Promissory Action of Trichoderma spp. and Fungicides in the Management of Fusarium Wilt of Gerbera

Novel Trichoderma harzianum isolate NVTH2, effective against Fusarium oxysporum f. sp. gerberae (FOG) causing wilt in Gerbera showed growth tolerance against azoxystrobin 23 % SC, kresoxim methyl 44.3% SC and carbendazim 50% WP at all the tested concentrations (50ppm, 100 ppm, 250 ppm, 500 ppm, 1000 ppm, 1500 ppm and 2000 ppm) followed by fosetyl Al 50% WP, difenoconazole 25% EC and tebuconazole 250 EC (effective against FOG) moderately tolerant at lower concentrations. The fungicides like propioconazole 25% EC, propineb 70 WP and tebuconazole 50%+ trifloxystrobin 25% WG (effective against FOG) completely resisted the growth of NVTH2. Finally, a field experiment was designed by utilizing 3 most efficient Trichoderma spp. and 4 fungicides in combination treatments. As a result, root dipping+ soil drenching with NVTH2 @5ml/ lit followed by soil drenching of tebuconazole @1ml/lit at fortnight interval alternately resulted in highest yield, growth promotion and percent inhibition of wilt incidence.

Gerbera jamesonii Bolus ex Hook has very huge demand globally.The production of cut flowers has gone from 2,071 million stems in 2007 to 6,667 million stems in 2011, which ultimately increased the growth of domestic and export markets in India 1 .Deterioration of soil health makes Gerbera highly susceptible to soil borne diseases under protected cultivation.The major soil borne diseases of Gerbera are foot rot, wilt, root rot complex and blight 2 .
Trichoderma harzianum is among the various species of the Trichoderma that is considered to be the utmost effective biocontrol agent 3 .The additive effects of Trichoderma and compatible fungicides were studied from many years.Arunasri et al. 4 reported that Trichoderma sp. which was effective against collar rot of crossandra were highly compatible with thiram (32% growth inhibition) and followed by captan (47.5%).Integration of Captan + Metalaxyl with Trichoderma harzianum and T. virens were found to be the superior to control the wilt complex of bell pepper caused by four wilt pathogens viz.Fusarium oxysporum, Phytopthora capsici, Rhizoctonia solani and Sclerotium rolfsii and promoted seedling growth 5 .

Isolation, pathogenicity and identification of FOG
Pathogen was isolated from the infected roots of Gerbera (var.Donavan yellow) on potato dextrose agar (PDA) medium amended with 1000 ppm of streptomycin sulphate.Surface sterilisation of infected root bits was done by 0.1% mercuric chloride (HgCl 2 ) solution for 30 seconds and subsequently washed in sterile distilled water and were incubated at room temperature (27±2°C) for 5 days.The phenotypic characterization was done according to Burgess et al. 6 using a light microscope (Labomed -IVU 5100) and photographed using a Labomed camera model LX400 with an image analyser -pixelpro programme.

Collection of fungal antagonists
Three most effective isolates of Trichoderma spp.against FOG were selected from the Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, India and were taken for further experimentation (Table 1).

Development of liquid formulation of Trichoderma spp
The fungal antagonists viz., T. harzianum strain NVTH1, T. harzianum NVTH2 and T. viride TV1 were cultured on 1000ml of Potato Dextrose Broth and incubated in an orbital shaker at 150 rpm at room temperature (28±2°C) for 48hr.Later the liquid biomass was mixed with 1% glycerol (10ml), tween 20 (10ml) and poly vinyl pyrrolidone -40000 ml.wt (10g) each separately 8 .The resultant mixture was kept in orbital shaker at 200 rpm for 5 minutes to ensure uniform blending and homogenization of the bacterial cells.Then the formulation was standardized to obtain one ml of formulation consists of 10 6 cfu/ml.The liquid formulation was stored at 5°C for further study.

Root dip and soil drenching with liquid formulation of Trichoderma spp. against wilt of Gerbera under protected cultivation
Field experiment was conducted during 2013-2014 in Gerbera (var.Donavan yellow) fields located at Spic Agro Biotech centre, Ooty, to assess the efficacy of liquid formulation of Trichoderma spp.(10 6 cfu/ ml) @ 5ml/litre and fungicides @ 1ml/ lit against wilt under protected condition (polyhouse).Thirty days old plants of Gerbera were used and the experiment was laid out with 7 treatments and 3 replications in RBD.The bed size of each replication was 5m 2 with 30 × 30 cm spacing (Table 2).

Statistical analysis
All the experiments were statistically analyzed independently.The treatment means were compared by Duncan's Multiple Range-Test (DMRT) 9 .The package used for analysis was IRRISTAT version 92-1 developed by the International Rice Research Institute, Biometrics unit, The Philippines.

Symptomatology of Fusarium wilt
The symptoms associated with wilt were yellowing of lower most leaves, and subsequently spread to entire plant.Affected leaves droop down and finally wilted.In later stages of the crop the presence of black discolouration in collar areas and brownish discolouration in petioles was observed.Wilting of the entire plant occurred within 3 to 4 weeks after infection (Figure 1).Similar report was made by Garibaldi et al. 10

Identification of the Pathogen
The mycelium of the fungal culture on PDA medium was initially white and later turned light pink to dark pink in different isolates.Macroconidia was sparse, and fusoid, 2-3 septate and measured 16.0-29.0x 2.5-4.2μm.Microconidia were abundant, hyaline, continuous, ovoid and measured 3.8-8.5 x 2.0-3.5 μm.Chlamydospores were hyaline and spherical, measured 4.0 -7.5 μm in diameter.Based on these phenotypic characters, the pathogen was confirmed as Fusarium SUNEETA et al.: STUDY OF Fusarium WILT OF GERBERA oxysporum f. sp.gerberae (KJ570974).The morphological characters were similar with the descriptions made by Booth 11 .

Pathogenicity
Inoculation of F. o. f. sp.gerberae (FOG) in to the healthy Gerbera seedlings of var.Bellwater white (30 days old) expressed the typical symptoms of wilt of Gerbera after 15 days of inoculation.Infected plants showed typical stunting of the plants and yellowing of leaves with brown to black streaks noticed in the crown portion and petioles of the plant.No symptoms were observed in uninoculated control plants.Similar pathogenicity results were recorded by Garibaldi and Minuto 12 .

Compatibility of Trichoderma harzianum isolate NVTH2 with fungicides
In the present study, tebuconazole 250 EC which was the most effective fungicide against FOG 13 was compatible with biocontrol agent T. harzianum isolate NVTH2 only at lower concentrations and was highly compatible with azoxystrobin 23 % SC, kresoxim methyl 44.3% SC and carbendazim 50% WP at all the tested concentrations.It was followed by fosetyl Al 50% WP, difenoconazole 25% EC and tebuconazole 250 EC moderately tolerant at lower concentrations.The fungicides like propioconazole 25% EC, propineb 70 WP and tebuconazole 50%+ trifloxystrobin 25% WG (effective against FOG) 13 resisted the growth of NVTH2 to 100% (Table -3).
Bhai and Thomas 14 reported that mancozeb and copper oxychloride were highly compatible with Trichoderma harzianum which was effective against rhizome rot of cardamom.Tapwal et al. 15 reported that T. viride effective against Armillaria mellea is compatible with the fungicide captaf and blue copper only to some extent.

Effect of Trichoderma spp. and fungicides on the management of Fusarium wilt and Plant growth promotion in polyhouse
Delivery of T. harzianum isolate NVTH2 through root dipping & soil drenching @ 5ml/litre+ soil drenching of Tebuconazole @ 1ml/lit recorded Likewise, treatment T1 also favored the early flowering and increased flower parameters, followed by treatment T6 over the untreated control (Table -4 and 5).
The efficiency of managing soil borne pathogen FOG can be maximised with the combined use of fungicides and antagonistic fungi but this can be achieved only if both are compatible with each other.Gaur and Sharma 16 integrated T. viride -1 or T. harzianum (TG-1) with metalaxyl and cymoxanil 8% + mancozeb 64% to control root rot in cotton.Seed treatment with T. harzianum + soil treatment with neem cake powder + foliar spray with carbendazim had reduced the Fusarium wilt of tomato and increased the the yield and growth parameters like shoot & root length and fresh weight & dry weight of the plants over the control 17 .
Many triazole compounds have good fungicidal and plant growth regulating activities.In the triazole fungicide (difenoconazole), thirteen novel triazole analogs of difenoconazole containing 1, 3-dioxolane rings have been synthesized and they express plant-growth regulatory activity 18 .Seedling inoculation with three isolates of Trichoderma spp.(1, 2 and 3) significantly increased the growth and controlled the wilt pathogen (F.o. f. sp.gerberae) of Gerbera plants as compared to control 19 .

CONCLUSION
Present experiment was undertaken by keeping in view of the hasty incidence of Fusarium oxysporum f. sp.gerberae in Gerbera plants.Initially the antagonistic fungi (NVTH2) was tested for its compatibility with the chemical fungicides at all types of dosages.Mostly it was compatible at lower dosages with fungicides.Later, the management module was developed at field level by combining both the Trichoderma spp.and fungicides against the FOG which finally resulted in the efficient module of RD + SD with NVTH2 @ 5ml/lit+ SD with Tebuconazole @ 1ml/lit at fortnight interval alternatively.

Table 1 .
Various isolates of Trichoderma collected from Department of Plant Pathology

Table 2 .
Treatment schedule for Fusarium wilt management

Table 3 .
Compatibility of Trichoderma harzianum (NVTH2) with fungicides S. Fungicides Mycelial growth (mm 2 )* *Values are mean of three replications.In a column, means followed by a common letter are not significantly different at the 5% level by DMRT Values in parentheses are percent inhibition over control (%).

Table 4 .
Effect of Trichoderma spp.+ fungicides on wilt incidence, growth characters and flower yield of Gerbera under protected cultivation *Values are mean of three replications Means followed by a common letter are not significantly different at 5% level by DMRT Data in the parenthesis are per cent reduction over control RD-Root dipping; SD-soil drenching; **SD applied alternate to ***SD at fortnight interval

Table 5 .
Effect of Trichoderma spp.+ fungicides on flowering in Gerbera under protected cultivation (wilt management trial) lowest wilt incidence of 3.28% with highest mean flower yield of 52.33 numbers/m 2 and also increased the root length and plant height.It was found significant with the application of treatment T6 in growth promotion and yield parameters.
*Values are mean of three replications Means followed by a common letter are not significantly different at 5% level by DMRT Data in the parenthesis are per cent reduction over control RD-Root dipping; SD-soil drenching; **SD applied alternate to ***SD at fortnight interval the