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Role of associative bacterial interactionin the induction of tuber rot incidencein cassava (Manihot esculenta Crantz)Lekshmi Saraswathi Pillai a & Raj Shekhar Misra ba Division of Crop Protection , Central Tuber Crops ResearchInstitute , Thiruvananthapuram , Indiab Regional Central Tuber Crops Research Institute , Bhubaneswar ,IndiaPublished online: 11 Jul 2013.

To cite this article: Lekshmi Saraswathi Pillai & Raj Shekhar Misra (2013) Role of associativebacterial interaction in the induction of tuber rot incidence in cassava (Manihot esculentaCrantz), Archives Of Phytopathology And Plant Protection, 46:13, 1540-1551, DOI:10.1080/03235408.2013.771459

To link to this article: http://dx.doi.org/10.1080/03235408.2013.771459

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Role of associative bacterial interaction in the induction of tuber rotincidence in cassava (Manihot esculenta Crantz)

Lekshmi Saraswathi Pillaia and Raj Shekhar Misrab*

aDivision of Crop Protection, Central Tuber Crops Research Institute, Thiruvananthapuram,India; bRegional Central Tuber Crops Research Institute, Bhubaneswar, India

(Received 18 January 2013; final version received 26 January 2013)

Cassava (Manihot esculenta Crantz), one of the leading staples in the world, hasconsiderable scope for integration into emerging markets through efficient andenvironmentally sound production of a diversified range of high quality, competitiveproducts for food, feed and industry. One of the major reasons attributed to the lowproductivity of cassava in India is the incidence of tuber rot in cassava incited byPhytophthora palmivora. Usually, an array of microorganisms is involved in rottingof cassava tubers in the field and in postharvest conditions. Unfortunately, the dis-ease management practices focus mainly on fungal pathogens. In this article, wetried to investigate the role of associative bacterial pathogens in induction of tuberrot of cassava, the potential of the bacteria with P. palmivora in causing disease inci-dence, spread and rotting. The bacteria and P. palmivora were originally isolatedfrom tuber rot infected cassava from the field having a disease incidence percentageof above 40%, checked for pathogenicity and were proven for Koch postulate. Thedetached tubers of disease resistant (cv. Sree Pathmanabha) and susceptible cultivars(cv. M4) of cassava were used for the study. The effect of bacterial association wasstudied in Cassava agar plate assay, in detached tubers and in in planta experimentsin in vitro glass house conditions. The bacterial isolates were identified as Gram-negative coco bacilli of class Pseudomonas, Erwinia amylovora and Achromobacterdenitrificans.

Keywords: P. palmivora; assosiative bacteria; tuber rot

Introduction

Cassava (Manihot esculenta Crantz), one of the leading staples in the world, hasconsiderable scope for integration into emerging markets through efficient and environ-mentally sound production of a diversified range of high quality, competitive productsfor food, feed and industry (Sree Kumari 2011). It is a native of Brazil and is widelycultivated in the tropics, especially in Nigeria, Brazil, Indonesia, Africa, Thailand andAsia. It serves as a staple food for more than 500 million people all over the world. Itis cultivated over 28m ha. FAO places it in the third most important source of caloriesin the tropics, after rice and maize. Cassava could become the raw material base for anarray of processed products that will effectively increase demand for cassava andcontribute to agricultural transformation and economic growth in developing countries.One of the major reasons attributed to the low productivity of cassava in India is the

*Corresponding author. Email: rajshekharmisra@gmail.com

Archives of Phytopathology and Plant Protection, 2013Vol. 46, No. 13, 1540–1551, http://dx.doi.org/10.1080/03235408.2013.771459

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incidence of tuber rot in cassava incited by Phytophthora palmivora. The causativeorganism was first identified as P. palmivora by Johnson and Palaniswami (1999).The disease affects the tuber part of the crop mostly in field conditions. Resistance tothe disease has not been reported in any of the cultivars (Edison 2004). It is transmittedthrough water and is a cut wound pathogen (Johnson and Palaniswami 1999).P. palmivora causes bud-rot of palms, fruit-rot of cocoa, coconut and arecanut. Thecrossinoculation potential of other species is not well studied in cassava (Johnson andPalanisamy 1999).

In India, as far as the reports are concerned the tuber rot of cassava is caused byP. palmivora only, there might be chances for other species of fungus and secondaryinvaders in causing infection in cassava. Usually, an array of microorganisms isinvolved in rotting of cassava tubers in the field and in post harvest conditions. Unfor-tunately, in disease management practices, the focus is mainly on fungal pathogens. Inthis study, we discuss the role of associative bacterial pathogens in induction of tuberrot of cassava, the potential of the bacteria with P. palmivora in causing diseaseincidence, spread and rotting. A good knowledge about the pathogen, their variabilityand adaptability may be helpful in understanding the disease scenario in the croppingsystem of cassava, so that a meaningful control schedule can be evolved for the bettermanagement of the disease in the system. With this view, a survey has been conductedto collect potential pathogens, associated with tuber rot of cassava in Kerala. Potentialisolates were studied for their morphological characters for confirmation, disease aetiol-ogy in relation to associative bacteria, and their role in disease induction in detachedtubers and in in planta trials to confer their interrelationship with P. palmivora incausing tuber rot in soil conditions.

Materials and methods

Biological material

Minisetts of healthy cassava nods of variety M4 and Sree Pathmanabha (M. esculentaCrantz) obtained from Farm house of the Division of Crop Production, Central TuberCrops Research Institute (CTCRI) (Sreekariyam, Thiruvananthapuram, India) were usedfor the study. The cassava setts were disease-free as the plants were grown in nurserytroughs containing sterile sandy loam soil. The planting material was 3–5 cm in thick-ness and contained two nods each.

Isolation and identification of the pathogen

The Infected tuber samples of cassava exhibiting typical symptoms of tuber rot wereobtained from the fields having report of a severe attack of P. palmivora in Kerala.(Table 3, Figure 1). The pathogens were isolated by tissue segment methods

Figure 1. The morphology of associative bacteria in tuber rot induction by P. palmivora.

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(Rangasami 1958). The tuber tissues selected for isolation contained a small portion ofdisease as well as healthy tuber part. Samples were washed in running tap water, cutinto small pieces of 3–5mm size and surface disinfected by immersing them in 70%alcohol for 2min, rinsed twice with sterile distilled water and placed in cassava agarmedia (M4 cassava starch; 15 gL�1, Sucrose; 5 gL�1, Agar 15 gL�1, without any antibi-otics amendments). Incubation for 5 days was carried out at 27 °C. The pathogens wereisolated into pure culture from radiant mycelia that emerged from the inoculated tissuesand were maintained in carrot agar medium (CDA; 250 gL�1 carrot, 20 gL�1 dextroseand 20 gL�1 agar) supplemented with rose bengal and streptomycin sulphate. Bacterialcultures surrounding the sample pieces were purified into pure culture by continuousstreaking and maintained in NA slands for further study. The P. palmivora isolates wereproved for its virulence and pathogenesis by Koch postulate in detached cassava tubersof variety M. esculenta M4.

Characterisation of isolates

The isolates were verified as P. palmivora by comparing the morphology of the isolateswith known culture of P. palmivora isolate in cultural library of CTCRI (Sreekariyam,Kerala India). Induction of sporangia was done in sterile water, suspended with cutpieces of culture disc of 3mm diameter, in Petri dishes for over night under normallight (Aragaki et al. 1967). Zoospore structures were studied by chilling sporangial sus-pension at 5 °C for 30min. The mating type was determined by pairing P. palmivoraisolates with A1 and A2 tester strains (PP-A1T1 , PP-A1T2 and PP-A2T1 , PP-A2T2) onV8 agar medium (50 gL�1 V-8 juice, 0.2 gL�1 CaCO3 and 20 gL�1 agar). Plates incu-bated at 14 °C in the light and examined for the presence of oospores after one month.The isolates elaborated oospores on pairing with A1 tester strain were designated A2

mating type. The four associative bacteria obtained from tuber rot samples were sub-jected to biochemical identification based on Bergey’s Manual of Systematic Bacteriol-ogy (Doudoroff and Palleroni 1974) and by BIOLOG Breath prints (BIOLOG GN(USA) microtitre plate analysis, for which the organism was grown on Biological Uni-versal Growth Medium (BUGM) (Biology Inc., California, USA) plates at 28 ± 2_C for24 h and observed for the ability of isolate to oxidise 95 different carbon sources(Figures 1 and 2).

The growth vigour of P. palmivora and bacterial association

The P. palmivora isolates were compared for their growth vigour in the presence andabsence of associative bacteria on cassava agar plates. The cassava agar plate (Cassavastarch 20 gL, sucrose 5.00 gL, agar 15 gL) were inoculated with 5-mm disc of activelygrowing P. palmivora culture of 5 days old maintained in carrot agar medium. The associ-ation of bacteria and P. palmivora was done by inoculating 10 μl of 106 dilution of asso-ciative bacteria on P. palmivora plates. The un-inoculated plate served as the control.

Characterisation of bacteria for its extracellular enzyme production

The extracellular hydrolytic enzymes of associative bacteria were detected on cassavamash broth [cassava mash (rind + flesh) –20 g, 0.1M phosphate buffer pH 7–100mL]by inoculating lag phase culture of the isolates on the medium. The production of dif-ferent lytic extracellular enzymes viz. cellulase, protease, pectinase, amylase and lipase

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by P. palmivora were carried out using solid-state fermentation. For SSF, 1mL ofconidia suspension (2� 109 conidiamL�1) of P. palmivora isolates were inoculated into250-mL Erlenmeyer flasks containing sterilised substrate [10 g cassava rind, 10 g cas-sava flesh and 10mL of mineral salt solution (g/l; KH2PO4, 2.5; KNO3, 5.0; MgSO4.7H2O, 1.0;Na2SO4, 1.0; FeCl2, 0.02; ZnSO4. 7H2O, 0.0015; CuSO4. 5H2O, 0.003 andMnSO4, 0.001) in distilled water] followed by an incubation at 35 °C for 7 days. Afterincubation, the crude extracellular enzyme supernatants were obtained by squeezeextraction, twice with 100mL of 50mm sodium acetate buffer pH 6.5 through a muslincloth. Further, the filtrate obtained were centrifugated at 15,000� g for 10min at 4 °C.The clear supernatant was used as the enzyme source for the determination of pectinase(Manachini et al. 1988), amylase (Akpan et al. 1999), β glucosidase, cellulase (Rayet al. 1993), lipase (Benjamin and Panday 1997) and protease (Kunitz 1947) (Figure 3).

In vitro in tuber assessment of virulence

Healthy cassava tubers of variety M4 (susceptible) and Sree Pathmanabha (resistant)grown at the CTCRI Farm (CTCRI, India) were used for the study. Tubers of the samesize were washed and surface sterilised with 1% sodium hypochlorite for 5min, rinsedtwice with sterile distilled water. Length and breadth of tubers were recorded priorto inoculation. For individual inoculations, 20 μl spore suspension (1�105mL) ofP. palmivora isolates and 20 μl bacterial suspension (1�105mL) of associative bacterialisolates were used. For association experiments, 20 μl microbial suspension from thecombination of respective bacterial and fungal suspensions [(1�105mL) associativebacteria +P. palmivora spore suspension of (1�105mL)] was used. Inoculation wasmade in the middle region of tuber via, 2-mm syringe and tubers were maintained in ahumid chamber (90–100% RH) with an incubation period of 7 days. The experimentswere conducted with five replicates with five tubers each.

In planta studies

The effect of associative bacterial strains on tuber rot induced by P. palmivora isolateswas carried out in pot experiments using sterile loamy sand in the greenhouse. Liveplant inoculation was done in 6months old cassava plants of variety M4 and Sree Path-manabha maintained in pots (1m� 1m) under glass house conditions. Previously disin-fected minisetts of healthy cassava nods of variety M4 and Sree Pathmanabha seedlingswere planted in pots (1�1M) in the green house of CTCRI at 30 °C, with a 16-h lightperiod followed by an 8-h dark period. A spacing of 90 cm� 90 cm was maintainedbetween the pots and were allowed to grow to attain maturity of 6months. For chal-lenge inoculation, individual bacterial strains were crossstreaked separately on NutrientAgar medium and incubated at 24 °C for 48 h. The cells were harvested by scrappingwith sterile glass spreader after drenching with 5m sterile water. Cell suspensions wereadjusted to OD 620 = 0.1 that correspond to cell density approximately 107–108 cellsmL�1, and they were also shown by comparison of dilution plate counts. Thespore suspension of P. palmivora isolates were reared individually in sterile distilledwater by inoculating 2–3 mycelial disc of 7 days old respective P. palmivora isolates.The spores were adjusted to 1�105mL.

The inoculation was performed in the middle rhizoplane region of the tuber via,2-mm syringe removing surface soil and sterilising the tuber surface with 70% alcoholmoistened cotton. About 20 μl each of bacterial (1�108 cfumL�1) and P. palmivora

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(1�105mL) suspensions and their combinations [(100mL bacterial (1�108 cfumL�1)and P. palmivora (1�105mL)] were used for inoculation. The lesions were coveredwith moist cotton and were sealed with sterile surgical tape to avoid other bacterialinfection. The surface soil was replaced, and the plants were maintained in a humidchamber (90–100% RH), and the symptoms developed were recorded after 5 days’incubation.

The pots were arranged in completely randomised block design with thirteen treat-ments, with single inoculation of associative rhizobacterias [AB-08, AB-09, AB-10,AB-11] and P. palmivora [PP-08, PP-09, PP-10, PP-11] and combinations of associativebacteria and P. palmivora isolates and an inoculated control [T1-Control, T2 -PP-08,T3-PP-09, T4-PP-10, T5-PP-11, T6-AB-08, T7-AB-09, T8-AB-10, T9-AB-11, T10-PP-08+AB-08, T11-PP-09 +AB-09, T12-PP-10 +AB-10, T13-PP-11 +AB-11]. Each treatmentconsisted of 20 plants each with three replications. The experiment was conducted inthe year of 2008. The planting was performed in the 2 February 2008, and the harvestwas carried out after 200 days (20 September 2008). The recommended dose of fertilis-ers was NPK @ 100:50:100 kg/ha [urea, single super phosphate and muriate of potash].The plants were irrigated twice in the morning and in the evening with sterile distilledwater. After harvest, all tubers per plant were collected and were analysed for tuberyield (gramme per plant and kilogramme per hectare) and the tuber rot incidence.

Relative virulence

Relative virulence was calculated by fixing a scale of virulence for in plants and intuber experiments in vitro, on the basis of rot infection induced on five tubers per plant.The area of infection per tuber is calculated based on the length and breadth of lesionformed when accessed horizontally (L�B). The control tuber area is fixed as50� 50mm (2500 cm2) which is the mean if 100 tubers of various varieties studied.The relative virulence is expressed as lesion area (mm2).

Disease incidence

The tuber rot incidence was calculated at 200 days after the study period (2008). Theplants with rot incidence were compared with plant inoculated with P. palmivora as acontrol. The disease incidence was observed by counting the number of plants withtuber rot and the total number of plants planted in the study. [Per cent Disease Inci-dence =Number of rotted plants in the plot/Total number of plants in the plot� 100]using the formula described by Rose (1974) (Table 1). All experiments were repeatedthrice. The tuber yield was calculated, and the pooled data were analysed statistically.

Figure 2. Morphology of P. palmivora isolates inducing tuber rot.

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Statistical analysis

The data were analysed statistically according to standard procedures for the analysis ofvariance (general linear model) and mean separation (least significant difference) (SASInstitute, Cary, NC). All differences referred to in the text were significant at the 0.05%level of probability.

Result

The survey conducted in tuber rot reported regions of Kerala revealed that tuber rot is aserious constraint causing low productivity of cassava. Severity of the disease rangedfrom 80 to 40%. A disease severity of 82% was noted in Palode, Thiruvananthapuram,Kerala, India (Table 1). Tuber rot of cassava caused by P. palmivora was characterisedby brown lesions and foul rotten smell. However, no disease symptoms were seenoutside the tuber, other than yellowing of leaves in some plants. A water-soaked appear-ance has also been seen in some tubers. Gram-negative, mucoid, rod shaped bacteriawere found associated with P. palmivora infection. The organisms were lytic enzymeproducers, pectin and a starch degrader. An increase in virulence was noted in the pres-ence of the associative bacteria. Four strains of P. palmivora, isolated from the infectedtuber samples and were identified by spore morphology and morphology in culturalmedia, compaired with known P. palmivora strains maintained in the Phytophthoraculture library, of CTCRI (CTCRI, India) (Table 2). All isolates reproduced pathogenic-ity and virulence and has proven Koch postulate, in detached tuber and in live plantspecimens.

00.5

11.5

22.5

33.5

44.5

5

The Extra cellular enzyme profile of P. palmivora and Associative Bacteria

Amylase

Cellulase

Pectinase

Protease

Lipase

Polygalacturonase

PP-08 PP-09 PP-10 PP-11 AB-08 AB-09 AB-10 AB-11

P.palmivora Associative Bacteria

Glucosidase

Figure 3. The extracellular lytic enzyme profile of P. palmivora and associative bacteria.

Table 1. Disease severity of P. palmivora isolates from different geographical areas.

Sl No. Isolates Geographical origin Latitude and longitude PDI (%)

1 PP-08 Aryanadu, Thiruvananthapuram, Kerala 08:34:42, 77:05:06 802 PP-09 Palode, Thiruvananthapuram, Kerala 08:43:32, 77:01:29 853 PP-10 Aluva, Ernakulum, Kerala 10:00:58, 76:18:12 754 PP-11 Kizakkumbhagom, Ernakulum, Kerala 09:01:36, 76:45:11 45

PDI- Percentage disease index.

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Table2.

Identificatio

nof

theP.

palmivoraandassociativebacteria.

Fun

galisolates

Colon

ycolour

Spo

rang

ialmorph

olog

y

Oospo

res

form

ation

Mating

type

Molecular

identificatio

nShape

Size(L

�B)

Pedicel

leng

thPapilla

PP-08

Offwhite

Sym

podial

32�21

3μm

Ellipsoidal

Heterothallic

A2

P.pa

lmivora

PP-09

Offwhite

Sym

podial

33�24

4μm

Ovo

idal

Heterothallic

A2

P.pa

lmivora

PP-10

Offwhite

Sym

podial

35�22

3μm

Ellipsoidal

Heterothallic

A1

P.pa

lmivora

PP-11

Offwhite

Sym

podial

32�24

5μm

Ovo

idal

Heterothallic

A2

P.pa

lmivora

Bacterial

isolates

Colon

ymorph

olog

yMicroscop

ical

observations

Pheno

typicalandbiochemical

identificatio

nBiologidentificatio

nGramme

reactio

nShape

Motility

End

oSpo

reAB-08

White

mucoid

G�v

eCocco

bacillu

sMotile

–Pseud

omon

assp.

Pseud

omon

assp.

AB-09

White

mucoid

G�v

eCocco

bacillu

sMotile

–Pseud

omon

assp.

Pseud

omon

assp.

AB-10

White

mucoid

G�v

eBacillus

Motile

–Erw

inia

sp.

Erw

inia

amylovora

AB-11

Brownmucoid

G�v

eBacillus

Motile

–Achromob

actersp.

Achromob

acter

denitrificans

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Table 3. Growth rate of P. palmivora and associative bacteria isolates on cassava agar plates(mm/h).

Treatments 0 12 24 36 48 60 72 84 96 108 130

Control 0 0 0 0 0 0 0 0 0 0 0PP-08 0 10 22 30 42 50 55 58 60 62 65PP-09 0 09 20 27 38 46 52 54 57 59 60PP-10 0 09 19 27 37 47 51 54 57 60 61PP-11 0 08 20 28 40 48 52 56 58 60 62PP-08 +AB-08 0 10 36 46 64 88 90 90 90 90 90PP-09 +AB-09 0 09 30 42 59 84 89 90 90 90 90PP-10 +AB-10 0 08 32 40 60 82 88 90 90 90 90PP-11 +AB-11 0 10 33 44 62 86 90 90 90 90 90

aThe study is the mean of five replications with five plates each.bScale of growth in cassava agar plate experiments in vitro is fixed on development of mycelium in mm inaccordance with time (12 h).

Table 4. Virulence of P. palmivora, associative bacteria isolates and their combinations.

Treatments

In tuber (mm2)aIn planta(mm2)b

Virulence classcM4 SPA M4 SPA

Con 0000 0000 0000 0000PP-08 1472 0020 1082 0009 4PP-09 1568 0016 1120 0004 4PP-10 1200 0016 1000 0009 4PP-11 1600 0020 1088 0012 4AB-08 0280 0200 0200 0189 3AB-09 0200 0180 0180 0162 3AB-10 0180 0166 0182 0160 3AB-11 0220 0190 0200 0186 3PP-08 +AB-08 2500 0100 2500 0025 5PP-09 +AB-09 2500 0025 2500 0030 5PP-10 +AB-10 2500 0032 2500 0025 5PP-11 +AB-11 2500 0090 2500 0090 5

Class 1 : Non pathogenic Mean area < 25mm2 on 7th dayClass 2 : Less virulent Mean area between 25 and 100mm2

on 7th dayClass 3 : Moderately virulent Mean area between 100 and 400mm2

on 7th dayClass 4 : Virulent Mean area between 400 and

1600mm2 on 7th dayClass 5 : Highly virulent Mean area between 1600 and

2500mm2 on 7th day

aThe study is the mean of five replications with five tubers each, the results are given as whole figures omit-ting decimal values.bThe study is the mean of five replications with 20 plants each.cScale of virulence for in planta and in tuber experiment in vitro is fixed on the based of rot infection inducedon five tubers per plant. The area of infection per tuber is calculated based on length and breadth of lesionformed when cut horizontally (L�B). The control tuber area is fixed as 50� 50mm (2500 cm2).

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Characterisation and identification of tuber rot pathogens

Cultural morphology of P. palmivora isolates showed off white thick colony withmoderate surface growth, Sporangial morphology varied with isolates. The myceliumwas smooth without hyphal swelling and diameter varied from 3–5 μm. Sporangialsize and shape varied according to conditions under which it is formed. Variation insize and shape was observed within the isolates. Sporangia were formed readily in2–3 days and were caducous and sheared with a small short and stout occluded pedi-cel. Pedicel length varied from 3–5 μm. Sporangia were palpitate and papilla promi-nent. The shape of sporangia was ellipsoidal or ovoid with round base. Size ofsporangia ranged from 33–35� 21–25 μm to 25–52� 22–36 μm in agar culture andwater respectively in an L.B ratio of 1.3–1.5 in agar culture or 1.13–1.52 in water.All the four isolates of P. palmivora were tested for mating type by pairing withknown A1 tester strains. Three of the isolates (PP-08, PP-09, PP-11) could formoospores in these pairings plates. Thus, the isolates were designated as A2 matinggroups and the isolate PP-10 as A1 mating group. The associative bacterial Isolatesviz., AB-08, AB-09, AB-10 and AB-11 were identified as Pseudomonas sp. as perBergey’s Manual of Systematic Bacteriology (Table 2, Figure 2). The BIOLOG GNbreath prints confirmed the isolates as Pseudomonas spp (Figure 1).

Growth rate of P. palmivora and associative bacteria on cassava sucrose plates

The bacteria found associated with P. palmivora infection showed a positive correlationwith growth rate when inoculated in combination than as single inoculations. Thecombination attained full growth (89.25mm) in 72 h compared with control, whereP. palmivora was inoculated alone (62mm in 130 h). The isolates showed an almostsimilar growth rate when inoculated alone (Table 3).

The extracellular lytic enzyme profile of P. palmivora and associative bacteria

The extracellular lytic enzyme profile of P. palmivora and associative bacteria revealedthat the organisms are potent lytic enzyme producers. The bacteria produced enzymeamylase, cellulase, protease, pectinase, poly galacturonase and β glucosidase rangingfrom 2.99–4.62UmL�1, 1.33–2.44U gmL�1, 1.65–1.99UmL�1, 1.90–2.11UmL�1,1.24–1.87UmL�1, 2.01–2.90UmL�1, 2.30–2.99UmL�1 and P. palmivora elaboratedlytic enzymes to the tune of 3.06UmL�1, 2 UmL�1, 3.46U gmL�1, 1.98UmL�1,1.99UmL�1, 1.89UmL�1, 1.92UmL�1, respectively (Figure 3).

Relative virulence of P. palmivora, associative bacterial isolates and theircombinations in vitro

In in planta assay, the virulence and relative growth vigour of P. palmivora incombination with associative bacteria was high compared with infection rate caused byP. palmivora alone. The combination attained full rot within 4 days from the date ofinoculation. The bacterial association showed the typical tuber rot symptom, character-ised by full rot with obnoxious odour, oozing out and watery appearance, where as theP. palmivora rot was characterised by dry rot. The oozing of internal fluids, water-soaked appearance and obnoxious odour, might be contributed by bacterial partner.However, when inoculated with bacteria alone, the infection rate was not severe. Incassava, soft rot symptoms appeared in response to infection with foul estery odour, but

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not intense as in the case of tuber rot by P. palmivora. The rot was more confined tocassava rind region with watery appearance.

Discussion

In tropics, many fungal diseases cause severe crop loss, but their modes of action arenot yet identified. Most of the time, the causal agents and associative organisms play asignificant role in disease aetiology and many a time their symbiotic association endsup in severe yield loss. In India, P. palmivora induced tuber rot disease of cassava isbecoming a serious issue in total production of cassava where the role of bacteria asso-ciated with tuber rot disease was investigated in this study. The results clearly confirmthe effect of bacterial association with P. palmivora in causing tuber rot infection. Fourisolates that emerged from rot samples were identified as P. palmivora by comparingthem with known isolates of P. palmivora maintained in CTCRI culture collectionlibrary (CTCRI, India). Basically, P. palmivora is characterised by the production ofsympodial, ovoid, ellipsoid or fusiform, semi-papillate sporangia that are caducous andwith a medium pedicel (2–6 μm). The associative bacterium has been found along withP. palmivora while isolating the pathogen from infected sample. The bacterial associa-tion accelerated virulence in cassava sucrose agar plates. Increased mycelia growth rateif P. palmivora was grown in Cassava dextrose media was described by Weststeijn andOkafor (1971). In vitro studies in detached cassava tubers of disease susceptible (cv.M4) and resistant cultivar (cv. Sree Pathmanabha) of cassava against virulent strain ofP. palmivora was used to evaluate the effect of bacterial association, since cassava tuberis the target site, were the pathogens attack and produce rot. The tuber rot area wasused as the marker for disease resistance imparted by cultivars, and the symptomsvaried with organisms and cultivar resistant types. In cv. M4, the tuber rot was charac-terised with classical rot symptoms involving severe rot, browning, watery appearance,oozing out of internal fluids and obnoxious odour as described by Johnson and Palan-iswami (1999), which might be due to the degradation of starch and related compoundsof cassava. In cv. Sree Pathmanabha, the rot is characterised as a black brown darkspot, shows the inability of P. palmivora to survive in the host tissue, caused the dyingof cells in the inoculated area and left the remaining part of the tuber uninfected. Thespot was dry, without odour. Mostly, five patterns of lesions are observed in tubers,cone-shaped lesion, spread lesion, lesion confined to one part with full rate of the part,rot confined middle part round the cambium with round spread and rot confined tocassava rind, while the middle part remaind uninfected (Lekshmi and Misra 2012).

In plants, physical barriers are a fundamental feature of defence mechanisms againstphytopathogen attack. In cassava tubers, the first such line of defence is the peridermallayer of cells (Cutter 1992). The pathogen must possess effective lytic enzyme to breakthe defence structure imparted by the plants. Basically, Phytophthora sp. are cut woundpathogens, lacking cell wall degrading enzymes in ample level. The associative bacteriapossessed an effective set of cell wall degrading enzymes which might have helpedP. palmivora in disease induction. The extra cellular enzyme profile detected the pres-ence of amylase, cellulase and pectinase in cassava extract. But the isolate failed togrow profusely. The lytic enzyme produced included pectinase, amylase etc. Wheninoculated in pure form P. palmivora produced radial mycelium, which covered thetuber piece with out rotting smell.

In in vitro in planta assay, the virulence and relative growth vigour of P. palmivorain combination with associative bacteria was high compared with infection rate caused

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by P. palmivora alone. The combination attained full rot within 4 days from the date ofinoculation. The association showed the typical tuber rot symptom, characterised by fullrot with obnoxious odours, oozing out and watery appearance, whereas the P. palmivorarot was characterised by dry rot. The oozing of internal fluids, water-soaked appearanceand obnoxious odour, might be contributed by bacterial partner. However, when inocu-lated with bacteria alone, the infection rate was not severe. In cassava, soft rot symp-toms appeared in response to infection with foul estery odour, but not intense as in thecase of tuber rot by P. palmivora. The rot was more confined to cassava rind regionwith watery appearance. Sturz and Matheson (1996) demonstrated that communities ofendophyte bacteria within potato tubers affected tuber resistance to bacterial soft rotdisease in vivo.

However, the bacterial agents alone could not cause infection in an alarming rate.P. palmivora inoculated tuber showed a dry rot confined to the rind region, with a delayin attaining full rot where as the associative bacteria associated P. palmivora infectionshowed classic symptoms of cassava tuber rot, with appearance of dark round, irregularshaped, watery lesions, oozing of internal fluids and shrivelling of the tubers withcharacteristic obnoxious odour and rot.

So, in this context, the associative bacterium found in connection with P. palmivoratuber rot incidence in Kerala assumes importance in disease aetiology. This associationmight be the reason for the endemic nature of tuber rot. There are several reportsstamping the role of secondary invaders and associative bacteria in pathogenesis offungus sp. (Afano and Collmer 1996). As our understanding of associative bacteria con-tinues to grow, the potential to capitalise on the unique characteristics and close associa-tion with tubers also grow. Further study in this area is necessary for progress indeveloping control measure against tuber rot of cassava. To conclude, the variety SreePathmanabha can be used as tuber rot resistant line in high tuber rot infested areas,which would alleviate to a certain extent the problem which the farmers face owing tothe endemicity of the disease. We are now assessing combined action of P. palmivoraand associative bacteria in field conditions to prove the endemic effect of the combina-tion in exposed environmental conditions. This would, we are certain, will bring upadditional information, which may lead to the development of effective controlmeasures to alleviate the endemicity induced by tuber rot to cassava.

AcknowledgementsThe funding provided for conducting this research work by the Indian Council of AgriculturalResearch, New Delhi, is gratefully acknowledged. The authors thank the Director, Central TuberCrops Research Institute, Thiruvananthapuram, for providing infrastructure facilities.

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