Browsing by Author "Nimisha, T"

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    Abiotic stress tolerance in native isolates of Beauveria bassiana (Balsamo) Vuillemin (Hypocreales :Cordycipitaceae)
    (Department of Agricultural Entomology, College of Agriculture,Vellanikkara, 2024-07-22) Nimisha, T; KAU; Deepthy, K B
    Environmental factors such as high temperature, low humidity and soil acidity often limit the biological control potential of the entomopathogenic fungus (EPF), Beauveria bassiana. Identifying abiotic stress-tolerant B. bassiana isolates represents a possible strategy to overcome this problem. Native isolates of EPF tend to adapt more to the environmental stress conditions in the locality than the exotic isolates, which are exposed to a different microclimate and microbiota. Thus, this study aimed at identification of the abiotic stress tolerant native isolates of B. bassiana and elucidation of the biochemical and molecular mechanism of stress tolerance. Survey was conducted in ten districts of Kerala, across different agroclimatic zones (159 locations). Soil samples as well as field infected cadavers were collected during the survey. The physicochemical properties of the soil samples were analysed to understand prevailing abiotic stress conditions in the area of sample collection, from which the entomopathogenic fungi isolated. For soil isolation of EPF insect bait method and serial dilution and plating method were followed. Beauveria bassiana was not obtained from any of these soil samples. However, out of the 12 field infected cadavers collected, three were infected with B. bassiana. Beauveria bassiana was identified based on the morphological characters and later confirmed by molecular characterization. Sequencing of the ITS region (550 bp) revealed genetic differences among the isolates. The sequences were submitted to NCBI GenBank (National Center for Biotechnology Information) and the accession numbers were generated (BTL1 - OP271760, BTL2 - OP290199 and PKDE - OP292066). A maximum likelihood phylogenetic tree was built, and the evolutionary relationship among the isolates were also studied. Beauveria bassiana isolates (BTL1, BTL2 and PKDE) were grouped in a single cluster confirming their genetic relationship. Bioassay against third instar nymphs of cowpea aphid (Aphis craccivora Koch) revealed that at lower concentration of 105 spores/ml, only the PKDE isolate recorded cent percent mortality compared to other two native isolates as well as NBAIR (National Bureau of Agricultural Insect Resources) strain (Bb13). As the concentration of spore suspension increased to 107 spores/ml the PKDE and BTL2 isolates were on par with NBAIR strain in terms of LT50 values. The growth and biochemical parameters of the three native isolates of B. bassiana were studied under different abiotic stress conditions. The effects of temperature (28 - 40 oC), pH (2 - 6), salinity (0.5 - 2 M) and water stress induced by polyethylene glycol (PEG 10 - 45 %) on the growth of B. bassiana were assessed. Beauveria bassiana isolate PKDE (collected from Palakkad district) tolerated a temperature stress upto 40 oC. It also survived the extreme acidity (pH 2) and salinity (1.5 M) conditions. The B. bassiana isolate, PKDE was compatible with most of the commonly used insecticides viz., chlorantraniliprole, imidacloprid, thiamethoxam and spinosad. Among the fungicides tested, hexaconazole and carbendazim completely inhibited the growth of all the three isolates, while copper oxychloride showed 89 per cent compatibility with the isolate PKDE. The PKDE isolate of B. bassiana isolated from Palakkad district has shown exceptional resistance to the effects of temperature and drought stresses. Hence, biochemical characterization of this isolate was performed to confirm their ability of stress tolerance. Significant levels of trehalose content were recorded on exposure to heat (40 oC) and drought stress (45 % PEG) (20.33 mg/g of mycelia and 20.43 mg/g of mycelia, respectively) in the multiple stress tolerant PKDE isolate. A significant activity of catalase and peroxidase was also observed in response to heat stress at 40 oC in PKDE isolate (0.0072 EU/min/mg protein and 0.0602 EU/min/g tissue weight respectively), while activity was not significant with respect to drought. In PKDE isolte the mannitol dehydrogenase (MTD) and mannitol -1-phosphate dehydrogenase (MPD) displayed significantly increased activity upon exposure to temperature stress of 40 oC (0.363 and 0.317 EU/ min/ mg protein respectively) and drought stress (0.289 and 0.364 EU/min/ mg protein respectively) induced by 45% polyethylene glycol concentration compared to the control. Field studies concluded that two sprays of B. bassiana (PKDE, BTL2 and NBAIR strain) at a spore concentration of 1x108 spores/ml at 10 days interval, suppressed the cowpea aphid incidence. No mortality of natural enemies (coccinellid beetles and spiders) was observed in the treated plots. The protein profiling of PKDE isolate under stress conditions and without stress (control) was carried out to identify the molecular basis of stress tolerance. The results revealed that there was over-expression of proteins at high-temperature stress, and the molecular weight of proteins ranged between 11-17, 35-48, 48-63 and 100- 135 kDa. The relationship between heat shock proteins and thermotolerance in fungal biocontrol agents suggests a new approach for improving entomopathogenicity by enhancing the expression of thermotolerance-related proteins in conidia. This can be achieved by identifying fungal isolates with greater thermotolerance or by optimising the components of substrate for the growth of fungi to produce more thermotolerant conidia. The nucleotide sequence analysis in the neutral trehalase gene (Bb Nth1) and high osmolarity glycerol gene (Hog), which are known to be associated with multiple stress tolerance, revealed that Bb Nth1gene sequence of the three native isolates of B. bassiana contains a coding sequence (CDS) of 2232 bp which codes for 743 amino acids and the hog gene consists of 1077 bp which codes for 358 amino acids. The polymorphism analysis in the Bb Nth1 gene revealed that seven single nucleotide polymorphisms (SNPs) in the exon region and six SNPs in the intron region, in the BTL1 isolate. In the exon3 region of the BTL1 isolate one SNP was observed with G➔C transition and codon changed from GAG to GAC. The non-synonymous variation resulted in the substitution of glutamic acid to aspartic acid at 363rd position of amino acid sequence. In BTL2 isolate eight SNPs were found in the exon region and seven SNPs in the intron region. BTL2 isolate displayed two non-synonymous variations at the 363rd position (glutamic acid to aspartic acid substitution) and at 542th position (lysine to glutamic acid) of amino acid sequence. The second SNP observed in BTL2 isolate was with G➔A transition and codon changed from GAG to AAG. In the PKDE isolate, six SNPs were found in the exon region and none of which were non-synonymous variations. Only five SNPs were found in the intron region of PKDE isolate. Variant Effect Predictor software was used to determine the functional consequences of the observed SNPs. The two mutations observed in the Bb Nth1 gene resulted in a missense variant. The Protparam and HOPE software results also revealed that the mutation resulted in protein instability. While analysing the multiple sequence alignment of susceptible and multiple stress tolerant isolates, there was no non-synonymous variation in the Hog gene. The results of bioinformatics software such as Variant Effect Predictor and Protparam supported the above findings. The present study identified multiple stress tolerant isolate of B. bassiana (PKDE isolate) isolated from Palakkad district of Kerala which is safer to natural enemies and compatible with synthetic pesticides. This isolate may be successfully integrated as a microbial control agent in IPM programme.
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    Elucidating the biochemical basis of interaction between tea mosquito bug, helopeltis antonii signoret (hemiptera:miridae) and cashew (anacardium occidentale)
    (Department of Agricultural Entomology, College of Horticulture, Vellanikkara, 2018) Nimisha, T; Deepthy, K B
    Cashew is one of the important foreign exchange earning crops of India with an export value of Rs. 5,077 crores during 2016-17.Cashew production is not in tune with increase in area under its cultivation. Low productivity in cashew is mainly due to the incidence of pests and diseases. Among the insect pests, tea mosquito bug (TMB), Helopeltis antonii Signoret (Hemiptera: Miridae) is the most important. It causes 30-50 per cent yield loss and during outbreak situations even up to 100 per cent loss has been reported. Cashew varieties exhibit wide variation in response to TMB infestation. Hardly any variety has ever been recorded as resistant to TMB till date. However, a few accessions are reported to be capable of withstanding TMB infestation and hence have been grouped as less susceptible category. Understanding the basis of interaction between the bug and the cashew is a prerequisite in developing varieties resistant to TMB. The present study entitled “Elucidating the biochemical basis of interaction between tea mosquito bug, Helopeltis antonii Signoret (Hemiptera: Miridae) and cashew (Anacardium occidentale)” was undertaken at the Department of Agricultural Entomology, College of Horticulture, Vellanikkara during September 2017 to July 2018. The objective of the study was to understand the variation in secondary metabolites and defense enzymes in selected cashew varieties induced by tea mosquito bug infestation and to elucidate the secondary metabolite detoxification mechanisms in the pest. Three months old grafts of four cashew varieties viz., two from the highly susceptible category (Anagha, Madakkathara-1) and two from less susceptible category (Damodar, Raghav) were used to conduct the experiment. Female adult bug (0-24 h old) was allowed to feed on each variety for different time intervals of 6, 24, 48 and 72 h. The biochemical parameters such as protein, phenol, tannin and defense enzymes viz., polyphenol oxidase (PPO) and phenyl alanine ammonia lyase (PAL) in leaves of TMB infested and non infested cashew grafts were analysed before release (0 h) as well as at different intervals of release. Biochemical analysis of the released TMB (crude homogenate of whole insect) viz., variation in total protein, SDS PAGE profiling of TMB crude protein, detoxifying enzymes viz., carboxyl esterase, cytochrome P450, and glutathione-s-transferases (GST) were carried out before releasing on the plant and at different intervals after release. The total leaf protein was found to be highest in the less susceptible variety Damodar (0.9925 mg g-1) and the lowest in the highly susceptible Madakkathara-1 (0.6729 mg g-1). Total phenol content was highest in the less susceptible Damodar (69.834 mg g-1) and Raghav (67.207 mg g-1) and the lowest was recorded in Anagha (29.625 mg g-1). Regardless of the varieties, highest phenol content was recorded in samples taken after 72 h of TMB infestation. Tannin content was also high in the less susceptible varieties Raghav (4.420 mg g-1) and Damodar (4.276 mg g-1) while highly susceptible varieties Anagha and Madakkathara-1 recorded lower values. Irrespective of the varieties, the highest tannin content was recorded in samples before the release of TMB (6.662 mg g-1). Results of the present study revealed that, detoxifying enzyme specific activity of PPO was highest in Damodar (0.003158 EU g-1min-1) and observed lowest value in Anagha (0.001406 EU g-1min-1). Irrespective of the varieties, enzyme activity reached highest at 48 h of release (0.00367 EU g1min-1). PAL specific activity was highest in Madakkathara-1 (0.575 μg g-1 min-1) and lowest in Anagha and was on par with Raghav and Damodar. In SDS PAGE, the whole body homogenate of adult female TMB revealed presence of bands in the range of ̴ 63 kDa and 35-48 kDa in the early stage of infestation. However, as the exposure time increased, protein bands ranging from 17-75 kDa appeared. Defensive enzymes viz., carboxyl esterase and GST expressed elevated activity in TMB that fed on less susceptible Raghav (258.117 μmol min-1mg-1 protein, and 365.262 μmol min-1mg-1 protein respectively) and Damodar (208.916 μmol min-1mg-1protein, and 501.879 μmol min-1mg-1protein respectively) when compared to the highly susceptible varieties. Cytochrome P450 showed highest activity in TMB fed on Damodar (0.372 nmol min-1mg-1 protein) and it was having lowest activity in insect fed on other three varieties. The study revealed that, the secondary metabolites viz., phenol, tannin and PPO have a definite role in imparting resistance in cashew to TMB attack. These metabolites have potential use in early detection of resistance in cashew against TMB. The enhanced levels of detoxification enzymes in TMB indicate plasticity of the pest against host plant defense and chance of resistance development against synthetic insecticides.