Biological Control of Alternaria Fruit Rot of Chili by Trichoderma Species under Field Conditions

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1 Mycobiology 38(2) : (2010) The Koren Society of Mycology DOI: /MYCO Biologicl Control of Alternri Fruit Rot of Chili by Trichoderm Species under Field Conditions Most. Ferdousi Begum, M. A. Rhmn nd M. Firoz Alm Biotechnology nd Microbiology Lbortory, Deprtment of Botny, University of Rjshhi, Rjshhi 620, Bngldesh (Received Februry 17, Accepted Februry 22, 2010) Trichoderm strins were evluted under field conditions to ssy their efficcy in suppressing Alternri fruit rot disese nd promoting chili plnt growth. The experiment ws conducted t the Botnicl Grden, Rjshhi University, Bngldesh from July 2006 to Mrch Appliction of Trichoderm hrzinum IMI significntly (p = 0.0) suppressed the disese compred to Alternri tenuis (T2) tretment nd improved both growth nd yield. The tretment T4 (T. hrzinum IMI A. tenuis) ws most effective in reducing disese percentge (72.27%) compred to A. tenuis (T1) tretment. The highest seed germintion rte (8.6%) nd the highest growth nd yield (12. g/plnt) ws lso recorded in the sme tretment (T4), followed by T (T. hrzinum IMI A. tenuis), T6 (T. hrzinum IMI A. tenuis), T2 (T. virens IMI A. tenuis), nd T3 (T. pseudokoningii IMI A. tenuis) tretment, while single tretment with A. tenuis significntly decresed these vlues. KEYWORDS : Alternri-fruit rot, Biologicl control, Chili, Trichoderm, Vigor index, Yield components *Corresponding uthor <E-mil : ferdrit@yhoo.com> Chili (Cpsicum nnum L.) is n importnt spice in Bngldesh, in 2000~2001, the totl re under chili cultivtion ws recorded s 166 h, with totl production of 141 metric tons [1]. Alternri fruit rot is seed-borne, widespred nd highly destructive disese tht infects chili plnts, nd yield loss cused by these diseses hs been recorded up to 100 percent under congenil environment conditions [2]. Biologicl control represents nturl nd ecologicl pproch to disese control tht reduces chemicl input nd their effects [3]. The fungi Trichoderm hs been n exceptionlly good model to study biocontrol becuse it is ubiquitous, esy to isolte nd culture, grows rpidly on mny substrtes, ffects wide rnge of plnt pthogens, is rrely pthogenic on higher plnts, cts s mycoprsite, competes well for food nd growth sites, produces ntibiotics nd hs n enzyme system cpble of ttcking wide rnge of plnt pthogens [4]. Furthermore, Trichoderm inhibit or degrde pectinses nd other enzymes tht re essentil for plnt-pthogenic fungi, such s Botrytis cinere, to penetrte lef surfces []. Although some chemicls re known to control Alternri tenuis, they re not lwys effective. Furthermore, becuse chili is vegetble crop, using chemicls for disese control is nonidel in view of the residue problems. Biocontrol of plnt pthogens using ntgonistic fungi nd bcteri, therefore, ssumes more significnce. Among ntgonistic fungi, Trichoderm hrzinum hs shown promise s biocontrol gent [6]. Although Trichoderm species re probbly the most widely used fungi in biologicl control of plnt pthogens, no in vivo experiment hs been conducted to test whether it cn control Alternri fruit rot of chili. Therefore, the objectives of this investigtion were to ssy the effects of Trichoderm strins ginst fruit rot pthogen A. tenuis. Plnt growth, yield components nd disese percentge were recorded to evlute their performnce under field conditions. Mterils nd Methods To evlute the efficcy of Trichoderm species t controlling Alternri fruit rot disese in chili, experiments were conducted t the Botnicl Grden of Rjshhi University, Rjshhi, Bngldesh from July 2006 to Mrch Seed collection. Locl chili vriety Bogr ws collected from the Spices Reserch Centre, Bogr, Bngldesh. Pthogen-free helthy seeds were selected for use in this experiment. Sources of Trichoderm. Five Trichoderm strins, including T. virens IMI , T. pseudokoningii IMI nd T. hrzinum IMI , T. hrzinum IMI , nd T. hrzinum IMI were used in this study which ws collected from the Biotechnology nd Microbiology Lbortory, Deprtment of Botny, Rjshhi University, Bngldesh. These strins were previously verified [7] by CABI Bioscience, Surrey, UK. Isoltion of A. tenuis. A. tenuis ws isolted from infected fruit prts of chili which were collected fter proper 113

2 114 Begum et l. recording of the symptoms of the disese. Following stndrd phytopthologicl methods [8], pthogen ws isolted from the trnsitionl zone of helthy nd infected tissues on potto dextrose gr (PDA) medium. The pthogenicity of the A. tenuis isolte ws confirmed on locl chili cultivr. All the cultures were stored t 4 o C for further study. Preprtion nd ppliction of spore suspensions. Mycelil discs ( mm dimeter) of Trichoderm isoltes nd A. tenuis were obtined from 4~ dys old culture nd seprtely trnsferred to 0 ml PDA in 20-mL conicl flsk nd incubted t 28 o C. After incubtion, 30 ml of sterile distilled wter ws dded to ech culture nd the flsks were shken t 0 rpm for 30 min in n orbitl shker. Then the content of ech conicl flsk ws filtered through sterile muslin cloth. The culture filtrte, contining the spores, ws collected, nd concentrtion of 10 spores/ml ws obtined by dilution with sterilized distilled wter. For seed tretment, 10 to 1 seeds were dipped in the spore suspension ( 10 spores/ml) of 4~ dys old Trichoderm strins for bout 20 min, nd the treted seeds were dried by lminr ir flow. After tht, both Trichoderm treted nd untreted seeds were gin dipped in the spore suspension (3 10 spores/ml) of 7 dys old culture of A. tenuis for bout 20 min nd then dried by lminr ir flow. After germintion of the treted seeds, the pot soil ws treted with 30 ml of conidil suspension (combintion of Trichoderm strins nd A. tenuis) ccording to respective tretment. The tretment ws continued up to hrvesting with seven dys intervl. Tretments. The experiments were designed with the following combintions: T0 = Control (untreted soil nd untreted seeds) T1 = A. tenuis (3 10 spores/ml) T2 = T. virens IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml) T3 = T. pseudokoningii IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml) T4 = T. hrzinum IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml) T = T. hrzinum IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml) T6 = T. hrzinum IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml) Steriliztion of soil. Soil ws collected from the reserch field of Rjshhi University nd sterilized with formldehyde (formlin : wter = 1 : v/v) nd covered with polythene. After 30 dys of steriliztion, soils were put in the erth pots (30 20 cm). To minimize the loss of excess wter, 2 cm hole ws mde from the bottom of ech pot. Seed germintion nd vigour index. All treted seeds were sown seprtely in ech pot with soil previously inoculted with A. tenuis (3 10 spores/ml). Untreted seeds were sown in un-inoculted soil s positive control. At lest 10 seeds were sown in ech pot. After 7 dys of germintion, seed germintion percentges were recorded. Vigour index for ech tretment ws determined ccording to the following formul from Abdul-Bki nd Anderson [9]. Vigour index = [Men of root length (cm) + Men of shoot length (cm)] percentge of seed germintion Collection of dt on yield nd yield contributing chrcteristics. Yield nd yield contributing dt were collected t different stges of plnt growth fter sowing. Observtions were recorded for plnt height, lef number, node number, primry nd secondry brnch numbers, number of flowers t mximum flowering, number of leves t mximum flowering, totl number of fruit, fresh fruit weight, dry fruit weight, totl number of seeds, yield per plnt nd percentge of infected fruit. Percentge of infected fruit. Percentge of infected fruit ws recorded by the following formul: No. of infected fruit Percentge of infected fruit = Totl No. of fruit Experimentl design nd sttisticl nlysis. The experiment ws crried out following Rndomized Block Design with three replictions. Dt on growth yield nd yield Tble 1. Seed germintion (%) nd vigour index of chili under different tretments Tretment % of seed germintion Shoot length (cm) Root length (cm) Vigour index T d 2.01 d 1.84 e f T1 3.6 e 1.69 e 1. f g T b 2.6 c 2.34 c d T c 2.34 c 2.1 d e T T b b T b 2.87 b 2.77 b 41 c Vlues within column followed by the sme letters re not significntly different (p < 0.0) by DMRT nlysis. T0 = Control, T1 = Alternri tenuis (3 10 spores/ml), T2 = Trichoderm virens IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml), T3 = T. pseudokoningii IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml), T4 = T. hrzinum IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml), T = T. hrzinum IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml), T6 = T. hrzinum IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml). Vlues re the mens of three replictions.

3 Biologicl Control of Alternri Fruit Rot of Chili by Trichoderm Species under Field Conditions 11 contributing chrcteristics were recorded nd sttisticlly nlyzed by DMRT test with the help of the computer pckge progrm SPSS (SPSS Inc., Chicgo, IL, USA). Results nd Discussion Germintion percentge nd vigour index. Seed germintion nd the vigour index were significntly (p 0.0) ffected by the tretments (Tble 1). The highest percentge of seed germintion (8.6%) nd vigour index (69.67) were recorded for the seeds treted with T. hrzinum IMI (T4), nd the lowest ws recorded for pthogen tretment lone (T1). Seed germintion ws drsticlly reduced for T1 (A. tenuis) nd control. These results reveled tht T. hrzinum might promote chili seed germintion. Trichoderm spp. hve evolved numerous mechnisms like mycoprsitism, production of inhibitory substnces, inctivtion of pthogen enzymes nd induction of resistnce to ttck other fungi nd enhnce plnt nd root growth [10]. Seedling vigour ws found to be higher when seeds were treted with T. hrzinum IMI (T4) spore suspension, wheres control (T0) nd A. tenuis (T1) showed the worst seedling vigour. Consistent with these results, Mukhtr [11] observed the highest vigour index when okr seeds were treted with T. hrzinum. Lo nd Lin [12] screened Trichoderm strins on plnt nd root growth of bitter gourd, loofh nd cucumber nd noted tht Trichoderm strins significntly incresed seedling height by 26 to 61%, root explortion by 8~209%, lef re by 27~38% nd root dry weight by 38 to 62% 1 dys of showing. Shke [13] observed the highest percentge of seed germintion nd vigour index when rice seeds were treted with T. hrzinum. Growth nlysis. After 30, 60, nd 90 dys, plnt height, lef number, node number, primry brnch number, secondry brnch number, nd lef nd flower numbers t mximum flowering were highest for T4 (T. hrzinum IMI ) nd lowest for T1 (A. tenuis) tretment, with Tble 2. Effect of seed tretment with Trichoderm strins on chili growth chrcteristics Tretment Plnt height (cm) No. of lef No. of node DAS No. of primry brnch es No. of secondry brnch es * No. of lef No. of flower t the mximum flowermum flower- t the mxiing stge ing stge T d d e 0.89 e 4.89 cd d 3.11 de 1 e de 2.89 e 3.6 f e cd e 08.6 e T1 2.3 e e f 04 f d e 2.22 e f e 2.22 f 2.44 g f d f e T2.28 b bc 2.08 cd cd b bc 4.67 bc 19.6 d bc 4.22 d d 3 c 77.6 b d 29 c T c 14.7 cd de 06.6 de bc cd 3.78 cd e cd 3.44 e 4.22 e d 4.67 c d d T T.96 b 19.7 b 30.6 b b 78 b b.6 b 2.78 b b 6.22 b 7.22 b b b b 44.6 b T6.49 b b c bc 7.44 b 121 b b c bc c 6 c 68.6 b 81.6 b c c Vlues within column followed by the sme letters re not significntly different (p < 0.0) by DMRT nlysis. T0 = Control, T1 = Alternri tenuis (3 10 spores/ml), T2 = Trichoderm virens IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ mll), T3 = T. pseudokoningii IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml), T4 = T. hrzinum IMI ( 10 spores/ ml) + A. tenuis (3 10 spores/ml), T = T. hrzinum IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml), T6 = T. hrzinum IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml). Mens of three replictions. Tble 3. Effect of Trichoderm strins on chili yield nd yield contributing chrcteristics Tretment Totl no. s of fruit Fresh fruit Dry fruit No. of seed/fruit 100 seed Yield (gm)/plnt T ef e 0.19 e e 0.44 cd de T1 03 f f 0.13 e e 0.37 d 01.9 e T d cd 0.4 cd c 0.4 bcd bc T de de 0.3 d d 0.48 bcd cd T T b b 0.66 b 177 b 0.63 b T c 08.9 c 0.3 c 18.6 bc 0. bc b Vlues within column followed by the sme letters re not significntly different (p < 0.0) by DMRT nlysis. T0 = Control, T1 = Alternri tenuis (3 10 spores/ml), T2 = Trichoderm virens IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ ml), T3 = T. pseudokoningii IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml), T4 = T. hrzinum IMI ( 10 spores/ ml) + A. tenuis (3 10 spores/ml), T = T. hrzinum IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml), T6 = T. hrzinum IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml). Mens of three replictions.

4 116 Begum et l. significnt difference (p < 0.0) (Tble 2). These results indicte tht T. hrzinum IMI hs growth-promoting effects on chili. Growth promoting effects by T. hrzinum hs been reported in other crops [14, 1] nd by T. koningii [1], but growth promotion hs not been demonstrted by T. virens on cotton [16]. Yield nd yield contributing chrcters. The highest totl number of fruit, fresh fruit weight, dry fruit weight, nd number of seeds per fruit, 100 seed weight nd yield/ plnt ws highest for T tretment nd lowest for A. tenuis (T1) tretment (Tble 3). T. hrzinum IMI incresed yield nd yield contributing chrcteristics by 74.77% for totl number of fruit, 8.33% for fresh fruit weight, 74.26% for dry fruit weight, 40.2% for number of seeds/ fruit, 33.33% for 100 seed weight nd 73.01% for yield/ plnt compred to A. tenuis (T2) tretment. The results revel tht the yield nd yield contributing chrcteristics were significntly ffected by the ppliction of T. hrzinum IMI With T. hrzinum tretment of the seeds, mny workers found much higher yields compred to control. Sultn [17] obtined up to 81.60% higher len4 Fig. 1. Effect of Trichoderm strins on percentges of infected chili fruit. Brs mrked by the sme letters re not significntly different (p < 0.0) by DMRT nlysis. T0 = Control, T1 = Alternri tenuis (3 10 spores/ ml), T2 = T. virens IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml), T3 = T. pseudokoningii IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml), T4 = T. hrzinum IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml), T = T. hrzinum IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml), T6 = T. hrzinum IMI ( 10 spores/ml) + A. tenuis (3 10 spores/ml). Fig. 2. Photogrphs show the effects of Trichoderm strins on suppressing Alternri fruit rot of chili; T0 = infected fruit of chili in control; T1 = infected fruit of chili treted with A. tenuis; T2 = helthy fruit of chili treted with T. virens IMI A. tenuis, T3 = infected fruit of chili treted with T. pseudokoningii IMI A. tenuis, T4 = helthy fruit of chili treted with T. hrzinum IMI A. tenuis, T = helthy fruit of chili treted with T. hrzinum IMI A. tenuis, T6 = helthy fruit of chili treted with T. hrzinum IMI A. tenuis.

5 Biologicl Control of Alternri Fruit Rot of Chili by Trichoderm Species under Field Conditions 117 til seed yield when they were treted with T. hrzinum. Sumitr nd Gikwrd [18] opined tht T. hrzinum incresed shoot nd root length in Trichoderm treted plots. Hrmn [19] reported tht T. hrzinum (T22), when pplied s seed tretment on pottoes, frequently incresed both size nd yield. d Luz et l. [20] lso observed tht yields of whet seeds infected with Pyrenophor triticirepentis, were significntly incresed fter ppliction of T. virens (1.66 kg/h). Percentge of infected fruit. The highest percentge of the lowest percentges of infected fruit ws recorded in T4 tretment (Fig. 1). In the control (T0), remrkble percentge of infected fruit ws lso observed; in this cse infection my be due to the seeds or environment. Appliction of T. hrzinum IMI ws significntly (p 0.0) suppressed the disese (72.27%) compred to the A. tenuis (T1) tretment (Fig. 2). Bisws nd Ds [21] reported tht ginst seedling disese Trichoderm is superior s seed coting. Prsd et l. [22] found tht soil treted with T. hrzinum showed 61.% disese control in chickpe while Kshem et l. [23] observed seed < 30% disese control in lentil. From the bove findings it my be concluded tht T. hrzinum IMI is more effective t controlling Alternri fruit rot disese in chili, nd this strin lso showed promising results on chili germintion, growth nd yield chrcteristics. The results suggest tht this strin my be used s n effective biocontrol gent to control fruit rot disese of chili. Acknowledgements The uthors re highly grteful to the Ministry of Ntionl Science Informtion nd Communiction Technology, Bngldesh for finncil support. The uthors lso express their specil thnks to the Spices Reserch Centre, Bogr, Bngldesh. References 1. Bru H, Rhmn MM, Msud MM. Effect of storge continers environment t different storge period on the qulity of chili seed. Int J Sustin Crop Prod 2009;4: Mhl MF. Study of fruit rot disese of chili cused by Alternri tenuis nd its control in Bngldesh [disserttion]. Rjshhi: University of Rjshhi; Lewis JA, Ppvizs GC. Biocontrol of plnt diseses: the pproch for tomorrow. Crop Prot 1991;10: Islm MS, Sh AK, Mosddeque HQ, Amin MR, Islm MM. In vitro studies on the rection on fungi Trichoderm to different herbicides used in te plnttion. Int J Sustin Crop Prod 2008;3: Zimnd G, Elnd Y, Chet I. Effect of Trichoderm hrzinum on Botrytis cinere pthogenicity. Phytopthology 1996;86: Bunker RN, Mthur K. Antgonism of locl biocontrol gents to Rhizoctoni solni inciting dry root of chili. J Mycol Plnt Pothol 2001;31: Rhmn MA. Screening of Trichoderm spp. nd their efficcy s bioconversion gent of municipl solid wste through pproprite technique of solid stte fermenttion [disserttion]. Rjshhi: University of Rjshhi; Booth C. The genus Fusrium. Surrey: Commonwelth Mycologicl Institute; Abdul-Bki AA, Anderson JD. Vigour determintion in soyben seed by multiple criteri. Crop Sci 1973;13: Ozby N, Newmn SE. Biologicl control with Trichoderm spp. with emphsis on T. hrzinum. Pk J Biol Sci 2004; 7: Mukhtr I. Influence of Trichoderm species on seed germintion in okr. Mycopth 2008;6: Lo CT, Lin CY. Screening strins of Trichoderm spp. for plnt growth enhncement in Tiwn. Plnt Pthol Bull 2002;11: Shke MU. Studies on sheth blight disese of Rice (Oryz stiv L.) cused by Rhizovtoni solni nd its control [disserttion]. Rjshhi: University of Rjshhi; Chng YC, Chng YC, Bker R, Kleifield O, Chet I. Incresed growth of plnts in the presence of the biologicl control gent Trichoderm hrzinum. Plnt Dis 1986;70: Windhm MT, Eld Y, Bker R. A mechnism for incresed plnt growth induced by Trichoderm spp. Phytopthology 1986;76: Hnson LE. Reduction of verticillium wilt symptoms in cotton following seed tretment with Trichoderm virens. J Cotton Sci 2000;4: Sultn N. Biologicl control of foot nd root rot of lentil with Trichoderm hrzinum [disserttion]. Mymensingh: Bngldesh Agriculturl University; Sumitr R, Gikwrd SJ. Checking Fusrium wilt of pigeonpe by biologicl mens. J Soil Crop 199;: Hrmn GE. Myths nd dogms of biocontrol: chnges in perceptions derived from reserch on Trichoderm hrzinum T-22. Plnt Dis 2000;84: d Luz WC, Bergstrom GC, Stockwell CA. Seed pplied bioprotectnts for control of seed-borne Pyrenophor triticirepentis nd gronomic enhncement of whet. Cn J Plnt Pthol 1998;19: Bisws KK, Ds ND. Biologicl control of pigeon pe wilt cused by Fusrium udum with Trichoderm spp. Ann Plnt Prot Sci 1999;7: Prsd RD, Rngeshwrn R, Anuroop CP, Rshmi HJ. Biologicl control of wilt nd root rot of chickpe under field conditions. Ann Plnt Prot Sci 2002;10: Kshem MA, Nznin HA, Islm MM, Jlluddin M, Hossin I. Effect of different methods of ppliction of Trichoderm in controlling foot nd root rot of lentil. Bngldesh J Seed Sci Tech 200;9:39-42.