P. Suneeta*, S. Vinod Kumar, K. Eraivan Arutkani Aiyanathan and S. Nakkeeran
Department of Plant Pathology, Tamil Nadu Agricultural University,
Coimbatore – 641 003, Tamil Nadu, India.
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.
Keywords: Fungicides, Gerbera, growth promotion, Trichoderma.
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 India1. 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 blight2.
Trichoderma harzianum is among the various species of the Trichoderma that is considered to be the utmost effective biocontrol agent3. 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 growth5.
MATERIALS AND METHODS
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 (HgCl2) 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.
The pathogen F. oxysporum multiplied in potato dextrose broth, consisting of 107conidia/ml was inoculated @ 1% to sterilized potting mixture (laterite soil: sand: compost) in 3:1:1 ratio filled @ 5kg/pot.
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).
Compatibility of Trichoderma harzianum NVTH2 with fungicides
Compatibility of Trichoderma harzianum NVTH2 with fungicides viz. difenoconazole 25% EC (Score), tebuconazole 50% + trifloxystrobin 25% WG (Nativo), azoxystrobin 23 % SC (Amistar), propineb 70 WP (Antracol), fosetyl aluminium 50% WP (Alliete), propioconazole 25% EC (Tilt), tebuconazole 250 EC (Folicur), kresoxim-methyl 44.3% SC (Ergon) and carbendazim 50% WP (Benfil) was tested by Poisoned food technique7. The minimum inhibitory concentrations (50ppm, 100 ppm, 250 ppm, 500 ppm, 1000 ppm, 1500 ppm and 2000 ppm) of systemic and contact fungicides were amended in 100 ml of Potato Dextrose Agar medium @ 15 ml/plate. The medium without fungicide served as control. The plates were incubated at room temperature (28±2oC). Three replications were maintained for each treatment @ 10 plates per replication.
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 separately8. 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 106 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. (106 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 5m2 with 30 × 30 cm spacing (Table 2).
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.
RESULTS AND DISCUSSION
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 16.0-29.0 x 2.5-4.2 µm. 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 oxysporum f. sp. gerberae (KJ570974). The morphological characters were similar with the descriptions made by Booth11.
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 un-inoculated control plants. Similar pathogenicity results were recorded by Garibaldi and Minuto12.
Compatibility of Trichoderma harzianum isolate NVTH2 with fungicides
In the present study, tebuconazole 250 EC which was the most effective fungicide against FOG13 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 Thomas14 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 the lowest wilt incidence of 3.28% with highest mean flower yield of 52.33 numbers/m2 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. 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 Sharma16 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 control17.
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 activity18. 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 control19.
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.
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