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Crop Protection 29 (2010) 1452e1459

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Crop Protection

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Mycoparasitic Trichoderma viride as a biocontrol agent againstFusarium oxysporum f. sp. adzuki and Pythium arrhenomanesand as a growth promoter of soybean

Rojan P. John a, R.D. Tyagi a,*, D. Prévost b, Satinder K. Brar a, Stéphan Pouleur b, R.Y. Surampalli c

a INRS-ETE, Universite du Quebec, 490, Rue de la Couronne, Quebec G1K 9A9, CanadabAgriculture et Agroalimentaire Canada, Sainte-Foy, Quebec G1V 2J3, CanadacUSEPA, P.O. Box-17-2141, Kansas City, KS 66117, USA

a r t i c l e i n f o

Article history:Received 18 May 2010Received in revised form23 July 2010Accepted 2 August 2010

Keywords:Trichoderma virideSoybeanBiocontrol agentFusarium oxysporum f. sp. adzukiPythium arrhenomanesGrowth promoter

* Corresponding author. Tel.: þ1 418 654 2617; faxE-mail address: tyagi@ete.inrs.ca (R.D. Tyagi).

0261-2194/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.cropro.2010.08.004

a b s t r a c t

Trichoderma viride was proved as an effective biocontrol agent against two fungal pathogens, Fusariumoxysporum f. sp. adzuki and Pythium arrhenomanes, infecting soybean. During an in vitro biocontrol test,Trichoderma showed mycoparasitism and destructive control against the tested fungal pathogens. Boththe pathogens significantly influence the germination and P. arrhenomanes had a severe effect (only 5%germination). The root system of the soybean plant was poorly developed due to the infection and itexerted a negative influence on the nodulation and further growth phases of the plant. During pot assayalong with biocontrol activity, Trichoderma showed growth promoting action on the soybean plant.Trichoderma enhanced growth of shoot and root systems and fruit yield after 12 weeks of growth.Pythium and Fusarium infected plants treated with Trichoderma had w194% and 141% more height thanpathogens alone. The fruit yield treated with Trichodermawas w66 per plant whereas the yield was only41 for a control plant. The plants infected with Pythium and Fusarium and treated with Trichoderma hadfruit yields of 43 and 53 respectively and those were 5 and 1.6 times higher than plants infected withpathogens.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Soybean (Glycine max (L.) Merr. cv. Lotus, an early cultivar, Centreof reference in agriculture and agri-food, Quebec, Canada) is one ofthemost important crops (Herridge et al., 2008; Prévost et al., 2010)and a source of vegetable protein and oil. Soybean represents half ofthe global legume crop area and 68% of global production and fixes16.4 Tg N/year, it represents more than three fourths of the N fixedby the crop legumes (Herridge et al., 2008). Fungal diseases area main obstacle to obtain a high yield of soya during commercialcultivation and generally some chemical fungicides are appliedto control these diseases. Application of chemical fungicide hasbeen replaced by biocontrol agents because of the emergence offungicide-resistant strains and public concerns regarding the healthand environmental impacts of these chemicals. During the past fewdecades, several potential biocontrol organisms have been isolated,characterized and commercialized, and thus, biocontrol of plantdiseases has received more consideration in plant disease control(Shali et al., 2010). Trichoderma spp. are considered as potential

: þ1 418 654 2600.

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biocontrol and growth promoting agents for many crop plants(Verma et al., 2007; Bai et al., 2008; Savazzini et al., 2009). Thecompetition with pathogens, parasitism and the production ofantifungal compounds are the most important mechanisms inbiocontrol activity (Verma et al., 2007; Savazzini et al., 2009).Trichoderma populations can be established relatively easily indifferent types of soil and can continue to persist at detectable levelsfor months. Trichoderma viride (isolate obtained from Indian Insti-tute of Technology, New Delhi, India) is a biocontrol agent againstsoil borne plant pathogens and it can easily colonize in plantrhizosphere and help to promote the plant growth (Verma et al.,2007).

Fusarium spp. and Pythium spp. are two fungal pathogens thatheavily infect soybean and thus influence growth from germinationto all stages of plant development. Pythium spp. infections arecommon to corn and soybean and cause damping-off diseases ofboth crops. The damage caused by Pythium spp. can bring in majoreconomic losses to plant growers (Zhang and Yang, 2000). Fusariumspp., such as Fusarium solani f. sp. glycines infect the seedling rootand cause leaf symptoms (necrosis, chlorosis, defoliation, etc.),which usually begin slightly before or after flowering. Severe earlyinfections of this strain may reduce yield through seed and pod

R.P. John et al. / Crop Protection 29 (2010) 1452e1459 1453

abortion (Luo et al., 1999). Pythium arrhenomanes 3047 (receivedfrom AAFC Ottawa collection, Ontario) and Fusarium oxysporumf. sp. adzuki (isolate received from AAFC Ottawa collection, Ontario)have been isolated and identified as specialised pathogens onsoybean. In vitro bioassay can ensure the biocontrol action ofT. viride on these pathogens and further, in situ bioassay wouldensure eventual success of the antagonistic action (Verma et al.,2007). Our group previously tested the potential of T. viride strainfor biocontrol of Fusarium sp. and Cylindrocladium floridanum(isolate obtained from Laurentian Forestry Centre (LFC), Quebec,Canada) and their effect on growth of different plants. According toSivan and Chet (1989), strains of Trichoderma spp. can vary in theirbiocontrol activity from pathogen to pathogen. Trichodermaharzianum (isolates T-203 and T-35) was strongly mycoparasitic onisolated strains of Rhizoctonia solani and Pythium aphanidermatum,but non-parasitic to Fusarium isolates, such as F. oxysporum f. sp.vasinfectum and F. oxysporum f. sp. melonis (Sivan and Chet, 1989).In the present study, potential of T. viride ATCC 9275 was tested forthe biocontrol of above mentioned newly isolated two fungalpathogens and effect of pathogen and antagonist on seed germi-nation, growth and preliminary yield (fruit) on soybean plants.

2. Materials and methods

2.1. Microorganism used and maintenance

T. viride ATCC 9275 was used as a biocontrol agent on twosoybean pathogens, P. arrhenomanes 3047 isolated from corn, AAFCOttawa collection, Ontario and F. oxysporum f. sp. adzuki isolatedfromsoybean, AAFCHarrow,Ontario. Both species can cause root rotdisease in soybean and corn. In this study, Bradyrhizobiumjaponicum 532c (Nitragin Inoculants, Milwaukee, WI, USA), a nod-ulating bacterium, is used for biological nitrogen fixation bysymbiosis. Pathogens and biocontrol agent were cultivated onpotato dextrose agar (PDA) at 30 �C for 5e7 days and stored at 4 �Cuntil use. Bradyrhizobiumwas grown inyeast extractmannitol broth(YMB), concentrated by centrifugation (10,380 g for 20 min at 4 �C)and stored at 4 �C until use.

2.2. In-vitro test for biocontrol

In vitro antifungal activity of T. viride against Fusarium sp. andPythium sp. was tested on dual culturing method. Discs (5 mm indiameter) of fungal pathogen strains (one at a time) and Tricho-derma cut from advancing edge of 7-day PDA culture are placed5 cm from each other and incubated at 30 �C. Inhibition of radialgrowth of fungi and encroachment over pathogens by Trichodermawere measured and compared with the control.

2.3. Plant assay

Dorrance et al. (2004) reported that Pythium spp. recoveredfrom infected seed or seedlings of corn or soybean, or directly fromsoil had a range of pathogenicity and aggressiveness when tested inpathogenicity assays. So, these pathogens and biocontrol agentswere tested to find the effect on seed germination in soil.

2.3.1. Petri dish testFungal pathogens and Trichoderma transferred separately to 1%

(w/v) water agar (WA) and incubated at room temperature(25e30 �C) for 7 days before inoculation. Soybean seeds weresurface-sterilized and placed on fungal cultures (WA). Seedswere also placed on uninoculated WA plates as controls. The plateswere incubated at 10 �C in the dark for 6 days then moved to roomtemperature (25e30 �C) for another 2 days before disease ratings

were marked. Each fungus was considered as one treatment, andthere were three replicate plates for each strain. The rating scale foraggressiveness on soybeanwas: 0¼ seed germinatedwithout visibleinfection (a seed canbedefined as germinatedwhen the primary rootlength equals the seed length); 1 ¼ germinated with light discolor-ation on roots; 2¼ germinated with short, severely discolored roots;3 ¼ died after germination; and 4 ¼ died before germination.Measurement of aggressiveness of fungal strain on soybean wascalculated as a disease index from the following equation:

Disease index ¼X

xi=N (1)

where xi is disease rating of ith replicate (i ¼ 0e4) and N is the totalnumber of seedlings examined.

2.3.2. Fungal soil inoculums preparationAll fungal cultures were grown on potato dextrose agar and

incubated at 25e30 �C for 3 days. Six 1 cm2 plugs of 3-day-oldpotato dextrose agar grown cultures of each fungus were placed in500 ml flasks containing 200 ml of sand, 11.2 ml of corn meal, and80 ml of deionised water (autoclaved twice for 40 min and thenagain for 24 h). The fungi were allowed to colonize the sandecornmeal medium for 10 days. The flasks were shaken every alternateday to ensure uniform colonization. The number of propagules wasdetermined by plating soil dilutions on PDA or corn meal agarplates (Bates et al., 2008).

2.3.3. Emergence assayA base layer of pasteurized soil (mixture of silica:vermiculite

moisture, 1:1), followed by a layer of pathogen, approximately2 mm thick, were placed in plastic pots with bottom drainage. Theinoculum was covered with a thin layer of pasteurized soil and 10seeds were sown on soil. B. japonicumwas used as a biofertilizer inthis study. Seeds were pre-inoculated 1 h before sowing withB. japonicum. The seeds were again covered with 100 g of pasteur-ized soil. The pots were placed into plastic watering trays and wereplaced in a growth chamber (22/17 �C day/night temperatures, 16 hphotoperiod). The number of seedlings that emerged after 10 dayswas recorded. All seedlings and seeds separated from the soil werewashed under running water, and each seedling was observed fordisease by recording root discoloration, root destruction or death ofseedling before or after germination.

2.3.4. Biocontrol assay (long term)The soil sample (mixture of silica:vermiculite, 1:1 moisture with

N2-free plant nutrient solution, Hoagland solution) was used indifferent combinations: (1) without any treatment, (2) mixed withT. viride soil inoculum, (3) mixed with Fusarium soil inoculum, (4)mixed with Pythium soil inoculum, and (5) mixed with T. viride andFusarium sp., soil inoculum (1:1 ratio), (6) mixed with T. virideand Pythium sp., soil inoculums (1:1 ratio). Inocula containedw1500propagules/g soil for all fungal cultures. Eight replicateplasticpots for each experiment were tried. Initially 10 seeds (coated withBradyrhizobium 2� 106 cells per seed) of soya sown in separate potscontaining soil were incubated and after 10 days only 2 germinatedplantsperpotwere retained forbioassaypurpose. Plantsweregrownunder controlled environment conditions (22/17 �C day/nighttemperatures, 16 h photoperiod) in a growth chamber (ConvironModel PGR15, Controlled Environments Limited,Winnipeg, Canada)under ambient CO2 (390� 60 mmolmol�1 USA). Plantswerewateredregularly (twice a day) so that the soil was maintained near satura-tion and nitrogen free nutrient solution (200 ml) poured once ineveryweek.Differentparameters related toplant, such asnodulationindex, severity of disease, dry weight (shoot and root), plant height,carbon and nitrogenwere recorded.

Table 1Characteristics of nodule and parameters for determination of the nodulation index.

Factor Scale or characteristics

Nodule size (A)Large 3Medium 2Small 1

Nodule colour (B)Pink 2White 1

Nodule number (C)No nodules 0Few nodules 2Many nodules 3

Nodulation index ¼ A � B � C � 18

Qualitative charactersPosition of nodule 1. On primary root

2. On secondary root3. Both primary and secondary roots

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Plantswereharvestedat thebeginningof blooming, after 6weeksof growth. Roots and shoots were separated and roots were gentlywashedunder streamwater to remove thesoil.Nodulation indexwasmeasured according to Prévost et al. (2010), by visual examination(Table 1; abundance, position, colour and size of nodule). Roots andshoots were dried for 72 h at 70 �C for the determination of dryweight (dw), and shoot systemwas grounded for the determinationof total C and N concentration. Another set of plants (four replicateseach)was kept for another 6weeks to evaluate theyield as fruits. The

Fig. 1. Plate assay for antagonistic activity of Trichoderma on fungal pathogens; 1Ae1C: Trich(arrow marks show Trichodermal growth), 2Ae2D: Trichodermal growth and inhibition ofgrowth), 3: Trichoderma, 4: Fusarium, and 5: Pythium.

dry weight of the fruit, plant and number of fruits were recorded.Infection in shoot system also was monitored.

2.4. Pigment analysis

Pigment of a knownweight of shoot systemwas extracted usingacetone with 20% (v/v) water. Chlorophyll and carotenoids wereestimated using spectroscopy and concentration was calculated onthe basis of method by Lichtenthaler and Buschmann (2001). Thepigment concentration was represented as mg/g dry wt.

2.5. Statistical analyses

All the experiments were conducted in 4e8 replicates and datapresented were average of replicates along with the standard devi-ation. Database was subjected to an analysis of variance (ANOVA)(STATGRAPHICS Centurion, XV version 15.1.02 year 2006, StatPoint,Inc., USA) and the results which have P < 0.05 were considered assignificant. Discrimination among the means was conducted usingFisher’s least significant difference (LSD) procedure.

3. Results and discussion

3.1. In-vitro study

P. arrhenomanes showed initial faster growth and covered morethan half of the platewithin 48 h (Fig. 1; 2Ae2C). Later, Trichodermaovergrew on Pythium and destroyed it completely after 120 h of

odermal growth and inhibition of Fusarium growth after 24, 48 and 120 h respectivelyPythium growth after 24, 48 and 120 h respectively (arrow marks show: Trichodermal

Fig. 2. Germination of seeds with different treatment: Bradyrhizobium inoculated soyseeds were used in all treatments and value is average of 80 seeds.

R.P. John et al. / Crop Protection 29 (2010) 1452e1459 1455

incubation. F. oxysporum f. sp. adzuki had slower growth thanTrichoderma. Growth of F. oxysporum was inhibited by encroach-ment of Trichoderma as is evident from Fig. 1(1Ae1C). Though,Trichoderma inhibited growth, it had less effect on sporulated partsof Fusarium. Trichoderma grew on all possible sides of the patho-genic fungus (Fusarium) in plate to suppress further growth ofpathogen. No further growths of pathogens were observed inTrichoderma controlled plates. Both of the pathogens were grownfully on control plates after prolonged incubation. These initialresults indicated that present strain of Trichoderma can be used asa biocontrol agent against the tested fungal pathogens. Microscopicobservation of dual cultured plates showed different interactions,such as coiling of Trichoderma around the pathogens. It proved thedestructive mycoparasitic action of Trichoderma sp. against fungalpathogens mainly on Pythium.

Antibiotic production, mycoparasitism, the production of cellwall-degrading enzymes and competition for nutrients or space areconsidered as the actions involved in biocontrol of pathogen(Zeilinger and Omann, 2007; Vinale et al., 2008). During myco-parasitic interactions between Trichoderma and fungal pathogen,

Fig. 3. Seedlings of different treatments on 10th day of emergence rating (1) and effect of trapplied, C: Fusarium applied, D: Fusarium þ Trichoderma applied, E: Pythium applied, and F

a diffusible factor released from the host before physical contactwas responsible for induction of hydrolytic enzymes (Cortes et al.,1998; Zeilinger et al., 1999; Zeilinger and Omann, 2007). Duringdirect contact, lectins in the host’s cell wall can induce coiling of theTrichoderma around the host hyphae andmycoparasite can produceappressorium-like structures to destroy the pathogen (Zeilingerand Omann, 2007). According to Zeilinger and Omann (2007),both enzyme production and infection structure formation areinduced responses activated by the diffusible factor. Benhamou andChet (1993) illustrated many interactions of Trichoderma withpathogens (Rhizoctonia and Pythium), such as Trichoderma hadgrown parallel to pathogen, grown along the pathogen and grownaround the pathogen. Normal degradation of pathogen myceliawould take place after penetrating with appressorium-like struc-tures and they had observed the growth of Trichodermal hyphaewithin the pathogen (Benhamou and Chet, 1993).

3.2. Petri dish test on germination of soybean seeds

Disease level was recorded on the basis of infection on seeds ordiscoloration of roots. Both fungal pathogens had almost similareffect on seeds in plate assay and disease indices were 3.3 and 3.7for F. oxysporum and P. arrhenomanes, respectively (maximumdisease index is 4). During disease rating, F. oxysporum andP. arrhenomanes totally infected all seeds, before germination orjust after germination.

3.3. Emergence assay

Effect of pathogen can affect the nodulation of soybean plant asthe pathogens affect the root system. Control seeds (treated onlywith Bradyrhizobium) had higher germination rate in initial days(Fig. 2). Sixty percent of seeds were germinated within first 5 days,whereas, only 40% germination was observed in Trichodermaapplied soil and soil in which F. oxysporum was controlled byTrichoderma (Fig. 2). At the end of the germination test (10th day),Trichoderma applied soil achieved the highest percentage ofgermination (90e100%). F. oxysporum highly affected seed germi-nation and only w35% of seeds were germinated. Trichoderma

eatment on root and shoot system (2) of plants on same day. A: Control, B: Trichoderma: Pythium þ Trichoderma applied plants.

Fig. 5. Plant root system showing nodulation index of each treatment, A: Control,B: Trichoderma applied, C: Fusarium applied, D: Pythium applied, E: Fusarium þTrichoderma applied, and F: Pythium þ Trichoderma applied plants.

R.P. John et al. / Crop Protection 29 (2010) 1452e14591456

application could enhance the germination to w65% of total seeds.P. arrhenomanes almost completely arrested the germination ofsoybean and most seeds did not germinate or died just aftergermination. Only w0.5% germination was observed on the finalday of emergence rating. The growth of the shoot and root systemswas also influenced by pathogenic infection (Fig. 3), however,disease could be controlled or reduced by Trichoderma application.The treatment of P. arrhenomanes controlled by Trichoderma haddelayed germination (40% germinationwas observed after 7th day).Finally on 10th day it had 65% and 90e100% after 13th day. Soilcontaining P. arrhenomanes had no further germination and itproved the severity of disease by this strain. Dorrance et al. (2004)reported that Pythium spp. can cause disease individually and mayprovide infection courts for other pathogens to colonize tissueslater in the season pointing an example, Pythium irregulare, caus-ative organism of root rot in corn.

3.4. Long term growth effect

One set of the plants (8 pots; 2 plants/pot) were kept in a phy-totron for 6 weeks to observe the effect of the treatment on growthand another set for 12 weeks to measure the fruit yield. It wasobserved that the height of the plants infected by both thepathogens was less among all types of current treatments in plantbioassay than those of control plants (Fig. 4). Fusarium spp. infectedplants were healthier than the Pythium spp. infected plants. Only6% of Pythium infected plants survived during long period ofgrowth.

After 6 weeks, the root systemwas collected from all treatmentsand checked for its growth and root nodule index. Root noduleindices were 18 (maximum possible) for control plants and Tricho-derma treated plants (Fig. 5). Plants under above conditions hadmany big and pink coloured nodules all over the root system. Boththe pathogen-infected plants were having fewer root nodules andnoduleswere smaller in size. The density of the root systemwas poorin the case of pathogen-infected plants and which also reducednodulation. Trichoderma treatment controlled the pathogens,severity of the infectionwas reduced and improved the plant growthas it controlled the germination. Growth improvement of plants

Fig. 4. Plant growth after 6 weeks, 1: Control, 2: Trichoderma applied, 3: Fusarium applied,applied plants.

could be due to the synergistic activity of Trichoderma spp. on hostplant. Trichoderma spp. might have colonized around the root andincreased the root biomass and helped to increase the availability ofnutrients. Biocontrol agents might have interacted with the plant forexchange metabolites and that could cause significant changes inplant metabolism (Vinale et al., 2008). When Trichoderma spp.colonize plant roots, they invade only in the surface layers of theroot, further penetration can be controlled by the plant defencereactions. Therefore, Trichoderma spp. are usually avirulent irre-spective of their intrinsic ability to attack plants (Harman et al.,2004). Even though some Trichoderma spp. grow only on roots, theplant defence reactions can become systemic and protect the entireplant from a range of pathogens and diseases. Besides, the rootcolonization increases the growth of the entire plant and thus resultsin an increase in plant productivity and the yields. Symbiotic asso-ciation with rhizosphere of the plant helps to surmount abioticstresses and improve nutrient uptake (Harman et al., 2004).

Dry weight of plants infected by Fusarium spp. and Pythium spp.was minimum among all types of treatments in plant bioassay.However, Trichoderma along with pathogen treated plantsimproved in their root and shoot dry weight (Fig. 6). It can beconcluded that T. viride was capable of suppressing the growth ofpathogen even under soil conditions in a similar way as itcontrolled in plate assay. Total nitrogen, carbon and dry weightwere higher in the case of Trichoderma treated plants establishingthe positive effect of the biocontrol agent on enhancement of plantgrowth. The quantification of the pigments (Table 2) also illustrated

4: Fusarium þ Trichoderma applied, 5: Pythium applied, and 6: Pythium þ Trichoderma

Fig. 6. Total nitrogen and carbon content (A) and dry weight (B) of the plant after 6 weeks of growth.

R.P. John et al. / Crop Protection 29 (2010) 1452e1459 1457

the effect of infection by pathogens on growth of soybean. Totalchlorophyll pigment was lowest in Pythium infected plants.Pythium infected plants had a height of 16 � 0.2 cm, while Tricho-derma alone treated plants had the maximum height(62.5 � 1.8 cm). Pythium and Fusarium infected plants treated withTrichoderma had w194% and 141% more height than Pythium andFusarium infected plants, respectively. Vinale et al. (2008) sug-gested that the secondary metabolites such as auxin likecompounds or auxin inducing substances by Trichodermaeplantinteraction might be a reason for the improved growth. The inoc-ulation of seeds with Bradyrhizobium promoted nodulation andincreased nodulationwas achieved by the increased density of root.

Table 2The concentration of photosynthetic pigments in plants subjected to different treatment

Treatment Chla (mg/g dw) Chlb (mg/g dw) Total chl (Chla þ Ch(mg/g dw)

Plant (control) 10.82 4.37 15.19Trichoderma 13.84 3.97 17.81Fusarium 11.45 4.06 15.50Pythium 7.59 3.03 10.62Trichoderma þ Fusarium 11.98 4.32 16.29Trichoderma þ Pythium 10.98 4.22 15.20

Note: Chlorophyll (chl); Chlorophyll a (Chla); Chlorophyll b (Chlb).All the experiments were conducted in triplicates and values were presented as an averadifference between the mean measures from one level of treatments to another at the 9

Thus, nitrogen uptake, growth and yield response of crop plantswere positively influenced by Trichoderma treatment (Rudreshet al., 2005).

The plants infected with pathogen had effect on the total dryweight and fruit yield (both number and dry weight) (Fig. 7).The root systems of plants infected with pathogens were lessdense than the control and Trichoderma treated plants haddenser root system. The growth in root system reflected on thegrowth of shoot and in fruit yield. Pathogen-infected plants,especially Pythium infected plants were bearing one or a fewfruits however, the other plants had many fruits on their nodalregion.

s.

lb) Total chl/pot(mg/shoot dw)

Chla/Chlb Carotenoid (x þ c) (Chla þ Chlb)/(x þ c)

71.58 2.47 4.00 3.8090.12 3.48 5.00 3.5654.72 2.82 3.67 4.220.66 2.51 2.69 3.95

82.47 2.78 4.02 4.0543.51 2.60 3.97 3.82

ge. P-values of the F-test were less than 0.05 and there was a statistically significant5% confidence level.

Fig. 7. Fruit yield (A) and plant dry weight (B) after 12 weeks of plant growth.

Table 3Discrimination among the means was conducted using Fisher’s least significantdifference (LSD) procedure (method: 95% LSD).

Treatments Average value (homogeneous groups)

Fruit dryweight (g)

Fruit wetweight (g)

Fruitnumber

Stem dryweight (g)

Root dryweight (g)

Pythium 2 (a) 10 (a) 9 (a) 5 (a) 1 (a)Fusarium 11 (b) 46 (b) 34 (b) 16 (b) 5 (b)Control 13 (b) 53 (bc) 41 (bc) 22 (c) 8 (b)Trichoderma þ Pythium 15 (bc) 44 (b) 44 (bc) 22 (c) 19 (cd)Trichoderma þ Fusarium 18 (c) 69 (cd) 53 (cd) 26 (c) 16 (c)Trichoderma 27 (d) 79 (d) 67 (d) 32 (d) 22 (d)

Different letters (a, b, c and d) within the same group (fruit, shoot and root dryweight or wet weight and fruit number) indicate the significant differencesamong these values determined by Fisher’s least significant difference (LSD) test(P � 0.05).

R.P. John et al. / Crop Protection 29 (2010) 1452e14591458

The pathogen treated plants had many aerial infections likeyellow spots on leaf, wilting of leaf, etc. The Trichoderma treatedplants were resistant to the other infections and may be due to theinduced resistance (Harman et al., 2004).

Significance of the values was statistically analyzed and proved.The ANOVA table decomposed the variance of measure into twocomponents, a between-group component and a within-groupcomponent. Since the P-values of the F-test were less than 0.05,there was a statistically significant difference between the meanmeasures from one level of treatments to another at the 95.0%confidence level. Discrimination among the means was conductedusing Fisher’s least significant difference (LSD) procedure and isshown in Table 3.

Further, the strain of Trichoderma used in current study can betested for the economical production using low cost medium, suchas agro-industrial wastes in solid or liquid media. Further study canbe aimed on the search of suitable inert and eco-friendly

R.P. John et al. / Crop Protection 29 (2010) 1452e1459 1459

ingredients for novel formulations to make the production tech-nology more promising.

4. Conclusion

The current study assures the efficiency of Trichoderma asbiocontrol agents against fungal soil pathogens and indicates theneed of production and development of Trichoderma basedbiocontrol agents to serve as a model for environment friendlybiocontrol agent. The enhanced root system in soybean treatedwith Trichoderma directly enhanced the nodulation and more bio-logical nitrogen fixation helps in the photosynthetic activity ofplants. Thus, the Trichodermal infection helps in the growth andyield of soybean providing its own metabolites and helps in themetabolism of nodulating bacteria. Trichoderma effectivelycontrolled the pathogen and simultaneously increased the growthof plants and proved as avirulent opportunistic symbiont inrhizosphere of soybean plant. Antagonistic Trichoderma enhancedresistance against the secondary infection of pathogen in soybean.

Acknowledgement

The authors are sincerely thankful to the Natural Sciences andEngineering Research Council of Canada (Grants A4984, CanadaResearch Chair), MAPAQ (807150), INRS-ETE for financial support.One of the authors, RPJ, is sincerely thankful to The Quebec fund forresearch on nature and technology (FQRNT, Quebec, Canada), forthe postdoctoral fellowship under the programme “ScholarshipPrograms of excellence for foreign students”. Authors are thankfulto Mr. Amine for his help in statistical analysis and Ms. Carole forher help in phytotron study.

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