Piriformospora indica- mediated degradation of strobilurin and triazole fungicides in tomato plants

Abstract

The research work entitled “Piriformospora indica-mediated degradation of strobilurin and triazole fungicides in tomato plants” was carried out at the Department of Plant Pathology, College of Agriculture Vellayani, Thiruvananthapuram during 2022-24. The objective was to evaluate the beneficial fungal root endophyte, P. indica on degradation or retention of strobilurin and triazole fungicides used for the management of black leaf mold of tomato. P. indica-colonization in tomato var. Vellayani Vijai was done in the standardized medium. The presence of the chlamydospores was observed in roots of tomato at 5 days after colonization (DAC). P. indica-colonization was further confirmed using fungus-specific primer Pi-tef by PCR. Compatibility of P. indica was studied with fungicides such as azoxystrobin, pyraclostrobin, propiconazole and tebuconazole. P. indica is compatible with azoxystrobin and to some extend with pyraclostrobin; and incompatible with propiconazole and tebuconazole. P. indica exhibited 72 per cent compatibility with azoxystrobin at 50 ppm; while 19.72 per cent compatibility was observed with pyraclostrobin. P. indica could not grow in triazole fungicides, propiconazole and tebuconazole even at 50 ppm. Leaves with characteristic symptoms of black leaf mold of tomato were collected from Coconut Research Station, Balaramapuram. The symptoms were yellow and chlorotic spots on the upper leaf surface and black sooty mold on the corresponding lower surface. The fungus was isolated on potato dextrose agar (PDA) medium. The mycelial growth was grey on the upper side with concentric zonations, and black on the rear side. The margin of the growth was regular. The growth rate was observed as 1.1 mm per day. Koch’s postulate of the pathogen was proved by detached leaf assay. Symptoms started appearing from 5th day onwards. Symptoms were chlorotic lesion on the upper surface and black sooty growth on the lower surface of the leaf. In vitro evaluation of the pathogen against azoxystrobin, pyraclostrobin, propiconazole and tebuconazole was done by poisoned food technique. Both strobilurin and triazole fungicide significantly inhibited mycelial growth of 165

Pseudocercospora fuligena. Triazole fungicides such as propiconazole and tebuconazole were more effective in inhibiting the mycelial growth of P. fuligena than strobilurin fungicides. At 50 ppm, P. fuligena has grown 3.80 cm in azoxystrobin, 3.80 cm in pyraclostrobin, 1.37 cm in propiconazole and 1.21 cm in tebuconazole fungicides. P. fuligena growth was completely inhibited at 100 ppm of triazole fungicides; but at least 25 per cent fungal growth was observed in strobilurin fungicides even at 1000 ppm. A field experiment was conducted during the summer season (2023) at Coconut Research Station Balaramapuram. The first spray of fungicides was given at the vegetative stage ie., 40 days after sowing. The second spray of fungicides was given after fruit set stage ie., 70 days after sowing. The leaves and fruit samples were collected at 2 h, 24 h, 3, 5 and 10 days after spraying and residues were assessed by LC-MS/MS. P. indica enhanced the retention of the fungicide residues in leaves. The fungus also enhanced the degradation of the fungicides in tomato fruits which makes the fruits safer to eat. P. indica-colonization decreased the incidence and severity of diseases such as black leaf mold, Alternaria leaf spot, leaf curl and mosaic under natural incidence. When azoxystrobin was used, 64.05 per cent reduction of black leaf mold severity was observed over control at 60 days after transplanting. When propiconazole was used, 55.49 per cent reduction was observed. When azoxystrobin was used along with P. indica, the disease severity has been further reduced to 71.89 per cent; and when propiconazole was used along with P. indica, a reduction of 69.25 per cent was observed. Moreover, P. indica enhanced the growth parameters such as plant height, number of flowers per plant, number of fruits per plant and average yield per plant. Further P. indica decreased the number of days taken for flowering, 50 per cent flowering, fruit set and 50 per cent fruit set. Molecular basis of P.indica-mediated degradation of strobilurin and triazole fungicides was done at 3 and 7 days after the application of the fungicides. RNA was extracted and reverse transcribed into cDNA. GAPDH was used as the house keeping gene. Expression of genes involved in the degradation of strobilurin fungicides such as strH and Carboxyl esterase and Ergosterol gene involved in the degradation of triazole were studied. P. indica-colonization significantly enhanced the expression of Carboxyl esterase and Ergosterol genes in the fruits, thus increasing the fungicides degradation in fruits; whereas it repressed the expression of strH and Ergosterol genes 166

in leaves, thereby increasing the retention of fungicides in leaves under field conditions. Relative expression of the genes at 7th day after the application the fungicides was less compared to that at 3rd day. P. indica-colonization together with triazole or strobilurin fungicides application could effectively control the black mold disease caused by P. fuligena with enhanced biometric and yield characters of tomato. P. indica-colonization significantly enhanced the retention of the fungicides in leaves by down regulating the fungicide degrading genes; and degradation of fungicides in fruits by upregulating the fungicide degrading genes under field conditions with ecological sustainability and also to produce safe-to-eat tomatoes.