PhD

The study entitled “Tolerance of entomopathogenic fungi Lecanicillium
saksenae (Kushwaha) Kurihara and Sukarno and Lecanicillium lecanii (Zimm.) Zare
and Gams to abiotic stress” was conducted at Department of Agricultural
Entomology, College of Agriculture, Vellayani, Thiruvananthapuram, during the
period 2017-22. The objective of study was to assess the tolerance level of
L. saksenae and L. lecanii to insecticides, fungicides, temperature and UV radiation
and explore the possibilities of developing tolerant strains.
Results of the experiment to study the tolerance of L. saksenae and L. lecanii
to insecticides revealed that the new generation insecticides viz., flubendiamide
39.35% SC, chlorantraniliprole 18.5% SC, imidacloprid 17.8% SL and
thiamethoxam 25% WG were compatible with both the fungi in half dose,
recommended dose and double the recommended doses. Among the old generation
insecticides, only dimethoate 30% EC was found to be compatible at half and
recommended doses. Malathion 50% EC, quinalphos 25% EC and chlorpyrifos 20%
EC were found to be toxic to both the fungi at all the test doses. Among all the
insecticides quinalphos 25% EC was found to be highly toxic with a Biological Index
(BI) of 14 to 22.
Among the fungicides tested, azoxystrobin 23% SC was found to be
compatible with L. saksenae and it was moderately toxic to L. lecanii. Copper
oxychloride 50% WP was found to be moderately toxic to L. lecanii and toxic to L.
saksenae. Carbendazim 50 % WP, mancozeb 75% WP, hexaconazole 5% EC and
tebuconazole 25 % EC were found to be toxic to both the fungi based on BI values.
Artificial selection to induce tolerance to incompatible insecticides and
fungicides was attempted by subculturing fungi in respective poisoned media for ten
successive generations. No improved strain could be developed for quinalphos
tolerance in both the fungi. Induction of fungicide tolerance resulted in two tolerant
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strains in L. saksenae but no improved strain could be developed in L. lecanii.
Hexaconazole tolerant strain of L. saksenae was able to tolerate the recommended
field dose (0.15 %). Carbendazim tolerant strain exhibited increase in growth,
sporulation and germination at the recommended dose (0.2%) compared to those in
the non-selected culture. REP-PCR data revealed the genotypic variation of the
strains. The variation quantified based on Jaccard’s coefficient analysis revealed that
carbendazim tolerant strain and hexaconazole tolerant strain were 42 per cent and 38
per cent dissimilar from mother culture.
Temperature tolerance study carried out at varying regimes of 30, 32, 34, 36,
38 and 40 oC (8h exposure) revealed that increase in temperature significantly
affected the growth parameters of both the fungi. Growth of L. lecanii and
L. saksenae was completely inhibited at 36 and 40 oC, respectively. In terms of
sporulation, no exponential reduction (>10 7
spores mL-1
) was observed till 34 o C in
L. saksenae, while in L. lecanii it was reduced to 105
spores mL-1
and it was
terminated at 36 oC. Comparative tolerance between the species revealed that
L. saksenae performed better than L. lecanii. No significant reduction (<15 per cent)
in terms of germination was observed in L. saksenae till 36 oC, while in L. lecanii
more than 70 per cent reduction was observed at 34 oC. L. saksenae spores were able
to germinate till 40 oC while in L. lecanii germination was completely inhibited at
this temperature. Attempts to induce temperature tolerance by subculturing the fungi
at highest tolerant temperature for ten generations did not yield any tolerant strain.
UV tolerance study conducted with two irradiances wave length viz., UVA -
365 nm and UVB - 290 to 315 nm for 10, 20, 30 min, 1h, 2h, 3h and 4h exposure
durations, revealed that the growth of both the fungi was not significantly affected
when exposed for shorter durations (10, 20 and 30 min), whereas when exposed for
longer durations (1, 2, 3 and 4h), there was significant reduction in growth. In both
the wave lengths and across the exposure durations, L. saksenae exhibited higher
colony growth than L. lecanii. In UVA irradiance, the growth reduction ranged
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between 13 to 75 per cent in L. saksenae and it was 23 to 81 per cent in L. lecanii.
In UVB irradiance, colony reduction was between 12 to 85 per cent in L. saksenae
and 31to 89 per cent in L. lecanii. In terms of sporulation, no exponential reduction
was observed in both the fungi. There was germination inhibition in both the fungi
exposed to UVA and UVB irradiance. In shorter exposures, L. lecanii exhibited
higher germination (30 to 51 per cent) while in longer exposures L. saksenae
exhibited higher germination (13 to 16 per cent).
UV mutagenesis resulted in two UV mutants one each in L. saksenae and
L. lecanii. L. lecanii mutant was named as LlUVM1 and L. saksenae mutant was
named as LsUVM. Both the mutants were superior in terms of growth parameters
when tested under UVA and UVB irradiance when compared to their mother culture.
The REP-PCR analysis revealed that UV mutants LsUVM and LlUVM1 varied
genotypically from the mother culture. Based on Jaccard’s coefficient there was 32
per cent dissimilarity with their respective mother cultures.
Bioassay carried out to study the effect of artificial selection on the
pathogenicity of fungicide tolerant strains of L. saksenae on rice bug Leptocorisa
oratorius Fab. revealed that spore suspensions of carbendazim and hexaconazole
tolerant strains of L. saksenae (107
spores mL -1
) in the presence of respective
fungicide, exhibited higher virulence than the mother culture. Mortality caused by
the tank mix formulation of carbendazim tolerant strain + 0.2 % carbendazim 50 %
WP caused 86.67 per cent mortality, while its mother culture resulted in 36.67 per
cent mortality. Hexaconazole tolerant strain caused 60 per cent mortality while the
mother culture did not cause mortality with tank mix of hexaconazole 5 % EC
0.15 %.
Pathogenicity assay of L. saksenae UV mutant revealed that there was no
significant difference in mortality of L. oratorius between the mother culture and the
mutant. In L. lecanii the UV mutant (LlUVM1) exhibited higher virulence on the
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brinjal mealybug, Coccidohystrix insolita Green. LlUVM1 caused 100 per cent
mortality on the eighth day, while the mother culture of L. lecanii caused 83.75 per
cent mortality.
Therefore, the study concludes that the indigenous isolate L. saksenae is
comparatively more tolerant to insecticides, temperature and UV exposure than
L. lecanii. Strains of L. saksenae developed for carbendazim and hexaconazole
tolerance performed better in terms of phenotypic characters. Genotypically, they
expressed variation of 42 and 38 per cent respectively from mother culture. The UV
tolerant strain developed in both the species were superior to the mother culture
under UV irradiance. The findings pave way to the utilization of the indigenous
isolate of L. saksenae and its fungicide tolerant mutants and the UV mutant of
L. lecanii in pest management as they can combat abiotic stress in prevailing climatic
conditions.