����������Indian Journal of Plant Protection Vol. 38. No. 2, 2010 (176-182)

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Department of Plant Pathology, Agricultural Research Station, SK Rajasthan Agricultural University, Sriganganagar - 335 001,Rajasthan, India.E mail: dr.rbgaur@yahoo.com

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Systematic studies were conducted (2006 to 2009) to search for potential biocontrol agent against dry root rotpathogen (Rhizoctonia bataticola) of cotton and consequently compatibility of potential bioagents with fungicides.Among nine local and exotic bioagent isolates evaluated against R. bataticola under dual culture technique,Coimbatore isolate of Trichoderma viride-1 and local isolate of T. harzianum (TG-1) proved most effective inhibited76.53 and 72.78 per cent growth of R. bataticola respectively. These two bioagents also proved their superiorityover rest of the bioagents in controlling root rot disease in pots under cage house conditions. Minimum at par rootrot incidence of 35.78 and 38.98 per cent was recorded in pots where seeds and soil was treated with T. viride-1and T. harzianum (TG-1) respectively. These bioagents exhibited 56.28 and 52.53 per cent disease reduction overuntreated control. Metalaxyl, fosetyl-Al, mancozeb, cymoxanil 8% + Mancozeb 64% mixture and copper oxychloridefungicides were found compatible with bioagent T. harzianum (TG-1) and tolerance limits (ED

50) of these fungicides

were >1000 µg/ml. Metalaxyl proved their compatibility with another potential bioagent T. viride-1 also whereeven >1000 µg/ml concentration was under safe tolerance limit (ED

50). Copper oxychloride, mancozeb, fosetyl-

Al and cymoxanil 8% + mancozeb 64% mixture fungicides showed moderate to good compatibility with T. viride-1 by exhibiting tolerance limits (ED

50) of 848, 710, 578 and 448 µg/ml respectively.

�������� �Cotton, root rot, Rhizoctonia bataticola, bioagents, fungicides, compatibility

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Cotton (Gossypium spp.) is one of the most importantcommercial crops in India and occupies a major source offoreign exchange. Rajasthan plays a vital role in cottoncultivation covering 3.36 lac ha area with 7.50 lac balesproduction with a productivity of 379 lint kg/ha (Pundir etal., 2005). Root rot caused by Rhizoctonia bataticola (Taub.)Butler [Macrophomina phaseolina (Tassi.) Goid] is the mostdestructive disease of cotton in the area (Gaur et al., 2005)causing considerable loss to the yield depending on theGossypium species, the environment and cultural practices.The pathogen is seed borne and lives in soil for long periodwhich are accordingly difficult to control either by chemicalmeans or resistance breeding (Singh and Verma, 1988).Application of chemical alone is apparently visible andeffective technique to manage the disease, but at the sametime it has now become controversial because of rising costinvolved and its polluting effect on the environment andliving beings. Thus, the harmful effect of fungicides on soilmicroflora, acquiring of resistance of fungicide bypathogenic fungi, high cost of chemicals and a great demandfor residue free products in the domestic and international

market, necessitated development of eco-friendlymanagement system for disease control.

It is now widely recognized that biocontrol of seed and soilborne plant diseases i.e. wilt, root rot, collar rot etc. is adistinct possibility for future and can be successfullyexploited in the modern agriculture especially with in theframe work of integrated pest management system(Natarajan and Manibhushanrao, 1996). The integration ofpotential bioagents along with chemical fungicides foreffective disease management is therefore urgently needed.It may be even better if a biocontrol agent besides beingeffective should be compatible with latest crop productionpractices including use of fungicides. Therefore, thefungicides compatible with potential bioagents needs to beidentified for integration in disease management strategies.Under present study different antagonistic flora includingspecies of Trichoderma, Gliocladium and Chaetomium wereexplored against the R. bataticola and incited root rot diseasein cotton. The compatibility of potential bioagents withrecommended and newly developed fungicides was alsoinvestigated.

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The pathogen (R. bataticola) was isolated from root portionof diseased cotton plants, pure cultured and maintained onpotato dextrose agar (PDA) medium. The native bioagentculture of T. harzianum (TG-1) was isolated from soilthrough ‘Dilution plate count method’ and maintained onPDA. Other isolates of T. harzianum-1, T. viride-1 andGliocladium virens were received from Department ofMicrobiology, Coimbatore whereas T. viride-2, T. virens, T.koningii, G. deliquescens and Chaetomium globosum werefrom Department of Biotechnology, Bangalore.

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The antagonistic activity of different bioagents against R.bataticola was studied for two consecutive years (2006 &2007) by dual culture technique (Morton and Stroube, 1955).Mycelial discs of 5 mm diameter cut from margin of 7 daysold actively growing culture of the R. bataticola and fungalbioagent were placed on potato dextrose agar (PDA) in thesame Petri plate opposite to each other (approximately 6 cmapart). Plates having only pathogen served as control. Eachtreatment was replicated thrice. The inoculated plates wereincubated at 25 ±1°C in a BOD chamber. The radial growthof the pathogen from the disc towards the center of theplate was recorded when the control plates were completelycovered with mycelial growth of the pathogen. The per centinhibition in growth of pathogen by bioagents over controlwas then calculated (Vincent, 1947).

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The bioagents which tested in vitro against R. bataticolawere further explored for their bioefficacy in controllingroot rot of cotton under cage house conditions during 2008using artificial soil inoculation technique.

���� ������� ' Autoclaved potting mixture of soil :FYM (3:1) was filled in sterilized (4% formalin) earthenpots of 50 cm diameter (5 kg/pot). The R. bataticola wasgrown on potato dextrose broth medium for seven days at28 + 2 oC. Mycelial mat was harvested and macerated inhomogenizer at the rate of 10 g/lit of sterilized distilledwater. Mixed mycelial suspension was inoculated in pot @100 ml/kg soil and pots were kept in shade for 24 h beforesowing.

���� ����������� ����������������� ' Forsoil treatment, mass multiplication of antagonists was doneon potato dextrose broth at 25 ± 1 oC for 10 days. Themycelial mat was harvested and macerated in sterilized

distilled water with the quantity equal to the broth. Thishomogenized suspension was added in pots 48 h beforesowing @ 100 ml/kg soil.

The pots were watered immediately after inoculation toprovide moisture for pathogenic/antagonistic growthestablishment. Seeds of cotton (cv. RG-8) coated withmycelial culture of the bioagents by rolling on 7 days oldculture plates were air dried and subsequently sown in pots@ 20 seeds/pot. One set of control was maintained by sowinguntreated seeds in pots of sick soil having only pathogen.Each treatment replicated thrice in completely randomizeddesign. As and when required, pots were irrigated withequally measured amount of water and plants affected withroot rot were recorded till above 80 per cent plants showeddisease incidence in control pots.

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Nine fungicides viz., fosetyl-Al 80% WP (Aliette),Metalaxyl 35% WS (Apron), carbendazim 50% WP(Bavistin), copper oxychloride 50% WP (blue copper),Cymoxanil 8% + Mancozeb 64% WP (Curzate M8),tebuconazole 25.9% EC (Folicur), mancozeb 75% WP(Uthane M-45), Pyraclostrobin 20% WG (Insignia) andcarboxin 37.5% + thiram 37.5% WS (Vitavax power) weretested against the potential bioagents namely T. harzianum(TG-1) and T. viride (Tv-1) on active ingredient basis using‘poisoned food technique’ (Schmitz, 1930) during the year2009.

Different concentrations of fungicides i.e. 50, 100, 200, 500,1000 and 2000 µg/ml were prepared in 30 ml sterilizeddistilled water and aseptically mixed with 30 ml sterilizedaliquots of double strength PDA medium (all ingredientsexcept water being double in amounts to those of normalPDA) in flasks to finally give a concentration of 25, 50,100, 250, 500 and 1000 µg/ml in poisoned medium. ThreePetri plates of each concentration of the fungicides wereprepared by pouring 20 ml PDA aliquot in each plate of 90mm diameter. After solidification of PDA, each plate wasinoculated at the centre with 5 mm disc of actively growingculture of bioagent. Plates without any fungicides served ascontrol. Inoculated plates were incubated at 25 + 1 oC tillthe mycelial growth of the bioagent completely covered thePDA in control plates. The linear growth of the colony ineach treatment was measured in two directions at right anglesto each other. The size of inoculated disc was deducted fromeach and per cent inhibition in growth of the bioagent overcontrol was calculated (Vincent, 1947). Completelyrandomized design was followed for the analysis of the dataobtained.

ED50

and ED90

values of the fungicides were calculated by

"##Indian Journal of Plant Protection Vol. 38. No. 2, 2010 (176-182)

plotting the log concentration of the fungicides against theper cent probit inhibition of the mycelial growth(Nageswararao, 1983).

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Mean of two years data (2006 & 2007) revealed that allbioagent isolates reduced the growth of R. bataticolasignificantly as compared to control. Among variousbioagent, T. viride-1 was significantly superior providingmaximum inhibition of 76.53 per cent in mycelial growthof the R. bataticola. Local isolate T. harzianum (TG-1)proved next in antagonistic potential against the pathogenand exhibited 72.78 per cent growth inhibition insignificantlyfollowed by T. harzianum-1 and G. virens which providedreduction of 72.50 and 72.36 per cent respectively. Bioagentisolates of T. viride-2, T. virens and G. deliquescens werefound comparatively less effective against the pathogenand rendered at par growth inhibition (Table 1). Presentresults are in agreement with those of Gogoi and Ali (2005)and Pan and Bhagat (2007) who established the antagonisticbehaviour of Trichoderma harzianum, T. viride andGliocladium virens against Rhizoctonia spp. Theantagonistic potentiality of Trichoderma spp. andGliocladium virens might be attributed to their ability ofproduce chitinolytic enzymes and gliotoxin respectively(Howell et al., 1993; Arumugam et al., 2003).

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All the bioagents significantly checked the root rot incidencein pots under cage house conditions as compared to untreatedcontrol where 82.03 per cent incidence was recorded (Table1). Pots with soil and seeds treated by Coimbatore isolate T.viride-1 showed least incidence (35.78%) and highestreduction (56.28%) of root rot disease over untreatedcontrol. Local isolate of T. harzianum (TG-1) and aCoimbatore isolate of T. harzianum-1 proved to be next inorder of efficacy in controlling the disease which allowed38.90 and 40.83 per cent incidence of disease and provided52.53 and 50.15 per cent disease control respectively. Thesebioagent isolates differed insignificantly from each other.Other bioagents viz., Gliocladium virens, T. viride-2 and T.virens exhibited 46.01, 44.40 and 41.28 per cent reductionin incidence. Rest of the bioagents were more or lessineffective against the disease. Sharma and Gupta (2003)and Gaur et al. (2005a & b) have also achieved good controlof root rot disease in cotton and other crops through bioagentTrichoderma spp. Besides plant disease control, the biocontrol agents are also reported to possess some plant growthpromotion activity. T. harzianum has been reported toincrease plant growth consequence of seed and rootprotection against pathogen (Mukherjee et. al., 1989).

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Two prominent bioagent isolates viz., T. harzianum (TG-1)

Table 1. In vitro antagonism of bioagents against Rhizoctonia bataticola and their bioefficacy in controlling root rotof cotton

Growth inhibition of R. bataticola over control (%) Root rot Disease reductionBioagents 2006 2007 Mean incidence (%) over control (%)

Trichoderma harzianum (TG-1) 71.39 (57.67) 74.17 (59.47) 72.78 (58.57) 38.90 (38.58) 52.53 (46.46)

T. harzianum -1 71.11 (57.49) 73.89 (59.27) 72.50 (58.38) 40.83 (39.72) 50.15 (45.09)

T. viride-1 75.56 (60.37) 77.50 (61.70) 76.53 (61.04) 35.78 (36.71) 56.28 (48.62)

T. viride-2 69.45 (56.44) 68.06 (55.60) 68.76 (56.02) 45.57 (42.45) 44.40 (41.78)

T. virens 68.89 (56.10) 67.78 (55.43) 68.34 (55.77) 48.13 (43.93) 41.28 (39.98)

T. koningii 67.78 (55.42) 63.61 (52.90) 65.70 (54.16) 52.73 (46.57) 35.65 (36.65)

Gliocladium virens 70.83 (57.32) 73.89 (59.27) 72.36 (58.30) 44.16 (41.64) 46.01 (42.70)

G. deliquescens 68.06 (55.59) 66.95 (54.93) 67.51 (55.26) 52.40 (46.38) 36.06 (36.89)

Chaetomium globosum 62.50 (52.25) 60.28 (50.93) 61.39 (51.59) 73.10 (58.77) 10.74 (18.68)

Control 0.00 (4.05) 0.00 (4.05) 0.00 (4.05) 82.03 (64.98) 0.00 (4.05)

CD (P= 0.05) 1.14 1.23 1.19 3.03 5.04

CV (%) 1.54 1.66 1.60 3.86 8.20* Average of three replications

Arc sine transformed values in parentheses

"#* Biocontrol of Root Rot in Cotton and Compatibility of Potential Bioagents R B Gaur et al.,

and T. viride-1 selected on the basis of their ability to controlR. bataticola of cotton were further explored for theircompatibility with fungicides.

������������+,�-".' All the fungicides tested, eachat six different concentrations exerted varying degree ofinhibition in mycelial growth of bioagent T. harzianum (TG-1). Out of nine fungicides tested, metalaxyl and fosetyl-Alproved most compatible with the bioagent culture of T.harzianum (TG-1). These fungicides did not exert any lethaleffect on the bioagent growth up to the concentration of500 ppm and even >1000 µg/ml (0.1%) concentration seemsunder safe tolerance limit (ED

50) which exhibited least

growth inhibition of 6.70 and 7.07 per cent respectivelycompared to control. Culture of the bioagent also showed

good compatibility with the mancozeb, mixture ofCymoxanil 8% + mancozeb 64% and copper oxychloridefungicides. No inhibition of bioagent growth was recordedup to the concentration of 100 ppm under these fungicideswhile tolerance limits (ED

50) of these fungicides were >1000

µg/ml. These fungicides inhibited just 18.03, 27.47 and35.30 per cent growth of T. harzianum at 1000 ppmconcentration, respectively.

Pyraclostrobin was moderately compatible with bioagentand reduced maximum growth (ED

90) at the concentration

of >1000 µg/ml, while ED50

value of this fungicide was 155µg/ml. The mixture of carboxin 37.5% + thiram 37.5% wasless compatible with bioagent as its ED

50 and ED

90 values

were 40 and 248 µg/ml, accordingly. The local bioagent

Table 2. Non-target effects of fungicides on mycelial growth of Trichoderma harzianum (TG-1) under in vitroconditions

% Inhibition in mycelial growth over control Concentration (µg ml-1)

Fungicides 25 ppm 50 ppm 100 ppm 250 ppm 500 ppm 1000 ppm Mean ED50

ED90

Fosetyl-Al 80% WP (Aliette) 0.00 0.00 0.00 0.00 0.00 7.07 1.18 >1000 >1000(4.05) (4.05) (4.05) (4.05) (4.05) (15.40) (5.94)

Metalaxyl 35% WS (Apron) 0.00 0.00 0.00 0.00 0.00 6.70 1.12 >1000 >1000(4.05) (4.05) (4.05) (4.05) (4.05) (15.03) (5.88)

Carbendazim 50% 100.00 100.00 100.00 00.00 100.00 100.00 100.00 <25 <25WP (Bavistin) (90.0) (90.00) (90.00) (90.00) (90.00) (90.00) (90.00)

Copper oxychloride 0.00 0.00 0.00 9.03 19.60 35.30 10.66 >1000 >100050% WP (Blue copper) (4.05) (4.05) (4.05) (17.47) (26.27) (36.37) (15.38)

Cymoxanil 8% + Mancozeb 0.00 0.00 0.00 2.77 12.93 27.47 7.20 >1000 >1000 64% WP (Curzate M8) (4.05) (4.05) (4.05) (9.53) (21.07) (31.60) (12.39)

Tebuconazole 25.9% 98.80 100.00 100.00 100.00 100.00 100.00 99.80 <25 <25EC (Folicur) (84.93) (90.00) (90.00) (90.00) (90.00) (90.00) (89.16)

Mancozeb 75% 0.00 0.00 0.00 0.00 7.45 18.03 4.25 >1000 >1000WP (Uthane M-45) (4.05) (4.05) (4.05) (4.05) (15.83) (25.13) (9.53)

Pyraclostrobin 20% 23.53 32.93 45.50 57.63 66.30 79.23 50.85 155 >1000WG (Insignia) (29.00) (35.03) (42.40) (49.40) (54.50) (62.93) (45.54)

Carboxin 37.5% + Thiram 41.57 54.90 69.40 88.63 100.00 100.00 75.75 40 27837.5% WS (Vitavax Power) (40.13) (47.80) (56.43) (70.47) (90.00) (90.00) (65.81)

Control 0.00 0.00 0.00 0.00 0.00 0.00 0.00 - -(4.05) (4.05) (4.05) (4.05) (4.05) (4.05) (4.05)

Mean 26.39 28.78 31.49 35.81 40.63 47.38(26.84) (28.71) (30.31) (34.31) (39.98) (46.05)

CD (P=0.05)

Fungicides (F) 0.83

Concentrations (C) 0.64

F x C 2.03

CV (%) 3.65

* Average of three replications; Arc sine transformed values in parentheses

"#/Indian Journal of Plant Protection Vol. 38. No. 2, 2010 (176-182)

was highly sensitive to carbendazim and tebuconazole.Maximum inhibitory concentrations (MIC) i.e. ED

90 values

of these fungicides were recorded lowest (<25 µg/ml)

1-3. Fosetyl-AL, 4-6. Metalaxyl, 7-9. Carbendazim, 10-12.Copper oxychloride, 13-15. Cymoxanil 8% + Mancozeb 64%,16-18. Tebuconazole, 19-21. Mancozeb, 22-24. Pyraclostrobin,25-27. Carboxin 37.5% + Mancozeb 37.5%, 28. Control

Figure 1. Effect of fungicides on mycelial growth ofTrichoderma harzianum (TG-1) at differentconcentration

1-3. Fosetyl-AL, 4-6. Metalaxyl, 7-9. Carbendazim, 10-12.Copper oxychloride, 13-15. Cymoxanil 8% + Mancozeb 64%,16-18. Tebuconazole, 19-21. Mancozeb, 22-24. Pyraclostrobin,25-27. Carboxin 37.5% + Mancozeb 37.5%, 28. Control

Figure 2. Effect of fungicides on mycelial growth ofTrichoderma harzianum (TG-1) at differentconcentration

amongst all fungicides tested. Therefore, these twofungicides proved completely incompatible and are notsuitable for integration with bioagent (Table 2, Fig. 1).

"*0 Biocontrol of Root Rot in Cotton and Compatibility of Potential Bioagents R B Gaur et al.,

Table 3. Non-target effects of fungicides on mycelial growth of Trichoderma viride-1 under in vitro conditions

% Inhibition in mycelial growth over control Concentration.(µg ml-1)

Fungicides 25 ppm 50 ppm 100 ppm 250 ppm 500 ppm 1000 ppm Mean ED50

ED90

Fosetyl-Al 80% WP (Aliette) 0.00 0.00 0.00 21.57 47.10 65.50 22.36 578 >1000(4.05) (4.05) (4.05) (27.67) (43.30) (54.10) (22.87)

Metalaxyl 35% WS (Apron) 0.00 0.00) 0.00 0.00 0.00 10.60 1.77 >1000 >1000(4.05) (4.05 (4.05) (4.05) (4.05) (18.77) (6.50)

Carbendazim 50% WP 100.00 100.00 100.00 100.00 100.00 100.00 100.00 <25 <25(Bavistin) (90.00) (90.00) (90.00) (90.00) (90.00) (90.00) (90.00)

Copper oxychloride 50% WP 0.00 0.00 0.00 10.60 38.03 55.27 17.32 848 >1000(Blue copper) (4.05) (4.05) (4.05) (19.00) (38.07) (48.03) (19.54)

Cymoxanil 8% + Mancozeb 0.00 0.00 11.77 24.73 56.83 67.43 26.79 448 >1000 64% WP (Curzate M8) (4.05) (4.05) (19.67) (29.80) (48.97) (55.20) (26.96)

Tebuconazole 25.9% EC 39.20 65.90) 100.00( 100.00 100.00 100.00 84.18 35 85(Folicur) (38.73) (54.27 90.00) (90.00) (90.00) (90.00) (75.50)

Mancozeb 75% WP 0.00 0.00 0.00 16.07 41.57 61.57 19.87 710 >1000(Uthane M-45) (4.05) (4.05) (4.05) (23.63) (40.13) (51.70) (21.27)

Pyraclostrobin 20% WG 29.03 43.93 56.47 65.90 72.93 85.50 58.96 74 >1000(Insignia) (32.60) (41.50) (48.63) (54.27) (58.67) (67.67) (50.56)

Carboxin 37.5% + Thiram 98.03 100.00 100.00 100.00 100.00 100.00 99.67 <25 <2537.5% WS (Vitavax Power) (83.43) (90.00) (90.00) (90.00) (90.00) (90.00) (88.91)

Control 0.00 0.00 0.00 0.00 0.00 0.00 0.00 - -(4.05) (4.05) (4.05) (4.05) (4.05) (4.05) (4.05)

Mean 26.63 30.98 36.82 43.89 55.65 64.59(26.91) (30.01) (35.86) (43.25) (50.72) (56.95)

CD (P=0.05)

Fungicides (F) 1.13

Concentrations (C) 0.87

F x C 2.76

CV (%) 4.20

* Average of three replications; Arc sine transformed values in parentheses

�� ������-"' Nine fungicides which were used forcompatibility studies rendered their toxic effect on themycelial growth of this bioagent with varying degree.However, metalaxyl proved significantly more compatibleover rest of the fungicides with the bioagent culture of T.viride-1 (Table 3, Figure 2). This fungicide did not affectthe bioagent growth up to the concentration of 500 ppm andeven >1000 µg/ml concentration was under safe tolerancelimit (ED

50) exhibiting least growth inhibition of 10.60 per

cent compared to control. Copper oxychloride, mancozeb,fosetyl-Al and cymoxanil 8% + mancozeb 64% mixturefungicides which followed and showed moderate to goodcompatibility with bioagent. T. viride-1 exhibited full growthup to the concentration of 100 ppm under these fungicidesexcept cymoxanil 8% + mancozeb 64% mixture where full

growth was observed up to the 50 ppm concentration only.Tolerance limits (ED

50) of these fungicides were 848, 710,

578 and 448 µg/ml and at 1000 ppm concentration exhibited55.27, 61.57, 65.50 and 67.43 per cent growth inhibition,respectively. The bioagent fungus was found most sensitiveto carbendazim and carboxin 37.5% + thiram 37.5% mixture.Maximum inhibition concentrations (ED

90) of these

fungicides were less than 25 µg/ml which was least amongstall fungicides tested.

Under present investigation, T. harzianum (TG-1) showedmore resistance against the fungicides rather than T. viride-1 which might be due to their inherent resistance tofungicides and their ability to degrade chemicals as describedby Papavizas (1985). Similar to present findings the

"*"Indian Journal of Plant Protection Vol. 38. No. 2, 2010 (176-182)

compatibility of Trichoderma spp. with metalaxyl, fosetyl-Al and mancozeb fungicides has been reported earlier also(Vijayaraghavan and Abraham, 2004; Annonymous, 2008).Lal and Maharshi (2007) and Khosla and Gupta (2008)observed the toxicity of carbendazim against theTrichoderma spp.

On the basis of present investigation it may be concludedthat seed and soil may be treated with either T. viride-1 or T.harzianum (TG-1) for root rot disease control in cotton.Both these bioagents may be integrated with metalaxyl,mancozeb, fosetyl-Al, copper oxychloride or cymoxanil 8%+ mancozeb 64% mixture fungicides for controlling the seedand soil borne plant diseases in more effective mannerwithout adverse effect on soil, environment and humanbeings. This study also suggests for cautious approach aboutthe use of incompatible fungicides like carbendazim,tebuconazole and mixture of carboxin 37.5% + thiram 37.5%with these bioagents.

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The authors are grateful to the Director Research, S.K.Rajasthan Agricultural University, Bikaner for providingfacilities during the course of these investigations.

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Received : 22-07-2010 Accepted : 15-10-2010

"*2 Biocontrol of Root Rot in Cotton and Compatibility of Potential Bioagents R B Gaur et al.,