Novel strains of Metarhizium anisopliae sorokin.(Ascomycota:sordariomycetes) with enhanced abiotic stress tolerance
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Date
2023-08-02
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Department of agricultural entomology, college of agriculture, Vellanikkara
Abstract
Novel strains of Metarhizium anisopliae Sorokin. (Ascomycota: Sordariomycetes) with enhanced abiotic stress tolerance Abstract The green muscardine fungus, Metarhizium anisopliae Sorokin is widely used in the management of various crop pests. However the efficacy of M. anisopliae in the field is often inconsistent, mainly due to the environmental stresses such as temperature extremes, drought, UV radiation, etc. To circumvent these hurdles, the present study was undertaken to develop novel strains of M. anisopliae with enhanced environmental stress tolerance. The Metarhizium isolates, EKM2, CKD, and M4 were collected from the repository maintained at the Department of Agricultural Entomology and were screened for their temperature and drought tolerance. The isolate EKM2 recorded the highest mycelial weight (6.221g), while the isolate M4 recorded the least mycelial weight (0.328 g) and the least sporulation at the highest temperature of 380C. The screening for drought tolerance was conducted at various levels of Polyethylene glycol (PEG) concentrations in which the isolate CKD has shown higher biomass (2.172 g) with higher sporulation, while isolate M4 recorded the lowest mycelial weight (1.336 g) at the maximum PEG concentration of 39 per cent. The three selected isolates of M. anisopliae (CKD, EKM2 and M4) were subjected to further experiments. The Metarhizium isolates (CKD, EKM2 and M4) selected from the preliminary screening were continuously subjected to temperature stress treatments starting from 35oC to 42oC. At the highest temperature of 41oC, the isolate, EKM2 recorded the highest mycelial weight (1.336 g), number of colonies (18.667103 cfu/ml), number of spores (0.783 X 10 7 /ml), spore size (3.470 µm) and mycelial thickness (3.07 µm). At the same temperature, the isolate, M4 recorded the lowest mycelial weight (0.63g), number of colonies (3 cfu/ml), number of spores (0.217 X 10 7 /ml), spore size (1.637 µm), and mycelial thickness (2.65 µm). Hence EKM2 has been identified as the thermo-tolerant isolate and M4 as the susceptible one. The thermo-tolerant isolate EKM2 was grown continuously for 6 successive generations at the same temperature level of 41oC in order to stabilize the stress tolerance. The Metarhizium isolates (CKD, EKM2, and M4) selected from the preliminary drought screening experiment were also continuously subjected to drought-inducing treatments at PEG concentrations starting from 25 to 40 per cent. At the highest level of PEG concentration (40 %), the isolate CKD showed higher mycelial weight (3.365 g), spore count (0.323X 107 /ml)), number of colonies (4 X 103 colonies/ ml), spore size (3.15 µm), and mycelial thickness (2.723µm). The isolate, M4 recorded the lowest mycelial weight (0.808g), number of colonies (2 cfu/ml), spore count (0.243 spores/ml), spore size (4.493 µm), and mycelial thickness (2.543 µm) at the same PEG concentration. Hence isolate CKD is identified as drought tolerant and M4 as susceptible. The drought-tolerant isolate, CKD was grown continuously for 6 successive generations at the same PEG concentration level of 40 per cent in order to stabilize the stress tolerance. The selected tolerant isolates were evaluated against wax moth larva (Galleria mellonella) to identify their biocontrol efficacy. Isolate EKM2 has shown cent per cent mortality at the dosage of 108 and 109 spores/ml on the 9th day after treatment. At the highest dosage of 1x 109 spores/ ml, the isolate CKD recorded a lower LT50 (5.099 days) value, and isolate M4 recorded the highest LT50 (6.124 days) value. The isolates EKM2 and CKD were found to be more virulent while M4 was less virulent. The thermo-tolerant isolate, EKM2 recorded a total protein content of 0.354 mg/ml, and the drought-tolerant isolate (CKD) recorded a total protein content of 0.373 mg/ml. The thermo-tolerant isolate EKM2 has shown a trehalose content of 1.954 mg/min/g tissue weight which was significantly higher compared to the control (1.074 mg/min/g tissue weight). The trehalose content in the drought-tolerant isolate CKD (1.970 mg/min/g) was also higher when compared to the control (1.224 mg/min/g). The catalase activity ranged between 0.237 EU/min/mg proteins in the control to 0.386 EU/min/mg protein in the thermo-tolerant isolate EKM2, while the catalase activity of the drought-tolerant isolate, CKD was 0.384 c protein which was higher when compared to its control (0.240 EU/min/mg protein). The thermo-tolerant isolate recorded a peroxidase activity of 0.0230 EU/min/g tissue and that of droughttolerant isolate CKD was 0.0236 EU/min/g tissue weight. The protease activity of thermo-tolerant isolate CKD was 0.490 EU/min/mg protein and that of the droughttolerant isolate was 0.621 EU/min/mg protein. At 25 minutes of reaction, the thermo-tolerant and drought-tolerant isolates have shown higher lipase activity (10.000 µmol fatty acid/ml and 11.00 µmol fatty acid/ml respectively) when compared to their respective controls. On the 5th day, both the thermo-tolerant and drought- tolerant isolates recorded significantly higher chitinase activity (1.140 µg of N- acetyl glucose amine/min/ml of broth and 3.062 µg of N- acetyl glucose amine/min/ml of broth respectively) than their controls. The temperature induction had up-regulated heat shock proteins of 25 kDa and 35 kDa in heat-induced isolate EKM2. The isolate also expressed heat shock proteins in the range of 100 kDa molecular weight. Drought tolerant isolate (CKD) on the other hand did not show the presence of any heat shock protein when compared to the control. The study could develop isolates of M. anisopliae which are tolerant to temperature and drought stress, with high virulence as well as higher biochemical activity