MSc

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