PertanikaJ. Trop. Agric. Sd. 20(1): 35-41(1995) ISSN: 0126^6128© Universiti Putra Malaysia Press

Antagonistic Effect of Malaysian Isolates of Trichoderma harzianum andGliodadium virens on Sclerotium rolfsii

J.JOMDUANG1 and M SARIAH2

1 Agricultural Research and Trainning CentreP.O. Box 89, A. Maung

52000 Lampang, Thailand

department of Plant ProtectionFaculty of Agriculture

Universiti Putra Malaysia43400 UPM. Selangor, Malaysia

Keywords: antagonism, chilli, Trichoderma harzianum, Gliodadium virens, Sclerotium rolfsii

ABSTRAK

Trichoderma harzianum dan Gliodadium virens yang dipencilkan daripada sklerotia yang diparasitdinilai potensi antagonis mereka terhadap Sclerotium rolfsii. Berdasarkan kepada mekanisma antibiosis invitro; divikultur dan ujian degradasi koloni, interaksi hifa, antibiosis dan parasitisma sklerotia. kedua duaantagonis didapati berkesan terhadap S. rolfsii. Walaubagaimanapun, G. virens didapati lebeh berkesandaripada T. harzianum dalam dxvi-kultur dan ujian degradasi koloni. Di bawah SEM, keduadua antagonismengkoloni, menembusi dan mengeluarkan spora didalam sklerotia patogen yang diuji, mencadangkanparasitisma keatas sklerotia sebagai mekanisma utama yang terlibat. T. harzianum and G. virens adalahserasi didalam aktiviti antibiosisnya. Disamping itu, mereka boleh hidup bersama dan sinergis apabila di ujididalam dxoikultur.

ABSTRACT

Trichoderma harzianum and Gliodadium virens isolated from parasitized sclerotia were evaluated for theirantagonistic potential against Sclerotium rolfsii. Based on the mechanisms of antagonism in vitro, dual cultureand colony degradation tests, hyphal interaction, antibiosis and parasitism of sclerotia, both antagonists werefound to be effective against S. rolfsii. However, G. virens was more effective than T. harzianum in dualculture and colony degradation tests. Under SEM, both antagonists colonized, penetrated and sporulated insidethe sclerotia of the test pathogen, suggesting parasitism of sclerotia could be the principal mechanism involved.T. harzianum and G. virens were comparable in their antibiosis activity. In addition, they can coexist togetherand are synergistic in activity when tested simultaneously in dual culture.

INTRODUCTION

Foot rot of chilli {Capsicum annuum L. local var.LangkapJ caused by a soilborne fungus Sclerotiumrolfsii Sacc. is prevalent in almost all chilli grow-ing areas in Malaysia. Practical and usable meth-ods of control have yet to be developed. The useof fungicides and crop rotation is limited due tothe high cost, the wide host range and longpersistence of sclerotia in the soil. Flooding ofsoils has been reported to be effective in reduc-ing sclerotial viability (Sariah 1995). However,

difficulties can arise from climatic and regionalrestrictions, land use and management.

Fungi, bacteria and actinomycetes have beenshown to exhibit antagonistic effects on S. rolfsii(Brathwaite and Cunningham 1982; Punja 1985;Chamswarng and Sangkaha 1988). The moststudied and effective are Trichoderma harzianumRifai (Wells et al 1972; Elad et al 1980; Henisand Papavizas 1983; Chamswarng 1992) andGliodadium virens Miller, Giddens and Foster(Papavizas and Lewis 1989; Ristaino et al 1991).

J. JOMDUANG AND M. SARIAH

These two antagonistic fungi attack sclerotia ofS. rolfsii causing failure in germination (Henis etal 1983; Henis and Papavizas 1983; Papavizasand Collins 1990), hence reducing disease inci-dence. However, different isolates or strains ofT. harzianum and G. virens were found toparasitize sclerotia of 5. rolfsii with varying levelsof efficiency (Henis et c' 1983; Sreenivasaprasadand Manibhushanrao 1990) and they tend to bemore crop and soil-specific.

The most important factor governing theactivities of a fungal biocontrol agent in the soilis the qualitative and quantitative make-up ofthe soil microflora and the ability of the antago-nists to maintain themselves in the soil andplant environment. Interspecific and intraspecificcompetition between isolates of Trichoderma havebeen reported (Marois and Locke 1985; Vajna1985). Hence the objective of this research wasto isolate and evaluate the effectiveness of indig-enous isolates of Trichoderma and Gliocladiumfrom the chilli rhizosphere for controlling5. rolfsii.

MATERIALS AND METHODS

Fungal Isolates

Trichoderma harzianum and Gliocladium virenswere isolated from parasitized sclerotia of 5.rolfsii collected from Malaysian soils cultivatedwith chilli. Isolation was carried out by directlyplating the sclerotia on potato dextrose agar(PDA) plates without surface sterilization. Theplates were incubated at 28°C for 3-4 days forthe antagonists to recover. T harzianum and G.virens recovered on PDA were reisolated to freshPDA for pure culture. S. rolfsii was isolated fromnaturally infected chilli plants. Cultures weremultiplied and maintained on potato dextroseagar at 28°C.

Antagonism in Culture

Antagonism between the antagonists and thetest pathogen was evaluated by the dual culturetechnique and the colony degradation tests. Alltests were carried out at 28°C.

Pairings were carried out on PDA in fivereplicated petri plates. Antagonistic activity wasassessed four days after incubation by measur-ing the radius of S. rolfsii colony in the directiontowards the antagonist colony and transformingthe data into percentage of inhibition of radialgrowth in relation to radius of the uninhibitedS. rolfsii colony in the control plate (Royce and

Ries 1978). The number of days for the antago-nist to overgrow and degrade the whole colonyof 5. rolfsii was also recorded. The number ofsclerotia of S. rolfsii colonized by the antagonistswhich failed to germinate when transferred tofresh PDA, was counted twelve days afterincubation.

From the zone of interaction or overgrowthin dual culture, mycelial fragments were takenperiodically, stained with cotton blue inlactophenol and observed under the light andscanning microscope for hyphal interaction.

The colony degradation tests was assessedby inoculating 5-mm diameter agar discs takenfrom the edge of a 4-day-old PDA culture of theantagonists onto a fully grown culture of S.rolfsii. Each treatment was carried out on fivereplicated plates. The antagonistic activity wasexpressed as index of the lytic activity (zone ofclearing) of the antagonists on S. rolfsii. Thenumber of sclerotia colonized by the antagonistwas counted at twelve days after incubation.

Parasitism of Sclerotia

Sclerotia were placed on the edge of the colonyof a 2-day-old PDA culture of each of the antago-nists. Periodic observations on the colonizationof the sclerotia by the antagonists were re-corded. Sclerotia were examined for their viabil-ity 24, 48, 72, 96 and 120 h after incubation byplating them on PDA after surface sterilizationwith 1% sodium hypochlorite for 5 min.

The parasitized sclerotia were also fixed in3% glutaraldehyde prepared in 0.1 M phosphatebuffer, pH 7.2. Samples were dehydrated in agraded ethanol series followed by methylbenzoate and methylbenzoate + celloidin, em-bedded in paraplast and sectioned to 5-10|xmthickness. Paraffin sections of the sclerotia werethen processed for scanning electron microscopyfollowing the technique of Gaudet and Kokko(1984) and examined under scanning electronmicroscope (JEOL JSM-35C).

Antibiosis

The ability of the antagonists to produce vola-tile and non-volatile inhibitors was studied ac-cording to the method of Dennis and Webster(1971a,b). Each plate of a 48-hr-old culture ofS. rolfsii was inverted over the plate of the an-tagonist, sealed and incubated at 28°C. The di-ameter of S. rolfsii colony was measured 7 daysafter inoculation and the antibiosis activity was

PERTANIKAJ. TROP, AGRIC. SCI. VOL. 20 NO. 1, 1997

ANTAGONISTIC EFFECT OF MALAYSIAN ISOLATES

calculated as the percentage of inhibition ofradial growth in relation to the average diam-eter of the 5. rolfsii colony in the control plateswhich were inverted over the PDA plates.

The effect of non-volatile inhibitors of theantagonists on the aerial growth of S, rolfsii wasdetermined using the culture filtrates of theantagonists. Filter-sterilized (0.45 |Lim cellulosenitrate membrane) culture filtrate of the an-tagonists grown in Richard's solution for 10days was amended with PDA. The pathogen wascentrally inoculated on 20% filtrate amendedPDA plates. Radial growth of the pathogen wasobserved and recorded 72 h after incubation at28°C. The experiments were conducted in acompletely randomized design with Five repli-cates and were repeated twice.

Compatibility Studies between T. harzianum and G.virens

Dual culture of T. harzianum and G. virens wascarried out on five replicated plates which wereincubated at room temperature. Simultaneousantagonistic effects of both the antagonistsagainst S. rolfsii were assessed by pairing thecultures on the same petri plate. Observationswere also made seven days after incubation.

RESULTSAntagonism in Culture

In dual culture, G. virens exhibited strongerantagonistic potential than T. harzianum againstS. rolfsii (Table 1). At four days after incuba-tion, G. virens showed 70.48% inhibition ofradial growth compared with 64.44% for T.harzianum. Sclerotia parasitized by the antago-nists were covered with conidiophores andconidia which were white when young andgreen when mature. However, the percentage ofcolonized sclerotia was not significantly differentbetween the two antagonists tested. Within sevendays, G. virens completely overgrew the colony ofS. rolfsii while T. harzianum was able to colonize5. rolfsii after eight days of co-inoculation.

Both antagonists actively lysed the 5. rolfsiicolony, causing disintegration of the mycelia,which resulted in clear zones. Similar effectswere observed in the colony degradation test.On microscopic observation, T. harzianum wasfound to have parasitized the pathogen's hyphaeby coiling and producing hooks and short hyphalbranches, which penetrated into the pathogen'shyphae. G. virens produced several short

TABLE 1Antagonistic effects of Trichoderma harzianum and

Gliocladium virens on Sclerotium rolfsii in dualculture and colony degradation tests

Antagonisticeffects

% inhibition ofradial growth% colonizedsclerotiaNumber of days

Dual culture

T harzi-anum

64.44a

98.30b

8to colonize colony

G. virens

70.48b

100b

7

Colonydegradation

T. harzi-anum

_

97.50*

11

G.vir-ens

_

100b

9

Means in the same row with different letters aresignificantly different (p<.01) using DMRT

branches which coiled compactly around thehyphae of 5. rolfsii causing it to become granu-lated and malformed. However, penetration intothe pathogen's hyphae was not observed.

Parasitism of Sclerotia

Sclerotia of S. rolfsii exposed to either T.harzianum or G. virens for 24 - 120 h failed togerminate. Colonies of T. harzianum and G.virens were recovered from the colonized sclerotiawhen plated onto fresh PDA and incubated for48 h at room temperature. Infected sclerotiawere soft and collapsed easily.

The mode of parasitism of T. harzianum andG. virens on the sclerotia of S. rolfsii examined byscanning electron microscopy showed there wasno difference in the mode of parasitism of thesclerotia by either T. harzianum or G. virens, bothof which produced profuse growth andsporulation on the sclerotia. Sections of theparasitized sclerotia showed antagonist hyphaepenetrating the sclerotia, parasitizing the looselyarranged hyphae of the medullary region result-ing in the maceration and disintegration of themedullary hyphae and the eventual disintegra-tion and destruction of the resting structures.Extensive mycelial growth and chlamydosporesproduction of the antagonists in the damagedmedulla region were detected (Plate 1).

Production of Volatile and Non-volatile InhibitorsT. harzianum and G. virens were able to producevolatile inhibitors that inhibited radial growthof S. rolfsii on PDA although the percentageinhibition was low (Table 2). Age of antagonists

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 NO. 1, 1997 37

J. JOMDUANG AND M. SARIAH

Plate 1. Scanning electron micrographs o/S. rolfsii sclerotia A. Healthy sclerotia; B. Sclerotia parasitized byT. harzianum;C. Sclerotia parasitized by G. virens; D. Chlamydospores o/G. virens in the medulla region of the parasitizedsclerotia (cu cuticle; r rind; c cortex; m medulla)

had a significant effect (p<.01) on their abilityto produce volatile inhibitors: the younger thecultures, the greater the ability to producevolatile inhibitors.

T. harzianum and G. virens also producednon-volatile inhibitors that inhibited the radialgrowth of S. rolfsii (Table 3). The ability toproduce non-volatile inhibitors by T. harzianumincreased with the age of the culture; in con-trast that of G. virens decreased.

Compatibility Studies between T. harzianum andG. virensThe two antagonists were found to have noinhibitory effect on each other in plate culture.There was no inhibition zone in the intermin-gled area of the two colonies seven days afterco-inoculation. Although G. virens grew fasterthan T. harzianum, thus occupying more surfacearea of PDA in the petri plates, there was noovergrowing of one antagonist over the other.The appearance of colonies of the antagonistswas the same as when grown separately.

Results from pairing cultures showed thatthe antagonistic effect of the two antagonistsagainst 5. rolfsii could occur simultaneously, caus-ing degradation and lysis similar to those previ-ously described. Observation four days after

incubation showed that radial growth of S. rolfsiiwas totally restricted within the intermingledarea and eventually the two antagonists grewover 5. rolfsii colonies.

DISCUSSIONDestruction of sclerotia of S. rolfsii will result inthe reduction of disease incidence and there-fore antagonists which actively parasitizesclerotia have greater potential to control dis-eases caused by S. rolfsii. Results from dualculture and colony degradation tests showedthat both G. virens and T. harzianum exhibitedparasitic activity on the sclerotia.. Sclerotia werecolonized, demonstrating mycoparasitism of theantagonistic isolates on S. rolfsii. Mycelia of S.rolfsii were lysed and disintegrated by T.harzianum, as seen by the clear zone that devel-oped in the colony degradation test and thepenetration of its hyphae under SEM. G. virensovergrew the colony of S. rolfsii and compactlycoiled around the hyphae, causing them tobecome granulated and malformed. Parasitizedsclerotia have conidia and conidiophores ofthe effective antagonists growing on them;hence, when plated on fresh PDA the sclerotiafailed to germinate. This observation has notbeen demonstrated before in the screening for

38 PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 NO. 1, 1997

ANTAGONISTIC EFFECT OF MALAYSIAN ISOLATES

TABLE 2Effect of volatile inhibitors produced by Trichoderma

harzianum and Gliocladium virens on radialgrowth of Sclerotium rolfsii

TABLE 3Effect of non-volatile inhibitors produced byTrichoderma harzianum and Gliocladium virens

on growth of Sclerotium rolfsii

Antagonists

Trichodermaharzianum

Gliocladiumvirens

Age of culture(days)

17 .

142128

17

142128

% inhibition ofradial growth

21.76b

19.07c

8.47e

7.30ef

6.23f

24.12a

7.35d

2.8682.47*2.56*

Antagonists

Trichodermaharzianum

Gliocladiumvirens

Means in the

Age of culture(days)

61017

61017

% inhibition ofradial growth

9.37d

11.50^12.8b

30.25*11.75^10.38cd

same column with different letters aresignificantly different (p<.01) usingj DMRT.

Means in the same column with different letters aresignificantly different (p<.01) using DMRT.

effective antagonists. Furthermore, the colonydegradation test was found to be simple, quickand reliable as the lysis activity could be seenand the percentage of colonized sclerotia helpedto distinguish the ability of mycoparasitismamong the test antagonists. It appears thatthere could be a correlation between ability ofthe antagonist to parasitize sclerotia, and itsability to suppress disease. However, this needsfurther testing under glasshouse and field con-ditions.

Suggested mechanisms of antagonism of T.harzianum and G. virens are antibiosis, lysis, com-petition and mycoparasitism (Cook and Baker1983; Chet 1987). The two antagonists parasitizeboth the hyphae and sclerotia of S. rolfsii Theirmechanisms of mycoparasitism were in accord-ance with the previous findings by Henis et al(1983), Sariah (1986) and Papavizas and Collins(1990), Penetration was by the production ofshort-branched hyphae in the case of T. harzianum,while G. virens tightly coiled around the patho-gen's hyphae. The medulla region of sclerotiaexposed to either T. harzianum or G. virens showedsevere damage, while the cortical region appearedto be intact, suggesting that the preferred site ofinfection was the medulla tissues. Chet et al.(1969) found that the resistance of sclerotia tobiological degradation was dependent upon themelanin-rich rind of the wall structure and theorganization of cells comprising the inner layersof the sclerotium.

The principal mechanism of antagonism isantibiosis through the production of cell-walldegrading enzymes or inhibitors by the antago-nists, which results in the mycelia and sclerotiaof S. rolfsii becoming lysed, macerated and disin-tegrating. Enzymes secreted by Trichoderma mostcommonly associated with biocontrol arechitinolytic enzymes, B-glucanases and cellulases(Chet 1987). Recent studies found that T.harzianum produced a variety of chitinolytic en-zymes including N-acetl-B-D-glucosaminidases,chitin 1,4-B-chitobiosidases and endochitinase(Harman et al 1993; Lorita et al 1993a,b; 1994).The ability of T. harzianum and G. virens toproduce volatile and non-volatile inhibitorswhich could inhibit growth of micro-organismshas been described by Dennis and Webster(1971a,b) and, recently, by Ghisalberti andSivasithamparam (1991). The antibiotics pro-duced by T. harzianum are 6-N-pentyl-2H-pyran-2-one, 6-N-pentenyl-2H-pyran-2-one, pyridone,anthraquinones, butenolides, isonitrin D and F,trichorzianines (Ghisalberti and Svasithamparam1991) and furanone (Ordentlich et al 1992). G.virens was also shown to produce several antibi-otics such as gliotoxin, gliovirin, gliocladic acid,heptelidic acid, viridin, viridiol and valinotricin(Taylor 1986).

In addition, dual culture pl<» igs showedthat T. harzianum and G. virens are compatibleand complementary to each other and there-fore, it is possible to mix these two antagonistsfor use in biological control of plant diseasescaused by S. rolfsii

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 NO. 1, 1997 39

J. JOMDUANG AND M. SARIAH

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