and shoot dry weight by 67%, 59% and 57%, respectively. There was 27–

67% and 27–57% reduction in shoot length and shoot dry biomass due to various culture

filtrate treatments (Table 4, Plate 5).


The effect of growth medium on root length and biomass of A. fatua was found

significant. There was 18% reduction in length and dry biomass each due to original

growth medium. Culture filtrates of all the test fungi were found effective in arresting

various root growth parameters at both 100% as well as 50% concentration. There was

55–86% and 47–77% reduction in root length and root biomass due to various culture

filtrate treatments, respectively. The most noticeable effect was due to filtrate of D.

hawaiiensis causing 86%, 82% and 77% inhibition in length, fresh and root dry weight of

root, respectively. Second most important Drechslera species was D. holmii causing

81%, 82% and 74% inhibition in root length, root fresh weight and root dry weight,


3.1.5. Effect of Fungal Culture Filtrates on Germination and Growth of Wheat

3.1.5.1. Wheat var. Inqlab 91

3.1.5.1.1. Effect on Germination

The effect of original as well as diluted growth medium was nonsignificant on

germination of wheat var. Inqlab 91. Original culture filtrates of various Drechslera spp.

significantly reduced germination by 7-12%, whereas the effect of 50% concentration

was nonsignificant (Table 5, Plate 6).

39

3.1.5.1.2. Effect on Shoot Growth

The original growth medium significantly reduced shoot length by 17% whereas

its effect on shoot dry weight was nonsignificant. All the culture filtrates significantly

reduced various shoot growth parameters. There was 26–38%, 14–39% and 28–46%

reduction in shoot length, and fresh and dry weight over control, respectively. The

adverse effect of D. holmii was more pronounced than the effect of rest of the fungal

species (Table 5, Plate 6).

3.1.5.1.3. Effect on Root Growth

The effect of original growth medium was significant on length and biomass of

root. There was 14% and 17% reduction in length and dry biomass of root over control,

respectively. Root length and dry biomass were significantly reduced by 26–70% and 34–

56%, respectively, due to different fungal culture filtrate treatments. The highest adverse

effect on various root growth parameters was recorded due to culture filtrate of D.

hawaiiensis while filtrate of D. biseptata was found least toxic (Table 5, Plate 6).

3.1.5.2. Wheat var. Sehar 2006


The effect of original as well as diluted growth medium on germination was

nonsignificant as compared to negative control. Original culture filtrates of all the four

fungal species significantly reduced germination by 11–18%. The effect of culture

filtrates of D. hawaiiensis and D. holmii was more pronounced than the culture filtrates of

D. biseptata and D. australiensis (Table 6, Plate 7).


Growth medium exhibited variable effects on length and biomass of shoot. Shoot

length was significantly reduced by 14% and 10% due to original and diluted growth

medium, respectively, while there was a nonsignificant effect of growth medium on shoot

dry weight. D. hawaiiensis and D. holmii exhibited greatest reduction in shoot dry

40

biomass resulting in up to 24% and 22% reduction, respectively. Diluted culture filtrates

of D. australiensis, D. biseptata and D. hawaiiensis failed to induce any noteworthy

effect on shoot length, shoot fresh weight as well as shoot dry weight (Table 6, Plate 7).


The effect of original growth medium on root length as well as root dry biomass

was significant that resulted in 18% and 11% decline, respectively. Root growth was

found to be more susceptible to treatments of growth medium as well as culture filtrates

than shoot growth. The effect of all the culture filtrate treatments on root dry weight was

significant. D. holmii, D. biseptata and D. australiensis caused greatest suppression in

root dry biomass resulting in 33% reduction each, while D. hawaiensis was proved to be

the least effective one causing 30% reduction in root dry biomass (Table 6, Plate 7).

3.1.5.3. Wheat var. Uqab 2000


The effect of growth medium on the germination of test wheat variety was

nonsignificant when compared with control. The effect of 100% culture filtrates of D.

hawaiiensis and D. holmii was much pronounced on germination resulting in 17% and

15% reduction in germination, respectively. The effect of 50% culture filtrates of all the

test fungal species was comparable to that of original growth medium (Table 7, Plate 8).


Growth medium showed significant effect on shoot length at both original and

diluted concentrations causing 18% and 14% reduction respectively. All the fungal

culture treatments affected shoot length at both the concentration. The most effective

Drechslera species in this regard was D. holmii that caused 43% reduction in shoot

length when 100% culture filtrate was used. The least effective Drechslera spp. remained

D. biseptata that caused 31% decline in shoot length. In case of shoot biomass, all the

fungal culture filtrates caused deleterious effects both at 100 as well as 50%

41

concentration except 50% concentration of D. australiensis where effect on root dry

weight was similar to original growth medium (Table 7, Plate 8).


Growth medium showed significant effect on various root growth parameters at

original concentration causing 17% and 16% reduction in root length and root dry weight,

respectively. Root growth exhibited susceptibility to all the treatments of fungal culture

filtrates, D. hawaiiensis being prominent resulting in 78%, 61% and 58% reduction

followed by D. australiensis resulting in 67%, 47% and 37% reduction in root length,

root fresh weight and root dry biomass, respectively. The least effective fungal species

was D. biseptata causing 38% inhibition in root length and 33% reduction in root dry

weight (Table 7, Plate 8).

42

Table 1: Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera

species on germination and growth of Chenopodium album in laboratory

bioassays.

Fungal species Conc.

(%)

Germi-

nation

(%)

Shoot

length

(mm)

Shoot

fresh wt.

(mg)

Shoot

dry wt.

(mg)

Root

length

(mm)

Root

fresh wt.

(mg)

Root dry

wt. (mg)

Control 0 100 a 16.5 a 1.40 a 0.19 a 10.40 a 0.19 a 0.087 a

Growth medium 50 93 ab 14.4 b 1.05 b 0.16 b 9.15 b 0.18 a 0.090 a

100 88 bc 13.7 c 0.97 bc 0.15 b 8.75 b 0.15 b 0.080 a

D. hawaiiensis 50 66 e 1.9 h 0.37ef 0.04 gh 1.40 fg 0.09 de 0.040 c

100 50 f 1.5 h 0.29 f 0.03 h 1.23 g 0.08 e 0.030 c

D. holmii 50 81 cd 12.4 d 0.83 cd 0.10 cd 7.90 c 0.12 c 0.054 b

100 72 de 7.6 f 0.72 d 0.08 de 3.57 d 0.10 d 0.038 c

D. biseptata 50 82 bc 10.3 e 0.77 d 0.10 c 7.70 c 0.12 c 0.063 b

100 72 de 7.5 f 0.71 d 0.08 c-e 2.35 e 0.10 d 0.037 c

D. australiensis 50 56 f 3.3 g 0.54 e 0.07 ef 2.42 e 0.10 d 0.036 c

100 50 f 2.7 g 0.44 ef 0.06 fg 1.71 f 0.09 de 0.033 c


species on germination and growth of Rumex dentatus in laboratory bioassays.


(%)

Germi-

nation

(%)

Shoot

length

(mm)

Shoot

fresh wt.

(mg)

Shoot

dry wt.

(mg)

Root

length

(mm)

Root

fresh wt.

(mg)

Root dry

wt. (mg)

Control 0 100 a 19.0 a 5.4 a 1.20 a 18.8 a 0.87 a 0.155 a

Growth medium 50 97 a 17.7 a 4.9 ab 1.15 ab 18.3 a 0.78 a 0.140 b

100 95 a 16.2 b 4.5 b 1.02 b 16.1 b 0.77 a 0.145 ab

D. hawaiiensis 50 59 c 8.4 c 2.1 c 0.60 c 8.5 c 0.38 b 0.081 c

100 51 d 6.2 d-f 1.5 cd 0.38 de 5.9 d 0.29 bc 0.057 d

D. holmii 50 95 a 7.1 cd 1.7 cd 0.47 d 5.6 d 0.25 b-d 0.050 de

100 88 b 5.2 ef 1.2 de 0.34 d-f 3.4 e 0.36 b 0.035 ef

D. biseptata 50 48 de 4.9 f 1.1 de 0.27 e-g 2.7 ef 0.13 cd 0.034 ef

100 44 e 3.6 g 0.7 e 0.21 fg 1.8 fg 0.08 d 0.019 g

D. australiensis 50 59 c 6.5 de 1.1 de 0.28 e-g 3.1 e 0.15 cd 0.032 fg

100 60 c 2.9 g 0.7 e 0.14 g 1.2 g 0.08 d 0.019 g

In a column, values with different letters show significant difference (P≤0.05) as

determined by Duncan’s Multiple Range Test.

43

Plate 2: Effect of culture filtrates of four Drechslera species on germination and

growth of C. album in laboratory bioassays.

1: Control (water) 2: 50% Medium 3: 100% Medium 4: 50% Fungal

Culture Filtrates (FCF) 5: 100% FCF

D. hawaiiensis

1

2 3

4 5

1

2 3

4 5

D. holmii

D. biseptata

1

5

3

1

5

2 3

4 5

D. australiensis

A

C D

B

2 3

4

44


growth of R. dentatus in laboratory bioassays.



D. hawaiiensis

1

2 3

4 5

D. holmii

1

2 3

4 5

D. biseptata

1

2 3

4 5

D. australiensis

1

2 3

4 5

A B

C D

45


species on germination and growth of Phalaris minor in laboratory bioassays.


(%)

Germi-

nation

(%)

Shoot

length

(mm)

Shoot

fresh wt.

(mg)

Shoot

dry wt.

(mg)

Root

length

(mm)

Root

fresh wt.

(mg)

Root dry

wt. (mg)

Control 0 100 a 52 a 5.17 a 0.53 a 58 a 5.31 a 0.78 a

Growth medium 50 96 a 44 b 4.70 ab 0.45 b 44 b 4.31 b 0.63 b

100 96 a 42 b 4.35 b 0.41 bc 37 c 3.72 c 0.53 c

D. hawaiiensis 50 81 c 26 e 2.65 d 0.27 d 25 d 2.27 d 0.35 d

100 56 e 18 h 1.90 e 0.19 e 10 f 0.88 g 0.18 f

D. holmii 50 88 b 29 d 3.45 c 0.39 c 19 e 1.90 de 0.35 d

100 56 e 25 ef 2.62 d 0.29 d 11 f 0.96 g 0.19 f

D. biseptata 50 77 c 35 c 3.05 cd 0.38 c 12 f 1.40 f 0.28 e

100 65 d 23 fg 2.62 d 0.29 d 11 f 0.86 g 0.17 f

D. australiensis 50 13 f 21 g 3.62 c 0.37 c 16 e 1.50 ef 0.34 d

100 6 g 21 g 2.75 d 0.29 d 6 g 0.65 g 0.15 f


species on germination and growth of Avena fatua in laboratory bioassays.


(%)

Germi-

nation

(%)

Shoot

length

(mm)

Shoot

fresh wt.

(mg)

Shoot

dry wt.

(mg)

Root

length

(mm)

Root

fresh wt.

(mg)

Root dry

wt. (mg)

Control 0 100 a 100 a 59 a 4.7 a 140 a 45 a 6.8 a

Growth medium 50 88 b 87 b 53 b 4.5 a 120 b 41 b 6.0 b

100 84 b 84 b 49 c 4.3 ab 115 c 40 b 5.6 c

D. hawaiiensis 50 70 cd 51 g 34 f 3.1 d 54 g 19 e 3.4 f

100 46 e 41 h 29 g 2.5 ef 20 j 8 f 1.6 g

D. holmii 50 76 c 59 f 37 e 3.2 d 50 g 17 e 3.3 f

100 71 c 33 i 24 h 2.0 f 27 i 8 f 1.8 g

D. biseptata 50 71 c 80 c 41 d 3.9 bc 86 d 29 c 4.1 d

100 64 d 73 d 38 e 3.4 cd 62 f 21 e 3.5 f

D. australiensis 50 70 cd 67 e 37 e 3.5 cd 67 e 26 cd 4.0 de

100 52 e 48 g 31 g 2.6 e 35 h 23 d 3.6 ef



46


growth of P. minor in laboratory bioassays.



D. hawaiiensis D. holmii

D. biseptata D. australiensis

1

47


growth of A. fatua in laboratory bioassays.



D. hawaiiensis

1

2 3

4 5

D. holmii

1

2 3

4 5

5

2 3

4

1


1

2 3

4 5

A B

C D

48


species on germination and growth of wheat var. Inqlab 91 in laboratory

bioassays.


(%)

Germi-

nation

(%)

Shoot

length

(mm)

Shoot

fresh wt.

(mg)

Shoot

dry wt.

(mg)

Root

length

(mm)

Root

fresh wt.

(mg)

Root dry

wt. (mg)

Control 0 100 a 115 a 71 a 4.3 a 92.1 a 49 a 7.5 a

Growth medium 50 98 ab 104 b 70 ab 4.2 a 91.1 a 42 b 6.5 b

100 96 a-c 95 c 66 ab 3.9 ab 79.1 b 39 b 6.2 b

D. hawaiiensis 50 95 a-c 79 ef 49 ef 2.8 de 31.3 f 24 ef 3.6 fg

100 88 d 75 fg 46 f 2.6 de 28.0 f 21 f 3.3 g

D. holmii 50 94 a-d 89 cd 59 cd 3.4 bc 68.3 c 33 cd 4.7 cd

100 88 d 71 g 43 f 2.3 e 40.2 e 29 de 4.1 ef

D. biseptata 50 95 a-c 92 c 63 bc 3.8 ab 77.2 b 37 bc 5.4 c

100 92 cd 85 de 57 cd 3.1 cd 68.2 c 34 cd 4.9 cd

D. australiensis 50 94 a-d 92 c 63 bc 3.8 ab 65.0 c 32 cd 5.2 c

100 93 b-d 79 ef 54 de 3.1 cd 50.3 d 30 d 4.3 d-f


species on germination and growth of wheat var. Sehar 2006 in laboratory

bioassays.


(%)

Germi-

nation

(%)

Shoot

length

(mm)

Shoot

fresh wt.

(mg)

Shoot

dry wt.

(mg)

Root

length

(mm)

Root

fresh wt.

(mg)

Root dry

wt. (mg)

Control 0 100 a 105 a 85 a 4.9 a 98 a 63 a 9.0 a

Growth medium 50 96 ab 94 b 77 b 4.6 ab 96 a 59 a 8.2 b

100 96 ab 90 bc 71 bc 4.4 a-c 80 b 51 b 8.0 b

D. hawaiiensis 50 90 bc 85 c 66 cd 4.2 a-d 72 b 39 c 6.6 cd

100 82 d 60 e 43 f 3.7 d 38 e 43 c 6.3 d

D. holmii 50 87 cd 75 d 51 e 4.0 b-d 39 e 35 c 6.2 d

100 82 d 59 e 45 ef 3.8 cd 34 e 33 c 6.0 d

D. biseptata 50 93 bc 87 c 66 cd 4.2 b-d 50 d 46 d 7.0 c

100 89 bc 76 d 61 d 4.1 b-d 39 e 35 c 6.0 cd

D. australiensis 50 94 ab 87 c 67 cd 4.2 b-d 61 c 40 c 6.7 cd

100 87 cd 65 e 49 ef 3.9 a-d 56 c 39 c 6.0 cd


species on germination and growth of wheat var. Uqab 2000 in laboratory

bioassays.


(%)

Germi-

nation

(%)

Shoot

length

(mm)

Shoot

fresh wt.

(mg)

Shoot

dry wt.

(mg)

Root

length

(mm)

Root

fresh wt.

(mg)

Root dry

wt. (mg)

Control 0 100 a 131 a 90 a 5.0 a 109 a 65 a 9.6 a

Growth medium 50 97 ab 113 b 86 ab 4.8 ab 101 a 56 b 8.9 a

100 94 a-c 107 c 81 bc 4.5 bc 91 b 51 bc 8.1 b

D. hawaiiensis 50 88 c-e 91 f 58 e 3.5 fgh 33 f 27 gh 4.4 e

100 83 ef 78 gh 53 e 3.3 gh 24 g 25 h 4.0 e

D. holmii 50 90 b-d 100 d 76 c 4.0 de 81 c 42 de 6.6 cd

100 85 de 75 h 51 e 3.1 h 49 e 36 ef 6.4 cd

D. biseptata 50 92 b-d 96 de 67 d 3.9 def 76 c 43 de 6.7 cd

100 88 c-e 91 f 54 e 3.4 gh 68 d 39 ef 6.4 cd

D. australiensis 50 89 b-d 100 d 80 c 4.2 cd 82 c 48 cd 6.9 c

100 80 f 81 g 54 e 3.6 efg 35 f 34 fg 6.0 d



49


growth of Inqlab 91 in laboratory bioassays.



1

\2

4

5 1

4

2

1



1

2 3

2

4

1

2 3

4 5

1

3

5 5 4

1

3 2

4 5

A B

C D

50


growth of Sehar 2006 in laboratory bioassays.



4

2

D. holmii


D. biseptata

B A

D C 1

4

2 3

5

1

3 2

4 5

1

2

4 5

3

2 3

5

4

1

4

51


D. biseptata


growth of Uqab 2000 in laboratory bioassays.



A B

D

3 2

1

4

1

2

3

4

5

1

2 3

4 5

1

2 3

4 5

5

D. australiensis

C D

B

3

5

1

5

3

1

52

3.2. Foliar Spray Bioassays

3.2.1. Effect of Fungal Culture Filtrates on Growth of C. album


Foliar spray with growth medium had negligible effect on length as well as dry

biomass of C. album shoot as compared to control where water was used for spray. In

general, the effect of various fungal culture filtrates was more pronounced on 1-week

than on 2-week old plants. Culture filtrate of D. hawaiiensis exhibited the highest

herbicidal effect resulting in significant reduction of 9% and 20% in shoot length and

shoot dry biomass, respectively, of 1-week old plants, over control. Similarly, foliar spray

of culture filtrate of D. australiensis significantly suppressed shoot dry biomass of 1-

week old plants by 14% over control. The effect of all other fungal culture filtrate

treatments including 1-week as well as 2-week old plants was nonsignificant as compared

to control (Fig.1 A & B, Plate 9).


All the fungal culture filtrates reduced root biomass of C. album by 6–17% over

control in 1-week old plants. The adverse effect of culture filtrate of D. hawaiiensis was

more pronounced as compared to rest of the treatments. However, the effect of foliar

application of all the four fungal culture filtrates was nonsignificant over control both in

1-week as well as 2-week old plants (Fig.1C).

3.2.2. Effect of Fungal Culture Filtrates on Growth of R. dentatus


Statistical analysis of data demonstrated that foliar spray with growth medium had

nonsignificant effect on length as well as dry biomass of R. dentatus shoot. Foliar spray

with culture filtrates of all the four Drechslera species significantly reduced shoot length

of 1-week old R. dentatus plants. Similarly, spray with culture filtrates of all the four test

fungal species except D. holmii significantly declined the shoot length of 2-week old

53

weed plants. Culture filtrate of D. australiensis was found to be the most effective

causing 42% reduction in shoot length followed by filtrate of D. hawaiiensis resulting in

41% reduction in the studied parameter of 1-week old plants. Culture filtrates of D.

holmii and D. biseptata showed comparatively less pronounced herbicidal activity against

the test weed species causing 33% and 23% reduction in shoot length of 1-week old

plants, respectively. The adverse effect of culture filtrates of various test fungal species

on shoot biomass was generally similar to that of their effect on shoot length. The highest

decline of 60% in shoot biomass of 1-week old plants was recorded due to application of

culture filtrate of D. australiensis followed by D. hawaiiensis (56%), D. biseptata (56%)

and D. holmii (54%). Adverse effect of foliar spray on shoot biomass was more

pronounced in 1-week than in 2-week old plants (Fig. 2 A & B, Plate 10).


The data indicate significant herbicidal potential of culture filtrates of various

Drechslera species against root growth of R. dentatus. Root biomass of the weed was

severely suppressed in 1-week old plants by 68–82% due to foliar spray of different

Drechslera species. Culture filtrates of D. australiensis and D. hawaiiensis appeared to

be the most effective inhibiting root biomass by 82% and 80%, followed by 74% and

68% reduction in root biomass due to filtrates of D. holmii and D. biseptata, respectively.

Herbicidal activity of culture filtrates of various fungal species on 2-week old plants was

comparatively less pronounced where 58-73% reduction in root biomass was recorded

(Fig. 2C).

3.2.3. Effect of Fungal Culture Filtrates on Growth of P. minor


All the fungal culture filtrates reduced shoot length and shoot biomass of P. minor

by 2–7% and 4–9% over negative control, respectively. However, the effect of foliar

spray treatments of all the four Drechslera species was nonsignificant both in 1-week and

2-week old P. minor plants (Fig. 3 A & B, Plate 11).

54


All the fungal culture filtrates reduced root biomass of P. minor by 1–10% over

negative control. However, the effect of foliar spray treatments of all the Drechslera

species was nonsignificant both in 1-week and 2-week old P. minor plants (Fig. 3 C).

3.2.4. Effect of Fungal Culture Filtrates on Growth of A. fatua


Growth medium showed a nonsignificant effect on shoot growth in terms of

length and biomass. None of the fungal culture filtrate treatments exhibited significant

effect against the shoot length of A. fatua. By contrast, shoot biomass of the weed showed

a variable response to foliar application of culture filtrates of different Drechslera

species. Highest and significant reduction of 37% and 42% in shoot biomass was

recorded due to culture filtrates of D. australiensis and D. biseptata in both 1-week as

well as 2-week old plants. Similarly, culture filtrate of D. holmii significantly suppressed

shoot biomass by 15% in 1-week old plants. The effect of rest of the fungal culture

treatment was nonsignificant against shoot biomass of A. fatua (Fig. 4 A and B, Plate 12).


Data presented in Fig. 4 C reveals that foliar spray with culture filtrate of D.

hawaiiensis significantly enhanced root biomass over control. All other foliar spray

treatment exhibited nonsignificant effect on root biomass of the target weed species.

3.2.5. Effect of Fungal Culture Filtrates on Growth of Wheat


Data presented in Fig. 5a–8a reveals a variable response of shoot length in various

wheat varieties to the four fungal culture filtrates. Foliar spray of culture filtrates of D.

holmii and D. australiensis significantly reduced shoot length of 1-week old plants of

Inqlab 91. Similarly, culture filtrates of D. biseptata and D. australiensis also

55

significantly reduced shoot length of 2-week old plants. In contrast, the effect of all the

fungal culture filtrate treatments on shoot length of Sehar 2006 and Uqab 2000 was

nonsignificant. The effect of culture filtrates of all the four Drechslera species on shoot

biomass of all the three test wheat varieties was nonsignificant (Fig. 5b–7b, Plate 13-15).


Effect of foliar spray treatments of all the four Drechslera species was

nonsignificant on root biomass of all the three test wheat varieties, at 1-week as well as 2-

week growth stage (Fig. 5C, 6C and 7C).

56

Fig. 1: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of

1-week and 2-week old Chenopodium album plants. Vertical bars show standard

errors of means of four replicates. Values with different letters show significant

difference as determined by Duncan's Multiple Range Test at P ≤ 0.05.

A

0

3

6

9

12

15

1-week old 2-week old

Sh

oo

t le

ng

th (

cm

) a a

ba a a

ab ab abab abab

B

0

0.02

0.04

0.06

0.08

0.1

0.12


Sh

oo

t b

iom

ass (

g)

cd ab

bcabd

ab

ab

a aba ab

a ab

C

0

0.009

0.018

0.027

0.036

0.045


Ro

ot

bio

mass (

g)

ab ab ab ab ab ab ab

b ab

ab

a

ab

Control Growth medium D. hawaiiensis

D. biseptataD. holmii D. australiensis

57

Plate 9: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-

week and 2-week old Chenopodium album plants.

1: Negative control (water), 2: Positive control (M-1-D), 3: Culture filtrate (CF) of D.

hawaiiensis, 4: CF of D. holmii, 5: CF of D. biseptata, and 6: CF of D. australiensis

1

A

B

Spray started on 1-Week old plants


2 3 4 5 6

1 2 3 4 5 6

58


1-week and 2-week old Rumex dentatus plants. Vertical bars show standard errors of

means of four replicates. Values with different letters show significant difference as

determined by Duncan's Multiple Range Test at P ≤ 0.05.

A

0

2

4

6

8


Sh

oo

t le

ng

th (

cm

) a a

d

a a bbc

c c

a

cd

B

0

0.02

0.04

0.06

0.08


Sh

oo

t b

iom

ass (

g)

e

ccd

a a

e

ab

e

aa

e

b

d

C

0

0.02

0.04

0.06

0.08


Ro

ot

bio

mass (

g)

e

a

d cd

aa a

cd c

b

de



59

Plate 10: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of

1-week and 2-week old Rumex dentatus plants.



A

B



1 2 3 4 5 6

1 2 3 4 5 6

60

Fig. 3: Effect of foliar spray of culture filtrates of Drechslera spp. on growth of 1-

week and 2-week old Phalaris minor plants. Vertical bars show standard errors of



A

0

3

6

9

12

15

18

21


Sh

oo

t le

ng

th (

cm

) a a a

a aa

a a

a a aa

B

0

0.03

0.06

0.09

0.12


Sh

oo

t b

iom

ass (

g)

aa a a a a

a a a a a a

C

0

0.03

0.06

0.09

0.12


Ro

ot

bio

mass (

g)

a a

a a a a a a

a a a a



61


1-week and 2-week old Phalaris minor plants.



A

B



1 2 3 4 5 6

1 2 3 4 5 6

62


1-week and 2-week old Avena fatua plants. Vertical bars show standard errors of



A

0

7

14

21

28


Sh

oo

t le

ng

th (

cm

) aaaaaaaaaa a a

B

0

0.05

0.1

0.15

0.2

0.25


Sh

oo

t b

iom

ass (

g)

a

cccc

abb ab abaa a

C

0

0.04

0.08

0.12

0.16

0.2


Ro

ot

bio

ma

ss

(g

)

bbab

bbbbbbb b

a



63


1-week and 2-week old Avena fatua plants.



A

B



1 2 3 4 5 6

1 2 3 4 5 6

64


1-week and 2-week old plants of wheat var. Inqlab 91. Vertical bars show standard



A

0

8

16

24

32

40


Sh

oo

t le

ng

th (

cm

)

c c c a-c bc c a-c a-c ab a ab a

B

0

0.08

0.16

0.24

0.32

0.4


Sh

oo

t b

iom

ass (

g)

ab ab

a a a

a a a a a a

aa a

C

0

0.06

0.12

0.18

0.24

0.3


Ro

ot

bio

mass (

g)

a a a a a a a

a a a a a



65


1-week and 2-week old wheat var. Inqlab 91 plants.



A

B



1 2 3 4 5 6

1 2 3 4 5 6

66


1-week and 2-week old plants of wheat var. Sehar 2006. Vertical bars show standard



A

0

8

16

24

32

40


Sh

oo

t le

ng

th (

cm

)

ab

a ab ab ab b

a ab

ab ab

ab ab ab

B

0

0.08

0.16

0.24

0.32

0.4


Sh

oo

t b

iom

ass (

g) a a a a a a a a a a a a

C

0

0.06

0.12

0.18

0.24


Ro

ot

bio

mass (

g) a a a a a a a a

a a a a



67

Plate 14: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth

of 1-week and 2-week old wheat var. Sehar 2006 plants.



A

B



1 2 3 4 5 6

1 2 3 4 5 6

68


1-week and 2-week old plants of wheat var. Uqab 2000. Vertical bars show standard



A

0

8

16

24

32

40


Sh

oo

t le

ng

th (

cm

) a a a a a a a a a a a a

B

0

0.1

0.2

0.3

0.4


Sh

oo

t b

iom

ass (

g)

a a a

a a a a

a a a a

a

C

0

0.06

0.12

0.18

0.24


Ro

ot

bio

mass (

g) a a

a a

a a a

a a a

a a



69

A


1-week and 2-week old wheat var. Uqab 2000 plants.



.



1 2 3 4 5 6

1 2 3 4 5 6

B

70

3.3. Field Experiment

3.3.1. Effect of Fungal Culture Filtrates on Weed Biomass

Both recommended and half dose of the herbicide Bromoxynil+MCPA

completely checked the growth of the target weed species. Similarly, weed growth was

also completely checked when half dose of the herbicide was used in combination with

culture filtrates of different Drechslera spp. In general, culture filtrates of all the four

Drechslera spp. significantly reduced biomass of R. dentatus as compared to weedy

check. However, variability among the herbicidal activity of the culture filtrates of

different Drechslera spp. was evident. Among the four Drechslera spp., culture filtrates

of D. australiensis and D. hawaiiensis were found to be more effective against R.

dentatus than the culture filtrates of other two Drechslera species. There was 58, 57, 31

and 39% reduction in dry biomass of R. dentatus due to foliar application of culture

filtrates of D. australiensis, D. hawaiiensis, D. biseptata and D. holmii, respectively

(Table 8).

3.3.2. Effect of Fungal Culture Filtrates on Wheat Growth and Yield

Maximum and significant reduction of 14% in height of wheat plants was

recorded due to R. dentatus in weedy check as compared to weed free control. Plants

height was also significantly reduced due to presence of the weed in treatments where

only culture filtrates of different Drechslera species was used in foliar spray. There was

6, 8, 11 and 13% reduction in plant height due to foliar spray of D. australiensis, D.

hawaiiensis, D. biseptata and D. holmii, respectively as compared to weed free control.

The effect of weed competition on wheat plants height was nonsignificant over control

where synthetic herbicide Bromoxynil+MCPA was sprayed either alone or in

combination with the culture filtrates of the four Drechslera species (Fig. 8A). The

response of number of tillers and total above ground dry biomass to the weed competition

and foliar spray applications was generally similar to that of the response of plant height.

Maximum and significant reduction of 30% and 28% in tillering and above ground dry

matter, respectively, was recorded in weedy check as compared to control. Similarly,

71

there was 9–21% and 12–23% reduction in number of tillers and above ground dry

matter, respectively, due to weed competition in various treatments where only the fungal

culture filtrates were used as foliar spray. In contrast, in all the synthetic herbicide

treatments, either alone or in combination with fungal culture filtrates, the effect of R.

dentatus interference on the two studied parameters was insignificant as compared to

control (Fig. 8 B & C).

Data regarding the effect of weed competition and various types of foliar spray

applications on grain yield and 100 grains weight is presented in Fig. 9. Highest reduction

in grain yield (43%) was recorded in weedy check over control. Similarly, significant

reduction in grain yield was also recorded due to the weed competition over control in

treatments where only fungal culture filtrates were used in the foliar spray application.

There was 21, 22, 35 and 36% reduction in grain yield over control in treatments of foliar

spray applications of culture filtrates of D. australiensis, D. hawaiiensis, D. biseptata and

D. holmii, respectively (Fig. 9A). R. dentatus interference significantly reduced the

weight of 100 grains by 30, 25 and 27% in weedy check, and in treatments where culture

filtrates of D. biseptata and D. holmii, respectively, were used in the foliar spray

application (Fig. 9B). The effect of R. dentatus on grain yield and 100 grains weight was

insignificant in Bromoxynil+MCPA treatments, either alone or in combination with

fungal culture filtrates (Fig. 9 A & B).

72

Table 8: Effect of foliar spray of herbicide bromoxynil+MCPA and culture filtrates of

four Drechslera spp. on biomass of Rumex dentatus.

Treatments Weed dry biomass

(g plot-1

)

Reduction over

weedy check

(%)

Weed free 0 ± 0 d -

Weedy check 915 ± 45 a 0

Drechslera hawaiiensis 391 ± 12 c 57

D. holmii 642 ± 30 b 30

D. biseptata 630 ± 13 b 31

D. australiensis 384 ± 22 c 58

Bromoxynil+MCPA (Full dose) 0 ± 0 d 100

Bromoxynil+MCPA (Half dose) 0 ± 0 d 100

Bromoxynil+MCPA (Half dose) + D. hawaiiensis 0 ± 0 d 100

Bromoxynil+MCPA (Half dose) + D. holmii 0 ± 0 d 100

Bromoxynil+MCPA (Half dose) + D. biseptata 0 ± 0 d 100

Bromoxynil+MCPA (Half dose) + D. australiensis 0 ± 0 d 100

±, Indicates standard errors of means of three replicates.

In a column, values with different letters show significant difference (P ≤ 0.05) as


73

Fig. 8: Effect of foliar spray of full (FD) and half dose (HD) of Bromoxynil+MCPA

and culture filtrates of four Drechslera spp. on different growth parameters of field

grown wheat. Vertical bars show standard errors of means of three replicates. Bars

with different letters show significant difference (P ≤ 0.05) as determined by Duncan’s

Multiple Range Test.

A

0

21

42

63

84

105

Pla

nt

heig

ht

(cm

)

aaaaaabb

cdd bc

d

a

B

0

2

4

6

8

10

12

14

No

. o

f ti

llers

/p

lan

t

aa aa a a b

c c b

d

a

C

0

200

400

600

800

1000

1200

1400

1600

1800

To

tal d

ry b

iom

as

s (

g)

aaaaaa

abbcbc

a-c

c

a

Weed free

D. holmii

Bromoxynil+MCPA (FD)

HD + D. holmii

Weedy check

D. biseptata

Bromoxynil+MCPA (HD)

HD + D. biseptata

D. hawaiiensis

D. australiensis

HD + D. hawaiiensis

HD + D. australiensis

74

Fig. 9: Effect of foliar spray of full (FD) and half dose (HD) of Bromoxynil+MCPA

and culture filtrates of four Drechslera spp. on grain yield and 100 grains weight of

field grown wheat. Vertical bars show standard errors of means of three replicates.

Bars with different letters show significant difference (P ≤ 0.05) as determined by

Duncan’s Multiple Range Test.

A

0

200

400

600

800

Gra

in y

ield

/plo

t (g

)

dede

e

cd b-d

a-cab

ab a-c a-c aba

B

0

0.5

1

1.5

2

2.53

3.5

4

4.5

5

5.5

10

0 g

rain

s w

eig

ht

(g)

a

bbb

aaaa

aaaa

Weed free

D. holmii

Bromoxynil+MCPA (FD)

HD + D. holmii

Weedy check

D. biseptata

Bromoxynil+MCPA (HD)

HD + D. biseptata

D. hawaiiensis

D. australiensis

HD + D. hawaiiensis

HD + D. australiensis

75

T3 D. hawaiiensis

T5 D. biseptata T6 D. australiensis T4 D. holmii

T1 Weed free T2 Weedy check

T7 Herbicide (RD) T8 Herbicide (HD) T9 HD + D. hawaiiensis

T10 HD +D. holmii T11 HD +D. biseptata

Plate 16: Effect of foliar spray of recommended (RD) and half dose (HD) of

Bromoxynil+MCPA and culture filtrates of four Drechslera spp. on field grown weed

and wheat.

T12 HD +D. australiensis

76

3.4. Leaf Discs Bioassays Using Crude Organic Fractions

Positive reaction showing necrotic spots was observed on punctured R. dentatus

leaf surface, while no necrotic spot was observed in case of unpunctured leaf discs. In

punctured leaf surface, chloroform fraction was found to be highly effective in producing

necrotic spot followed by ethyl acetate fraction. n-hexane and n-butanol fractions did not

produce any necrotic spot. Crude chloroform fraction produced necrotic spot at minimum

concentration of 1.0 mg mL-1

, while ethyl acetate fraction produced necrotic spots at

minimum concentration of 2.0 mg mL-1

. Severe discoloration was also observed in case

of bioassays performed with punctured leaves. In case of chloroform fraction, severe

discoloration was observed up to concentration of 0.25 mg mL-1

followed by ethyl acetate

fraction, severe discoloration was observed only at highest concentration of 4.0 mg mL-1

.

In case of n-hexane and n-butanol fractions, discoloration was not much pronounced.

Leaf sections treated with DMSO as positive control exhibited only light discoloration,

while distilled water used as negative control had no effect on treated leaf sections. In

case of unpunctured leaf surface, although chloroform and ethyl acetate fractions did

produce moderate discoloration, but this effect was less pronounced when compared with

similar treatments using punctured leaf sections (Table 9 & 10, Plate17).

3.5. Leaf Discs Bioassays Using Purified Chromatographic Fractions

In these bioassays out of six purified chromatographic fractions from crude

chloroform fraction of culture filtrate of D. australiensis, four viz. A, C, D and F were

found effective in producing necrotic spot on punctured leaf discs of R. dentatus leaves.

Among these, A and F were found most bioactive in producing necrotic spots on

punctured leaf discs surfaces at minimum concentration of 0.5 mg mL-1

. Fraction C was

found active at minimum concentration of 1.0 mg mL-1

, while fraction D was found

active at highest concentration of 2.0 mg mL-1

. Chromatographic fractions B and E did

not produce any necrotic spot. In case of bioassays performed with 2,4-D, as positive

control, maximum bioactivity was observed at minimum concentration of 0.25 mg mL-1

.

Discolouration of cut leaf sections was also observed to variable extent in all the

treatments. Fraction C and F induced severe discolouration at concentration of 2.0 mg

77

mL-1

. Interestingly, only light discolouration was observed in case of 2,4-D, even at

highest concentration of 2.0 mg mL-1

(Table 11, Plate18).

In case of bioassays conducted with purified chromatographic fractions from

crude ethyl acetate fraction, only fraction H was found active in producing necrotic spot

on cut R. dentatus leaves. This fraction induced necrosis as well as severe discolouration

only at highest concentration of 2.0 mg mL-1

(Table 12, Plate18).

78

Table 9: Leaf discs bioassays using crude organic fractions on punctured leaf surface.

DMSO Conc.

(µL 1000µL-1

)

DMSO effect

Organic

fraction

Conc.

(mg mL-1

)

Effect of crude organic fractions

n-hexane Chloroform Ethyl acetate n-butanol

Colour Necrotic

Spot Colour

Necrotic

Spot Colour

Necrotic

Spot Colour

Necrotic

Spot Colour

Necrotic

Spot

Water 0 4 Water 0 4 0 4 0 4 0 4

1.560 0 4 0.0625 0-1 4 1-2 4 0 4 0 4

3.125 1 4 0.1250 1 4 2 4 1 4 0 4

6.250 0-1 4 0.2500 1 4 2-3 4 1 4 1 4

12.500 0-1 4 0.5000 1-2 4 3 4 1-2 4 1 4

25.000 0-1 4 1.0000 2 4 3 5 2 4 1-2 4

50.000 0-1 4 2.0000 2 4 3 7 2 5 2 4

100.000 0-1 4 4.0000 2 4 3 10 3 7 2 4

Colour scale

0 = no change

1 = Light discoloration

2 = Moderate discoloration

3 = Severe discoloration

Necrotic spot scale

4 = No necrotic spot

5 = Necrotic spot ≤ 1mm

6 = Necrotic spot ≤ 2 > 1mm





79

Table 10: Leaf discs bioassays using crude organic fractions on unpunctured leaf surface.

DMSO Conc.

(µL 1000 µL-1

)

DMSO effect

Organic

fraction

Conc.

(mg mL-1

)

Effect of crude organic fractions

n-hexane Chloroform Ethyl acetate n-butanol

Colour Necrotic

Spot Colour

Necrotic

Spot Colour

Necrotic

Spot Colour

Necrotic

Spot Colour

Necrotic

Spot

Water 0 4 Water 0 4 0 4 0 4 0 4

1.560 0 4 0.0625 0 4 0 4 0 4 0 4

3.125 0 4 0.1250 0 4 0-1 4 1 4 0 4

6.250 0-1 4 0.2500 0-1 4 1 4 2 4 1 4

12.500 0-1 4 0.5000 0-1 4 2 4 2 4 1 4

25.000 0-1 4 1.0000 0-1 4 2 4 2 4 1 4

50.000 0-1 4 2.0000 0-1 4 2 4 2 4 1 4

100.000 0-1 4 4.0000 0-1 4 2 4 2 4 1 4

Colour scale

0 = no change




Necrotic spot scale








80

Plate 17: Effect of crude chloroform (A) and ethyl acetate (B) fraction

of culture filtrate of Drechslera australiensis on punctured leaf discs of

Rumex dentatus.

Necrotic spot

Necrotic spot

Concentration of

crude chloroform

fraction (mg mL-1

) A

B

4

2

1

2

4

Concentration of

crude ethyl acetate

fraction (mg mL-1

)

81

Table 11: Leaf discs bioassays using purified chromatographic fractions from chloroform fraction of D. australiensis on punctured leaf surface.

DMSO Conc.

(µL 1000 µL-1)

DMSO effect

2,4-D/

Compound

Conc.

(mg mL-1)

2,4-D effect

Effect of purified chromatographic fractions

A B C D E F

Colour N.S Colour N.S Colour N.S Colour N.S Colour N.S Colour N.S Colour N.S Colour N.S

Water 0 4 Water 0 4 0

4 0 4 0 4 0 4 0 4 0 4

0.780 0 4 0.0312 0-1 4 0

4 0 4 0 4 0 4 0 4 0 4

1.560 0 4 0.0625 0-1 4 0-1

4 0-1 4 0-1 4 0-1 4 0-1 4 0-1 4

3.125 0 4 0.1250 1 4 0-1

4 0-1 4 1 4 1 4 0-1 4 0-1 4

6.250 0 4 0.2500 1 6 1

4 0-1 4 1 4 1 4 0-1 4 0-1 4

12.500 0-1 4 0.5000 1 7 1

6 1 4 2 4 1 4 1 4 1 6

25.000 0-1 4 1.0000 1 10 1-2

7 1 4 2 7 2 4 1 4 2 7

50.000 0-1 4 2.0000 1 10 2

9 1 4 3 8 2 7 1 4 3 9

Colour scale

0 = no change




Necrotic spot scale








N.S = Necrotic spot

82

Table 12: Leaf discs bioassays using purified chromatographic fractions from ethyl acetate fraction of D. australiensis on punctured leaf

surface.

DMSO Conc.

(µL 1000 µL-1

)

DMSO effect

2,4-D/

Compound

Conc.

(mg mL-1

)

2,4-D effect Effect of purified chromatographic fractions

G H I

Colour N.S Colour N.S Colour N.S Colour N.S Colour N.S

Water 0 4 Water 0 4 0 4 0 4 0 4

0.780 0 4 0.0312 0-1 4 0 4 0 4 0 4

1.560 0 4 0.0625 0-1 4 0-1 4 0-1 4 0-1 4

3.125 0 4 0.1250 1 4 1 4 1 4 1 4

6.250 0 4 0.2500 1 6 1-2 4 2 4 1 4

12.500 0-1 4 0.5000 1 7 2 4 2 4 1-2 4

25.000 0-1 4 1.0000 1 10 2 4 2 4 2 4

50.000 1 4 2.0000 1 10 2 4 3 7 2 4

Necrotic spot scale








Colour scale

0 = no change




N.S = Necrotic spot

83

Plate 18: Effect of 2,4-D and chromatographic fractions (A), (C), (D), (F)

and (H) of culture filtrate of Drechslera australiensis on punctured leaf

discs of Rumex dentatus.

2,4-D

0.25

0.5

1

2

A

1

2

0.5

C

1

2

D

2

H

2

F

1

2

0.5

Necrotic spot

Necrotic spot

Concentration of

pure fraction

(mg mL-1

)

Concentration of

pure fraction

(mg mL-1

)

Concentration of

pure fraction

(mg mL-1

) Concentration of

pure fraction

(mg mL-1

)

Necrotic spot

Concentration of

pure fraction

(mg mL-1

)

Concentration of

pure fraction

(mg mL-1

)

84

3.6. Spectroscopic Data of Isolated Compounds

Chromatographic fractions A and F exhibited the highest herbicidal activity,

therefore spectroscopic analyses of only these two compounds were carried out.

Chromatographic fraction A was identified as compound 1 and that of F as compound 2.

3.6.1. Compound 1

Holadysenterine

Colorless amorphous powder.

[α]D28

14.6° (c = 1.0, MeOH).

M.p. 219-220.5 °C.

HREIMS m/z: [M]+

390.3030 (Calcd. for C23H38N2O3 390.3083).

EIMS m/z: [M]+ 390, 317, 354, 307, 289, 278, 154, 136, 115, 107, 85.

1H-NMR (CD3OD, 600 MHz) δH : 1.19 (1H, m, H-1), 1.33 (1H, m, H-1), 1.35 (1H, m, H-

2), 1.44 (1H, m, H-2), 2.99 (1H, brm, H-3), 5.44 (brs 1H, m, H-6), 1.84 (2H, m, H-12),

1.33 (1H, m, H-17), 4.01 (1H, d, J = 10.9, H-18), 3.70 (1H, d, J = 10.9 ), 1.29 (s, 3H, H-

19), 3.45 (1H, m, H-20), 1.38 (1H, d, J = 7.1 H-21), 2.01 (s, 3H, CO-Me).

H2N

H

HH3C

N

H H

H

OH

OCH3

HO

1

2

3

4

5

6

7

8

9

10

11

12

13

1415

16

17

18

19

20

21

Fig. 10. Chemical structure of holadysenterine

85

3.6.2. Compound 2

(Z)- docos-5-en-1-oic acid

Viscous oil.

HREIMS m/z: [M]+

338.5702 (calcd. 338.5727 for C22H42O2).

EIMS (rel int) m/z, [M]+ 338 (26), 324 (30), 294 (10), 293 (60), 279 (40), 265 (35), 225

(28), 117 (100), 113 (75), 87 (32).

1H-NMR (CH3OD, 500 MHz) δ: 0.88 ( 3H, t, J = 6.8 Hz, H-22), 2.89 ( 2H, t, J = 7.2 Hz,

H-2), 5.25 (2H, dt, J = 11.6 Hz, H-5 and H-6), 2.21 (4H, m, H-4 and H-7), 1.32-1.84 (28

H, br s, H-3, H-8-H-21).

13C-NMR (CH3OD, 125 MHz) δ: 180.1 (C-1), 130.1 (C-5), 127.5 (C-6), 14.1 (C-22),

22.1-38.8 (C-2-C-4, C-7-C-21).

HOOC1

2

3

4

5

6

7

8

9

10

11

1214

1315

16

17

18

19

20

21

22

Fig. 11. Chemical structure of (Z)- docos-5-en-1-oic acid

86

Chapter 4

Discussion Discovery of natural herbicidal compounds from plants and microbes is an

extraordinary challenge and is an area of intense research. In the past years, a number of

highly successful herbicidal compounds based upon fungal metabolites have been

discovered (Berestetskiy 2008). The genus Drechslera is well known as bioherbicides for

its use in weed control programs (Peng and Boyetchko 2006; Hirase et al. 2006; Casella

et al. 2010; Rabbani et al. 2011). Besides this, a number of herbicidal compounds have

also been isolated from culture filtrates of different species of Drechslera (Evidente et al.

2006c). However, studies regarding the herbicidal activity of metabolites of Drechslera

species from Pakistan are lacking. The present study was, therefore, carried out to

investigate the herbicidal potential of metabolites of various Drechslera spp. from

Pakistan against some problematic weeds of wheat. In general, metabolites of all the four

test Drechslera spp. namely D. australiensis, D. biseptata, D. hawaiiensis and D. holmii,

exhibited herbicidal activity to variable extent against various target weeds of wheat.

In laboratory bioassays, generally original concentration of the M-1-D growth

medium significantly reduced germination, length as well as fresh and dry biomass of the

seedlings. However, this effect was far less pronounced as compared to the effect of

fungal culture filtrates. Original growth medium reduced germination, shoot length, shoot

dry biomass, root length and root biomass by 4–16%, 15–19%, 9–23%, 14–36% and 6–

32%, respectively. On the other hand, original culture filtrates of various Drechslera

species suppressed germination, shoot length, shoot dry biomass, root length and root

biomass by 12–94%, 27–91%, 17–88%, 56–94% and 47–88%, respectively. Although

contents of the original growth medium exhibited adverse effect on germination and

seedlings growth to some extent, however, it is very likely that most of the medium

contents were used during the 28 days growth period of the test fungal species, and the

effect of these medium components was probably negligible in the fungal culture filtrate

treatments.

In laboratory bioassays, culture filtrates of all the test Drechslera spp. reduced

seed germination of the four target weed species by 12–94%. Earlier, Idrees and Javaid

87

(2008) have reported 23% reduction in germination of parthenium (Parthenium

hysterophorus L.) seeds due to culture filtrates of D. hawaiiensis. In a similar study,

Javaid and Adress (2009) reported 20%, 30% and 93% reduction in germination of

parthenium seeds due to culture filtrates of Drechslera biseptata, D. australiensis and D.

rostrata, respectively. Herbicidal activity of fungal metabolites against germination of

weed seeds is not restricted to the culture filtrates of Drechslera species only. There are

also reports of herbicidal activity of culture filtrates of Trichoderma spp., Fusarium spp.,

Cladosporium spp. and Alternaria alternata (Idrees and Javaid 2008; Javaid and Adrees

2009; Javaid and Ali 2011). Akbar and Javaid (2010) studied the effect culture filtrates of

presently tested Drechslera species using malt extract as growth medium. The results of

the two studies reveals that M-1-D is comparatively better growth medium than malt

extract for the preparation of fungal culture filtrates for management of weeds of wheat.

Recently, Javaid et al. (2012) have also reported similar differential effects of culture

filtrates of Trichoderma spp. prepared in M-1-D and malt extract growth media and

tested against parthenium weed. The variable herbicidal potential of the fungal

metabolites prepared in different growth media could be due to the formation of different

quantities of culture filtrates in different growth media (Zonno et al. 2008). In the present

study, seedling growth of various weed species was also adversely affected by culture

filtrates of the test Drechslera species. Similar inhibition in seedling growth of other

weed species such as parthenium has also been reported due to culture filtrates of

Drechslera and other fungal species (Javaid and Adrees 2009; Javaid et al. 2011b).

Findings of the present study reveals that culture filtrates of different test Drechslera spp.

showed variable herbicidal activity against the germination and seedling growth of target

weed species. Culture filtrates of D. australiensis and D. hawaiiensis were found more

effective in suppressing germination of the test weed species than the culture filtrates of

other two fungal species. Variation in herbicidal activity of different Drechslera spp. has

also been reported against germination of parthenium seeds (Javaid and Adrees 2009;

Javaid et al. 2011b). Variation in herbicidal activity of culture filtrates of different

Drechslera spp. could be attributed to the variation in chemical constituents of different

fungal species (Evidente et al. 2005; Zhou et al. 2008; Eneyskaya et al. 2009; Yang et al.

2009). In laboratory bioassays, generally root growth was more susceptible to various

88

employed culture filtrates of the four Drechslera species. As herbicidal compounds are

first absorbed by roots from the surroundings, resulting in reduced growth (Noor and

Khan, 1994).

In pot trials, the effect of M-1-D medium as foliar spray treatments on the growth

of weeds as well as wheat plants was generally nonsignificant. The effect of various

culture fultrates on the growth of the test weed species was highly variable with respect

to the target weed species. In laboratory bioassays, generally all the test plant species

showed pronounced susceptibility to various fungal culture filtrates. In contrast, in pot

trials, generally culture filtrates of all the four Drechslera species exhibited marked

herbicidal activity against R. dentatus while their effect on growth of rest of the test weed

species and wheat varieties was nonsignificant. Such differential herbicidal effects of

fungal culture filtrates in laboratory and pot bioassays has also been reported in other

similar studies (Javaid and Adrees 2009; Javaid et al. 2011b). The differential effect of

fungal culture filtrates in laboratory and pot trials may be attributed to the two factors.

First, in laboratory bioassays seeds were directly exposed to various culture filtrate

treatments. Consequently, the very delicate germinating seedling’s growth was severely

affected by the applied culture filtrates. In contrast, in pot trials spray was done on 1-

week and 2-week old seedlings which were comparatively more tolerant. Secondly, in

laboratory bioassays, seedlings were exposed to various fungal culture filtrate treatments

through out the experimental period while in pot trials, spray was done at some regular

intervals. Although culture filtrates of all the Drechslera species exhibited pronounced

herbicidal activity against R. dentatus, however, D. australiensis and D. hawaiiensis were

found to be the most effective fungal species causing 42% and 41% reduction in shoot

length of 1-week old plants. Adverse effects of foliar spray on shoot biomass of R.

dentatus was more pronounced in 1-week old than in 2-week old plants. Although roots

were not directly exposed to foliar spray application, however, root biomass in R.

dentatus was also severely suppressed in 1-week old plants by 68–82% due to foliar

spray of different Drechslera species. Earlier, Javaid and Ali (2011) studied the effect of

culture filtrates of various Trichoderma species on pot grown plants of some problematic

weeds of wheat and found that R. dentatus was the most susceptible weed species. The

differential response of the four weed species to the same or different culture filtrates

89

could be attributed to the different morphological and physiological characteristics of the

test plant species involved. Toxicity is assumed to be associated with the presence of

strong electrophilic or nucleophilic systems. Action by such systems on specific positions

of proteins or enzymes would alter their configuration and affect their activity (Macías et

al. 1992). Previously, various studies conducted regarding the effect of foliar spray of

culture filtrates of different pathogenic fungi including species of Fusarium, Alternaria

and Drechslera against parthenium weed support the findings of the present study and

suggested that fungal culture filtrates can be exploited as herbicides (Idrees and Javaid

2008; Javaid and Adrees 2010; Javaid et al. 2011b).

In both laboratory bioassays and pot trials, R. dentatus found to be the most

susceptible weed species to the application of culture filtrates of Drechslera spp. so this

species was selected for field trials. On the other hand, all the test wheat varieties

exhibited almost similar behaviour to the application of fungal culture filtrates thus only

one wheat variety Sehar 2006 was selected for field experiment. In the field study,

herbicidal activity of culture filtrates of four Drechslera spp. was compared with a

commercial herbicide Bromoxynil+MCPA. The chemical herbicide was used either alone

in recommended dosage or its half dose was applied in combination with culture filtrates

of various Drechslera species. R. dentatus reduced wheat grain yield by 43% over weed

free control. Application of recommended dose of chemical herbicide completely killed

the weed plants. Although none of the fungal culture filtrates treatments completely

eliminated the weed, however, these markedly reduced the weed biomass by 30–58%

over weedy check. Wheat grain yield losses in treatments where foliar spray of culture

filtrates of D. australiensis, D. hawaiiensis, D. biseptata and D. holmii was carried out

were 21, 22, 35 and 36%, respectively, as compared to 43% losses in weedy check. In the

present study, original culture filtrates were applied as foliar spray. It is highly likely that

efficacy of these filtrates can be enhanced markedly if these are applied in a concentrated

form because generally, quantity of active herbicidal constituents in fungal culture

filtrates is very low (Evidente et al. 2006bc). In the field trials, half dose of synthetic

herbicide also completely killed the R. dentatus plants. Consequently, the effects of

combined application of fungal culture filtrates and half dose of herbicide could not be

assessed. Further studies are required in this regards using lower concentrations of the

90

herbicide. To best of our knowledge, the present study is the first report of using fungal

toxins as herbicidal agents in true field conditions. Generally, earlier experiments were

carried out in trays or pots (Vurro et al. 2001; Javaid et al. 2011b).

In both laboratory and pot trials, the effect of culture filtrates of various test

Drechslera species was more severe on germination and growth of weeds than that of

wheat. In these bioassays, culture filtrates of the four Drechslera species reduced the

shoot length, shoot biomass and root biomass of various target weed species by 22–91%,

17–88% and 47–48%, respectively. By contrast, shoot length, shoot biomass and root

biomass of wheat varieties Inqlab 91, Sehar 2006 and Uqab 2000 was reduced by 0–

4.9%, 0–13% and 0–28%, respectively. In foliar spray bioassays culture filtrate

applications had variable effects on growth of the test weeds. Shoot and root growth of 1-

week old R. dentatus plants was significantly reduced by culture filtrates of all the fungal

species. Conversely, the effect of foliar application of culture filtrates of all the fungal

species was nonsignificant on growth of all the three test wheat varieties. Likewise, in

field trials culture filtrates of D. australiensis, D. biseptata, D. hawaiiensis and D. holmii

reduced the biomass of R. dentatus by 58%, 31% and 57% and 30%, respectively, while

had no adverse effect on growth of wheat. The differential response of the weeds

especially R. dentatus and wheat to fungal metabolites can be best exploited in the

management of R. dentatus and possibly other weeds by the culture filtrates of

Drechslera spp. under field conditions.

In the present study, culture filtrates of D. australiensis exhibited the best

herbicidal activity in laboratory bioassays, foliar spray pot experiments as well as under

field conditions. Therefore, this fungal species was selected for the isolation of active

herbicidal compounds. The crude culture filtrates of D. australiensis were fractionated

using four organic solvent viz. n-hexane, chloroform, ethyl acetate and n-butanol. Leaf

discs bioassays were carried out using different concentrations of these crude organic

fractions of culture filtrates of D. australiensis. Chloroform fraction exhibited the highest

herbicidal activity followed by ethyl acetate fraction in terms of necrotic spot formation

and causing leaf disc discolouration. Isolation and purification of compounds from

chloroform and ethyl acetate fractions using various chromatographic techniques

revealed the presence of six compounds from chloroform fraction and three compounds

91

from ethyl acetate fraction. Leaf discs bioassays using these purified compounds revealed

that the most active herbicidal compounds were present in chloroform fraction From

chloroform fraction, chromatographic fractions A and F showed the highest efficacy in

producing necrotic spot at punctured leaf surface at minimum concentration of 0.5 mg

mL-1

as compared to 0.25 mg mL-1

of reference compound 2,4-D. However, when

compared the efficacy of chromatographic fractions A and F with that of 2,4-D in terms

of discoloration of leaf discs, 2,4-D was found less bioactive than the two isolated natural

compounds as only light discolouration was observed in case of 2,4-D, even at highest

concentration of 2.0 mg mL-1

.

Structural elucidation of the most active chromatographic fractions A and F were

carried out using various spectroscopic techniques and these fractions were identified as

holadysenterine as (compound 1) and (Z)- docos-5-en-1-oic acid as (compound 2),

respectively. In many earlier studies, several herbicidal constituents has been isolated

from other species of Drechslera. Culture of Drechslera siccans (Drechsler) Shoemaker

is also reported to yield a phytotoxin named as 6,8-dihydroxy-3-(2’-hydroxypropyl)

isocoumarin (de-o-methyldiaporthin). Phyto-toxicity of this compound has been

estimated in terms of necrotic spot area when tested on Avena sativa, Echinochloa crus-

galli and Amaranthus spinosus (Hallock et al. 1988). Capio et al. (2004) isolated two

phytotoxic compounds namely cytochalasin B and dihydrocytochalasins from extracts of

dried mycelia and liquid culture filtrates of Drechslera wirreganensis Wallwork, Lichon

& Sivan. and D. campanulata (Lév.) B. Sutton. Similarly, another metabolite namely

drazepinone with broad spectrum herbicidal activity has been isolated from Drechslera

siccans. This compound was characterized as 3,5,12a-trimethyl-2,5,5a,12a-tetrahydro-1H

naphtha [20,30:4,5]furo[2,3-b]azepin-2-one (Evidente et al. 2005).

Compound 1 (holadysenterine) was isolated as amorphous solid from the

chloroform extraction. Molecular formula was obtained by HREIMS, showing peak at

m/z 390.3030 for C23H38N2O3 showing six degrees of unsaturation in the compound. Four

unsaturations were accounted for by a tetra-cyclic pregnane type skeleton, two were due

to the endocyclic double bond and was due to carboxyl function. The UV spectrum was

inconclusive. Inspection of 1H-NMR spectrum of compound 1 showed olefinic proton at

δ 5.44 (1H, br singlet). The spectra showed two doublets at δ 3.70 (1H, d, J = 10.9 Hz)

92

assignable to the hydroxyl methylene protons. At δ 2.99 and 3.45 two broad multiplets

were observed and were assigned to the H-3α and 20β, respectively. Two methyl singlets

were observed at δ 1.29 and δ 2.0 while at δ 1.38 a characteristic methyl doublet was

observed having J = 7.1 Hz. The downfield shift of methyl group at δ 2.0 indicated the

presence of acetamide functionality in the molecule. A solvent exchangeable proton

singlet due to N-hydroxy was observed at δ 4.98 (Bhutani, 1990), indicated its attachment

at amine side chain functionality. EIMS spectrum gave peaks at m/z 317 [M+H-

CH3CONOH]+ (-C-N-bond cleavage), 289 [M+H-C4H8NO2]+ (C17-C20 bond cleavage)

and 278 [M+H-C5H8NO2]+ (C13-C17 and C16-C17 bands cleavage). This proves the

attachment of hydroxyl methyl group at amine centre. On the basis of these evidences

and comparison with the literature data, compound 1 was assigned as (20S)- 20

acetylhydroxylamine, 3β- amino, 13β hydroxymethylenepregn-5-ene and named

previously as holadysenterine (Kumar et al. 2007). Due to lack of amount, 13

C spectrum

was unpredictable, however some 2D NMR spectra (HMBC, 1H-

1H COSY) were

showing some signals of 13

C and from which final structure of compound 1 was

concluded.

Compound 2, (Z)- docos-5-en-1-oic acid was isolated as viscous oil. Molecular

formula was calculated from HREIMS, which gave [M+] peak at m/z 338.5702 (calcd.

338.5727) corresponding to the molecular formula C22H42O2. The peaks in EIMS

spectrum differ each by 14 mass indicating the aliphatic hydrocarbon nature of the

molecule. The 1H-NMR spectrum indicate the characteristic peaks at δ 5.25 as a doublet

of triplet integrated for 2H having J value 11.6 showing olefinic moiety in the molecule.

Terminal methyl group was appeared at δ 0.88 as a triplet with J = 6.8 Hz. A

characteristic triplet integrated for two protons appeared at δ 2.89 with J = 7.2 Hz

indicating the attachment of methylene with carboxylic moiety. An envelop of 34 protons

was appeared as broad singlet inferred for 17 methylene units at δ 1.32-1.84. Another

prominent 4 proton integrated signal was appeared at δ 2.21 indicated the attachment of 2

methylene with the olefinic band which was further confirmed in HMBC correlation. The

13C-NMR spectrum displayed signal at 180.1 while the olefinic bands were appeared at δ

130.1 and 127.5. The terminal methyl group was appeared at 14.1 while the remaining 19

methylene units appeared in between δ 22.1-38.8. The exact location of the double bond

93

in the molecule was confirmed by the loss of peaks in EIMS spectrum at m/z 251 and

265, 225 and 211 due to α and β fission of the double bonds, which confirmed its location

at C-5. The stereochemistry of the molecule was obtained by the calculated coupling

constant value, which indicates small J value, 11.6 Hz. On the basis of these evidences

and comparison with the literature data the molecule was identified as (Z)- docos-5-en-1-

oic acid (Misra and Wagner 2006).

94

Conclusions

In laboratory bioassays, culture filtrates of all the four test Drechslera species

showed variable herbicidal activity against weeds and wheat. Weeds were

generally more susceptible than wheat varieties.

In foliar spray bioassays, fungal culture filtrates significantly reduced the

growth of R. dentatus while the effect on other weed species and wheat varieties

was generally nonsignificant.

Under field conditions, culture filtrates of D. australiensis and D. hawaiiensis

reduced biomass of R. dentatus by 58% and 57%, consequently increased the

wheat grains yield by 22% and 21%, respectively.

Culture filtrates of D. australiensis exhibited the highest herbicidal activity.

Two herbicidal compounds were isolated from chloroform fraction of culture

filtrate of D. australiensis and identified as holadysenterine and (Z)- docos-5-

en-1-oic acid.

Future Prospects

The two identified compounds can be used as structural lead for the total

synthesis of natural product based eco-friendly analogues to be used as

commercial herbicides.

The shelf life of the identified compounds should be investigated.

Studies are required to investigate the genes responsible for the production of

effective herbicidal compounds; this may help to the boost up the production of

these compounds.

95

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