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Evaluation of fungal epiphytes isolated from banana fruit surfaces forbiocontrol of banana crown rot disease

Dionisio G. Alvindia a,�, Keiko T. Natsuaki b

a Bureau of Postharvest Research and Extension (BPRE), CLSU Compound 3120, Science City of Munoz, Nueva Ecija, Philippinesb Department of International Agricultural Development, Faculty of International Agricultural Studies, Tokyo University of Agriculture, 1-1-1 Sakuragaoka,

Setagaya-ku 156-8502, Tokyo, Japan

a r t i c l e i n f o

Article history:

Received 6 October 2007

Received in revised form

25 February 2008

Accepted 27 February 2008

Keywords:

Native fungal epiphytes

Banana crown rot-causing pathogens

Clonostachys byssicola

Curvularia pallescens

Penicillium oxalicum

Trichoderma harzianum

a b s t r a c t

Fungi isolated from the surface of banana fruits were evaluated for in vitro antagonism towards

Lasiodiplodia theobromae. Thirteen fungi exhibiting pronounced growth inhibition of test pathogens

were further tested for antibiosis against Thielaviopsis paradoxa, Colletotrichum musae, and Fusarium

verticillioides. Clonostachys byssicola, Curvularia pallescens, Penicillium oxalicum, and Trichoderma

harzianum were antagonistic to all test pathogens. Inhibition by C. pallescens and P. oxalicum to

pathogens was at a distance, while C. byssicola and T. harzianum directly parasitized and killed the

pathogens. The metabolites of C. byssicola, C. pallescens, and T. harzianum significantly affected the

mycelial growth and conidial germination of the pathogens. In the artificial inoculation study, the

antagonists survived and colonized banana fruits after 3 d. Interfungal parasitic relationship was

observed between the antagonist and pathogen on artificial media and natural substrate. Postharvest

application in the packing house showed that the incidence of crown rot in antagonist-treated banana

was significantly lower when compared to fungicide and untreated control fruits.

& 2008 Elsevier Ltd. All rights reserved.

1. Introduction

In the Philippines, some banana fruits produced by small-scalefarmers without agricultural pesticide application during produc-tion and postharvest operations (hereafter referred to as ‘‘non-chemical bananas’’) are exported to Japan. Japanese consumersprefer non-chemical bananas from the Philippines because oftheir pesticide-free status. However, non-chemical bananas havebeen reaching the Japanese market with impaired quality due topostharvest diseases, particularly crown rot, due to the absence ofpesticide treatments and the long interval (19–21 d) from harvestto market (Alvindia et al., 2000b). Crown rot, the most importantpostharvest disease of banana, is a syndrome caused by severalfungi including Lasiodiplodia theobromae (Ogawa, 1970; Johansonand Blazquez, 1992), Colletotrichum musae (Finlay and Brown,1993), Thielaviopsis paradoxa (Alvindia et al., 2002), and a complexof Fusarium spp. (Hirata and Kimishima, 1997; Hirata et al., 2001;Alvindia et al., 2000a; Jimenez et al., 1993; Knight et al., 1977).These fungi infect the crown through fresh wounds created aftertrimming the crown of the banana hand into a crescent shape.However, disease symptoms only develop after harvest, usually

during fruit ripening. Crown rot is controlled commercially by apostharvest treatment which involves submerging hands orclusters of banana in fungicide solutions.

Quality improvement of non-chemical bananas from thePhilippines is an urgent concern to sustain the exportability ofthe product and compete with good-quality non-chemicalbananas from other countries saturating Japanese markets.Coating banana crowns with paraffin and the use of modifiedatmosphere packing are some interventions by other countries topreserve the quality of non-chemical bananas bound for Japanesemarkets. The control of postharvest diseases of non-chemicalbananas requires non-chemical intervention.

Considerable efforts have been made on biological control asan alternative to chemicals to control postharvest diseases.Biological control through the use of antagonistic microorganismshas recently emerged as a viable disease management strategy(Andrews, 1992; Janisiewicz et al., 1994; Wilson and Wisniewski,1989). It is a promising option for the control of postharvestdiseases of fruits and vegetables (Chalutz, et al., 1991; Chalutz andWilson, 1990; Hong et al., 1998; Janisiewicz et al., 1994; Korsten etal., 1995 and 1997; Mc Laughlin et al., 1992; Mortuza and Ilag,1999; Pusey and Wilson, 1984; Smilanick and Dennis-Arrue, 1992;Wilson and Wisniewski, 1989). The most viable approach toutilize antagonistic microorganisms to control postharvest dis-eases is the promotion and management of natural antagonists

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0261-2194/$ - see front matter & 2008 Elsevier Ltd. All rights reserved.

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� Corresponding author.

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that already exist on and are well adapted to the target plantsurfaces. The potential for resident microorganisms to be aneffective biological control agent depends on their ability tocolonize food sources in plants without damaging the cells(Janisiewicz et al., 1994). In vitro evaluation of saprophyticmycoparasites Gliocladium spp., Gliomatrix murorum, and Paecilo-

myces spp. from dry banana leaf trash, dry flower residues, greenbanana leaves, and green peel, as potential biocontrol agent, hasbeen reported to suppress crown rot complex pathogens C. musae,L. theobromae, and Fusarium moniliforme (Krauss et al., 1998). Inthe present investigation, however, we evaluated the antagonisticpotential of fungal epiphytes isolated from the banana fructoplaneagainst banana postharvest pathogens and crown rot disease.Moreover, the interparasitic fungal relationship of the antagonistwith the pathogen, mechanisms of combat, survival, and coloni-zation of natural habitats were elucidated.

2. Materials and methods

2.1. Screening and selection of candidate antagonistic native

fungal epiphytes

Fungi on the surface of developing banana fruits were collectedin the Philippines each week after flowering until the 11th week,during the rainy (August–October) and dry (January–March)seasons of 1998–2001 (Alvindia et al., 2006). Sterilized, water-wetted cotton balls (20 mm diameter) were used to swab thesurface of the fruits using sterilized tweezers. The swabs werethen placed in 100 ml distilled water and stirred thoroughly.Thereafter, 1 ml was pipetted from the solution and plated ontodichloran rose bengal agar, dichloran-18 glycerol agar and potatodextrose agar (PDA). Ten plates were used for each medium andincubated for 3–5 d at 25 1C. Fungi on the surface of matured fruitsimported to Japan from the Philippines were collected in Japanweekly from December 1997 to July 1998 by tissue and directisolation methods (Alvindia et al., 2000a). Tissue isolation wasapplied at the early stage when the disease symptoms on thefruits could be clearly distinguished from each other. Tissue piecesmeasuring 1 cm2 taken from the disease symptom were placed ina meshed metal container under running tap water for 5 min,blotted dry on sterile filter paper, and plated on water agar.Meanwhile, the direct isolation method was plating fungalmycelia, spores, and fruiting bodies picked from the banana fruitsurface using a sterile needle onto PDA plates. Plates wereincubated for 3–5 d at 25 1C. All fungi isolated from the bananafructoplane were evaluated for in vitro antagonism toward L.

theobromae, by the dual incubation method (Fokkema, 1978).Fungi with more than 25% growth inhibition (GI) toward L.

theobromae were further tested for antibiosis to T. paradoxa, C.

musae, and Fusarium verticillioides, the most active crown rotdisease-causing pathogens of non-chemical banana (Alvindia etal., 2002). Three mycelial plugs of each antagonistic fungus wereseeded equidistantly 1 cm from the plate periphery, while amycelial plug of the test pathogen was placed off-center on PDAplates. Mycelial plugs 4 mm in diameter were cut from the edge of7-d-old fungal colonies maintained on PDA+5% malt extract (ME).Plates inoculated only with test pathogen served as controls.Plates were incubated at 25 1C. The experiment was repeatedtwice with three replications of each treatment. Percent GI wasdetermined after 14 d using the formula described in Korsten et al.(1995): Kr�r1/kr�100 ¼ GI, where Kr represents the distance(measured in mm) of fungal growth from the point of inoculationto the colony margin of the control plates, r1 the distance of fungalgrowth margin in the direction of the antagonist, and the GI thepercent inhibition growth. Percent GI was categorized on a scale

from 0 to 4, where 0 ¼ no GI, 1 ¼ 1–25% GI, 2 ¼ 26–50% GI,3 ¼ 51–75%, and 4 ¼ 76–100% GI. Fungi that inhibited growth offour pathogens by more than 25% were selected as candidatebiocontrol agents for evaluation and optimization studies.

2.2. Interfungal parasitic relationship

Sterile glass slides were coated with a layer of PDA sufficientlythick to allow the fungi to grow but thin enough to enablemicroscopic examination. Each antagonistic fungus was placed onone end of the glass slide and the pathogen on the other andincubated for 14 d at 25 1C. After microscopic observations, fiveagar pieces (0.5 cm2) were excised from the agar where theantagonist and pathogen made contact. The agar pieces wereplated on PDA and incubated for 7 d at 25 1C. Fungal growth fromthe agar pieces was identified.

Matured banana fruit, yellow-green in color, were rinsed withsterile water and blotted dry. One side of a fruit was marked withthree squares (1�1 cm) well spaced from each other with a blackwaterproof pen. The first square received a 200 ml suspension ofantagonist spores (Trichoderma harzianum), the second a suspen-sion of the pathogen (C. musae), and in the third, a suspension ofan antagonist and pathogen mixture. This treatment was repeatedon other fruits with different antagonists (Clonostachys byssicola

and Curvularia pallescens) and the antagonist–pathogen mixture.The treatments were replicated in three fruits. The sporesuspension of antagonist and pathogen was calculated at106 ml�1. Fruits were kept in plastic boxes sprayed with sterilewater at 25 1C. Fruit tissue (36 mm2) was taken from the squareafter 3, 4, and 7 d for scanning electron microscope (SEM)observations. Samples were fixed for 24 h in 2% glutaraldehyde,rinsed in phosphate buffer, post-fixed in 2% OsO4 for 5 h, anddehydrated for 15 min in a series of ethanol solutions: 50%, 70%,80%, 90%, 95%, and 100%. Samples were immersed overnight inmethyl butyl acetate, dried in a Hitachi critical point dryer withCO2 as the transitional fluid, and mounted on aluminum stubs.Sample tissues were gold coated using a Hitachi E-102 ion sputterand examined with a SEM Hitachi S-400.

2.3. Production of metabolites

The effect of volatile metabolites on pathogens was donefollowing the procedure of Dennis and Webster (1971b). Twobottoms of PDA plates that were individually inoculated with adisk of pathogen and antagonist were joined together and firmlyattached by tape. The control sets did not contain the antagonist.

The effect of non-volatile metabolites on the pathogen wasevaluated by the method described by Dennis and Webster(1971a). A sterilized cellophane disk was aseptically laid on thesurface of PDA plate before a 4 mm diameter mycelial agar plugtaken from the edge of a young culture antagonist was seeded inthe center of the plate. The plate was incubated for 3 d and thecellophane containing the antagonist growth was removed. Onthe same medium, a disk of the pathogen was seeded. The controlplate had a pathogen growing similarly on a PDA plate previouslycovered with a cellophane disk without an antagonist.

The effect of direct-diffusible metabolites by antagonist topathogen was determined by transferring a 7-d-old mycelial plug(4 mm diameter) of the antagonist to the center of PDA platespreviously left overnight to allow excess water to evaporate. Theuninoculated PDA plate was placed over the antagonist-inoculatedPDA plate and attached firmly by tape. The plates were untapedafter 3 d. A mycelial plug (4 mm) of a pathogen was inoculatedat the center of the previously uninoculated plate. The controlsets did not contain the antagonist, but had the previously

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uninoculated plate placed over the no-antagonist plate andattached firmly by the tape.

The studies of volatile, non-volatile, and direct-diffusiblemetabolites were conducted in three replicates repeated twice.The cultures were incubated at 25 1C. Growth rates were recordeddaily by measuring colony diameter, and inhibition percentagewas obtained using the formula 1% ¼ [(C2�C1)/C2)]�100 (Eding-ton et al., 1971).

2.4. Effect of culture filtrate on conidial germination and mycelial

growth of pathogens

Mycelial disks of the fungal antagonist were inoculated in a250 ml Erlenmeyer flask containing 100 ml of nutrient broth(NB)+5% ME. The flask was incubated on a rotary shaker (70 rpm)at 25 1C for 15 d. The mycelial growth (MG) was separated usingsterile gauze and the filtrate was centrifuged for 25 min at13,000 rpm. The supernatant was filtered through a 0.45 mm filterunit. Ten milliliter of the filtrate was transferred to a sterile testtube and inoculated with a mycelial disk of the pathogen andincubated for 15 d at 25 1C. The control tube contained fresh NBinoculated with the pathogen mycelial disk. MG was categorizedon a scale from 0 to 2, where 0 ¼ no MG, 1 ¼ growth limitedaround mycelial disk, and 2 ¼ mycelia overgrows liquid medium.The experiment was repeated twice with three replications.

For the spore germination test, a 45 mm diameter Petri platewith 5 ml of the culture filtrate was inoculated with 100 ml ofpathogen spore suspension (106 ml�1) and incubated for 48 h at25 1C. Control plates received fresh NB instead of culture filtrate.Percent germination was calculated under the microscope bydividing the number of germinated conidia over the total numberof conidia counted. The experiment was repeated twice with threereplications.

2.5. Postharvest application of the antagonist in the packing house

Cavendish banana fruits (var. Bungulan) with 80% maturitywere obtained from non-chemical banana farmers from NuevaViscaya in Northern Luzon Island, Philippines. The fruits were de-handed and washed in water before the crown was trimmed.Thereafter, five banana hands of uniform size were selected andsprayed with each antagonist spore solution (106 spore ml�l). Thefruits were air-dried and stored in an incubator at 25 1C with90–95% RH. Crown rot index (CRI) was measured after 13 d and20 d using the index described by Alvindia et al. (2004) (Fig. 1).Fungicide (Maneb 2 ml l�1) and untreated control (water only)were provided as control checks. The experiment was repeatedtwice with three replications.

2.6. Statistical analysis

All of the comparisons of means were subjected to analysis ofvariance and the significant differences among treatments weredetermined with a least significant difference separation testusing Microsoft Corp. STATISTICAs Ver. 6.

3. Results

The identity and in vitro inhibitory effect of 13 fungal epiphytesagainst banana crown rot-causing pathogens are presented inTable 1. C. byssicola, C. pallescens, Penicillium oxalicum, andT. harzianum were the only species capable of inhibiting thegrowth of all test pathogens. Of these, C. byssicola and T. harzianum

were the most inhibitory toward all pathogens tested having the

highest mean GI category of 3.0, followed by C. pallescens (2.75)and P. oxalicum (2.25). The mode of action of C. pallescens andP. oxalicum to pathogens was mediated at a distance, while that ofC. byssicola and T. harzianum was by direct contact. Except for dualincubation, no further testing and optimization studies wereconducted on P. oxalicum because of the safety concern when thefungus is used as a biofungicide (Lugauskas, 2005). Lightmicroscopy and SEM observations of antagonist–pathogen inter-fungal parasitic relationship showed C. byssicola (Fig. 2a) andT. harzianum (Fig. 2b) coiled around the pathogen on agar andbanana fruit. Conidia of C. byssicola and T. harzianum germinatedand developed appressoria on the spores of C. musae (Fig. 3).Moreover, this study established that C. byssicola and T. harzianum

were parasitic nectrotrophs of banana fruits. The agar piecesexcised from the area where the antagonist and pathogengrew in contact for 14 d yielded only the antagonist whentransferred to fresh medium. No direct parasitism was observedby C. pallescens on the pathogen both on agar and banana fruit bylight microscopy and SEM observations. C. byssicola, C. pallescens,and T. harzianum colonized banana fruit 7 d after artificialinoculation.

The volatiles of T. harzianum inhibited the MG of L. theobromae,T. paradoxa, C. musae, and F. verticillioides by 49%, 65%, 90%, and43%, respectively (Table 2). However, the culture filtrates of theantagonist had varying effects on conidial germination and MG ofthe pathogens (Table 3). The culture filtrate of C. pallescens totallyinhibited conidial germination of the test pathogens. AlthoughC. byssicola and T. harzianum completely controlled conidialgermination of C. musae and T. paradoxa, only partial inhibitionwas noted for F. verticillioides. In the culture filtrate, conidia of thepathogens swelled abnormally with subsequent bulb formation.

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Fig. 1. Severity index of crown rot of banana hand (Alvindia et al., 2004). Crown rot

index (CRI) was categorized on a scale from 0 to 7, where 0 ¼ no discoloration or

mycelial growth on the crown, 1 ¼ discoloration or mycelial growth limited on

surface of the cut crown, 2 ¼ discoloration or mycelial growth less than 10% of the

crown area, 3 ¼ 11–40% discoloration or mycelial growth on crown area,

4 ¼ 41–70% discoloration or mycelial growth on crown area, 5 ¼ 71–100%

discoloration or mycelial growth on crown area, 6 ¼ discoloration or mycelial

growth advanced to finger stalks, 7 ¼ finger-stalk rot occurs causing the fingers to

drop-off when handled.

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The growth of F. verticillioides from germinating conidia wasabnormal with short, swollen mycelia.

The MG of F. verticillioides was not affected by the culturefiltrate of the antagonists tested. Meanwhile, the MG of otherpathogens was varyingly sensitive to the culture filtrate of theantagonist. The filtrate of C. pallescens completely controlled thegrowth of T. paradoxa and C. musae, but L. theobromae was onlypartly inhibited. The culture filtrate of T. harzianum entirelycontrolled the growth of T. paradoxa with a partial effect onL. theobromae, but was not successful against C. musae andF. verticillioides. In contrast, the filtrate of C. byssicola had no effecton the MG of any of the test pathogens.

Postharvest application of the antagonist in the packing houseshowed that C. byssicola, C. pallescens, and T. harzianum were thebest treatments after 13 d with CRI ranging from 1.4 to 1.8. Thefungicide (Maneb)-treated banana fruits were statistically similarwhen compared with the untreated controls with CRI’s of 3.0 and2.4, respectively (Table 4). After 20 d, CRI of C. byssicola- andT. harzianum-treated fruits was maintained at 1.4, whereas thoseof C. pallescens increased to 2.6. The CRI of fungicide treated anduntreated control fruits were both recorded as 4.4 after 20 d.C. byssicola and T. harzianum were the best treatments with 53%and 68% crown rot reduction after 13 d and 20 d, respectively,followed by C. pallescens with 50% after 13 d and 41% after 20 d.

4. Discussion

Control of banana crown rot with native fungal epiphytes couldbe advantageous compared to saprophytic mycoparasites. A singlespecies of native fungal epiphyte resulted in significant control ofrot in the packing house. The present investigation contrasts witha related study, signifying that though saprophytic mycoparasiteshad a reasonable number of desirable traits, no single strainexhibited consistent biocontrol activity (Krauss et al., 1998).Krauss reported the saprophytic mycoparasites Gliocladium spp.,G. murorum, and Paecilomyces spp. from dry banana leaf trash, dryflower residues, green banana leaves, and green peel as biocontrolagents of crown rot pathogens C. musae, L. theobromae, andF. moniliforme. Some of the fungi collected on banana fructoplanebelonged to genera already used as commercial biocontrol agentssuch as Clonostachys (previously Gliocladium), Trichoderma, and

Verticillium. The isolation of antagonsitic fungi from the bananafruit surface belonging to genus Acremonium, Aspergillus, Curvu-

laria, Penicillium, and Plectosporium was first reported in thisstudy. Except for a higher population of fungi on bananafructoplane during rainy season, no difference was notedbetween fungi collected during rainy and dry periods (Alvindiaet al., 2006).

An important attribute of a successful biocontrol agent is theability to efficiently control a range of pathogens. Four antag-onistic fungal epiphytes, namely, C. byssicola, C. pallescens,P. oxalicum, and T. harzianum conformed to this prerequisite bybeing generally effective against the various banana crown rot-causing pathogens in vitro. Whilst P. oxalicum had the qualities of agood biocontrol agent, further studies were terminated concern-ing safety of the organism for practical use and application(Lugauskas, 2005).

The susceptibility of pathogens to mycoparasitism has varyingdegrees of sensitivity (Elad et al., 1985; Foley and Deacon, 1986).The four most important active crown rot-causing pathogens ofnon-chemical bananas responded differently towards antagonists.Fortunately, C. musae the primary pathogen of the diseasecomplex was controlled by native antagonists in artificial andnatural substrates. C. musae drives the progress of the rot frominitially very low inoculum levels, while other pathogens requiremuch higher inoculum levels to elicit symptoms and are regardedas secondary invaders (Finlay and Brown, 1993). Nonetheless,other important pathogens of the disease complex were also bestcontrolled by the antagonists. It should, therefore, be relativelyeasy to target the important crown rot-causing pathogens by theantagonists in biocontrol of crown rot in the packing house.

Knowledge of the mechanism of antagonism may be used toimprove biocontrol (Spurr, 1981). In addition, an antagonist mayhave more than one mode of action. Exploiting all modes of actionwill increase the efficacy of the biocontrol agent. C. byssicola andT. harzianum antagonized pathogens by direct parasitism andproduction of extracellular antibiotics. Artificial inoculationrevealed that C. byssicola and T. harzianum can adapt easily tothe natural habitat, i.e., banana fruits, hence, survival andphysiological activity required for biocontrol is possible. Theefficacy of direct parasitism of antagonists to pathogens onbanana fruit was demonstrated when they are appliedas postharvest biocontrol agents. This investigation further

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Table 1Identity and effect of fungi isolated from the surface of banana fruits on in vitro growth of four crown rot-causing pathogens of banana

Fungal taxon Isolate code Growth inhibition (GI) categorya Mean GI

category

Pathogens

inhibited

Lasiodiplodia

theobromae

Thielaviopsis

paradoxa

Colletotrichum

musae

Fusarium

verticillioides

Acremonium strictum DGA005 1 0 1 0 0.5 2

Aspergillus caespitosus DGA006 2 0 0 1 0.8 2

Cylindrocarpon spp. 1 DGA007 1 0 0 1 0.5 2

Cylindrocarpon spp. 2 DGA008 1 0 1 1 0.8 3

Curvularia pallescens DGA003 2 4 3 2 2.75 4

Clonostachys byssicola DGA002 4 2 3 3 3.0 4

Penicillium oxalicum DGA004 2 2 3 2 2.25 4

Plectosporium

tabacinum

DGA009 1 1 1 0 0.7 3

Trichoderma

harzianum

DGA001 3 3 3 3 3.0 4

Ulocladium atrum DGA010 1 0 0 0 0.3 1

Verticillium tricorpus DGA011 1 1 1 0 0.8 3

Verticillium spp. 1 DGA012 1 1 1 0 0.8 3

Verticillium spp. 2 DGA013 1 1 1 0 0.8 3

a Percent growth inhibition was determined 14 d by using the formula of Skidmore (1976). Values were categorized on a scale from 0 to 4, where 0 ¼ no growth

inhibition, 1 ¼1–25%, 2 ¼ 26–50%, 3 ¼ 51–75%, and 4 ¼ 76–100%.

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elucidated the advantage of native fungal antagonists isolatedfrom the same habitat as that in which they are effective forbiocontrol. Likewise, though C. pallescens survived on the naturalsubstrate, no evidence of direct parasitism toward pathogens wasfound, suggesting that antibiosis was the only mode of action ofthis antagonist.

Our study did not examine how antibiosis helped directparasitism in the biocontrol activities of antagonist in the naturalsubstrate. Taking into account the important effects of metabo-lites on growth of pathogens in vitro, however, antibiosis could bea significant mechanism of action, giving the antagonists acompetitive edge in the natural substrate. Packing house experi-ments revealed that bananas treated with C. byssicola andT. harzianum had a lower incidence of crown rot compared with

C. pallescens. However, though better control of banana crown rotwas achieved with antagonists with direct parasitism andantibiosis as modes of action, this latter phenomenon has yet tobe investigated. Nevertheless, with C. pallescens showing inferiorperformance in the packing house, the significance of antibiosis asa mechanism involved in biocontrol on natural substrates iscontroversial (Fravel, 1988).

Future experiments should investigate the compatibility of amixed inoculum combining all the assets of the antagonists whichincludes saprophytic mycoparasites as well as epiphytic fungi asbiocontrol agents. Other future activities include the applicationof the antagonists in the field and further packing houseevaluations. Equally important is the commercialization of theproduct and examination of whether metabolites produced by the

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Fig. 2. Hyphal coiling of Clonostachys byssicola (pointed with arrow) on mycelial growth of Thielaviopsis paradoxa on PDA as seen under a (a) light microscope and a

(b) scanning electron microscope.

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Fig. 3. Germinating spores of (a) Trichoderma harzianum (Th) and (b) Clonostachys byssicola (Cb) showing germ tubes and appressoria on conidia of Colletotrichum musae

(Cm) 3 d after artificial co-inoculation on banana fruit surface as seen under scanning electron microscope.

Table 2Effect of metabolites produced by antagonists on mycelial growth of various pathogens

Pathogen Clonostachys byssicola Curvularia pallescens Trichoderma harzianum

V* NV** DD*** V* NV** DD*** V* NV** DD***

Lasiodiplodia theobromae � � � � � � 49% � �

Thielaviopsis paradoxa � � � � � � 65% � �

Colletotrichum musae � � � � � � 90% � �

Fusarium verticillioides � � � � � � 43% � �

V*—volatile metabolites, NV**—non-volatile metabolites, DD***—direct-diffusible metabolites.

Negative sign (�) indicates no effect on mycelial growth of pathogen.

Growth of L. theobromae and T. paradoxa was evaluated after 3 d, while those of C. musae and F. verticillioides was done after 7 d.

Value represents the percentage inhibition of a pathogen mycelial growth calculated using the formula 1% ¼ [(C2�C1)/C2)]�100 (Edington et al., 1971).

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antagonists are safe for human consumption. We deposited theisolates of C. byssicola DGA002 (MAFF 240262), C. pallescens

DGA003 (MAFF 240263), and T. harzianum DGA001 (MAFF240261) in Genebank, National Institute of AgrobiologicalSciences, Tsukuba, Japan, for safekeeping and as a source ofrelated future activities involving the biocontrol agents.

Acknowledgment

The authors gratefully acknowledge Japan Society for thePromotion of Science (JSPS) for granting research support todevelop non-chemical alternatives on postharvest disease man-agement of banana. Also, we thank Dr. Yukio Yaguchi, TokyoUniversity of Agriculture for the technical assistance duringelectron microscopy.

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Table 3Conidial germination and mycelial growth of various pathogens in antagonist culture filtrate

Culture filtrate Lasiodiplodia theobromae Thielaviopsis paradoxa Colletotrichum musae Fusarium verticillioides

%RCGa MGb %RCGa MGb %RCGa MGb %RCGa MGb

Clonostachys byssicola Nd 2 100 2 100 2 53 2

Curvularia pallescens Nd 1 100 0 100 0 100 2

Trichoderma harzianum Nd 1 100 0 100 2 82 2

Nd—not determined due to poor sporulation of L. theobromae in artificial media.a Reduction in conidial germination (%RCG) ¼ CGc�CGt/CGc�100; CGc—conidial germination of control, CGt—conidial germination of treated. Conidial germination

(%CG) was calculated by dividing the number of germinated conidia over the total number of conidia counted after 48 h at 25 1C.b Mycelial growth (MG) was categorized on a scale from 0 to 2, where 0 ¼ no mycelial growth, 1 ¼ growth limited around mycelial disc, and 2 ¼ mycelia overgrows

liquid medium, after 15 days at 25 1C.

Table 4Crown rot index and crown rot reduction (%) in treated and untreated bananas 13 d

and 20 d after treatment

Treatment Crown rot index� Crown rot reduction (%)

13 d 20 d 13 d 20 d

Clonostachys byssicola 1.4a 1.4a 53 68

Curvularia pallescens 1.8a 2.6b 50 41

Trichoderma harzianum 1.4a 1.4a 53 68

Maneb 3.0b 4.4c 0 0

Untreated control (water only) 2.4b 4.4c 0 0

Values with the same letter in a column are not significantly different according to

the LSD test (Pp0.01).

Crown rot reduction ð%Þ

¼crown rot index ðuntreated fruitsÞ � crown rot index ðtreated fruitsÞ

crown rot index ðuntreated fruitsÞ� 100

� See Fig. 1 for visual determination of index.

D.G. Alvindia, K.T. Natsuaki / Crop Protection 27 (2008) 1200–12071206

Author's personal copy

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