1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
Permanent URI for this communityhttp://localhost:4000/handle/123456789/1
Browse
2 results
Search Results
Item Physiological and molecular assessment of abiotic stress tolerance in selectively fertilized coconut hybrids (Cocos nucifera L.)(Department of Plant Physiology, College of Agriculture,Vellayani, 2024-03-14) Afna Mol, O P; KAU; Roy Stephen; Gladis, RThe present study entitled “Physiological and molecular assessment of abiotic stress tolerance in selectively fertilized coconut hybrids (Cocos nucifera L.) was carried out in the Department of Plant Physiology, College of Agriculture, Vellayani during 2018-2023. The objective of the study was physiological and molecular evaluation of selectively fertilized coconut hybrids for water stress and temperature stress tolerance and to assess genetic variability for temperature stress tolerance through pollen selection at critical temperature. The study consisted of four experiments. In the first experiment, 10 year old field grown coconut palms of the following varieties Kerasree (WCT X MYD)- 198 selectively fertilized, Keraganga (WCT X GB)-selectively fertilized, Kerasree (WCT X MYD) Hybrid, Keraganga (WCT X GB) Hybrid and West Coast Tall at Regional Agricultural Research Station, Pilicode, Kasaragod were used as the experimental material for the study. The experiment was carried out in Randomized Block Design (RBD) with four replications during 2019 March to 2019 April. Water stress was imposed by withdrawing irrigation from the field for one month and physiological and biochemical observations were made at 15 days interval .Then the palms were irrigated on 30th day and recovery responses were analysed on 15th day after the irrigation rescheduling. On physiological analysis, selectively fertilized Kerasree showed higher relative water content and epicuticular wax of the leaves. Keraganga selectively fertilized showed maximum photosynthetic rate and cell membrane stability index. Leaf temperature was minimum in Kerasree selectively fertilized hybrids followed by Keraganga selectively fertilized hybrid. WCT recorded higher transpiration rate followed by Kerasree selectively fertilized hybrid. Stomatal conductance was higher in WCT and then in Kerasree selectively fertilized hybrids. In all the parameters tested both the selectively fertilized hybrids responded positively towards drought than their normal hybrids and this was an indication of higher water use efficiency in them. Recovery response after rewatering was also higher in selectively fertilized hybrids than their normal hybrids. The biochemical parameters like chlorophyll content, antioxidant enzymes viz SOD and peroxidase activities were found maximum in Kerasree selectively fertilized hybrids under water stress condition. Recovery response of these parameters were also found elevated in Kerasree selectively fertilized hybrids. Also Kerasree selectively fertilized hybrids showed lowest membrane damage (lipid peroxidation value) compared with normal hybrids and WCT. Amount of proline in leaves was found higher in WCT, which was on par with Kerasree selectively fertilized hybrid and Keraganga selectively fertilized hybrid. 199 In the second experiment, molecular characterization was carried out in the above five treatments. Protein profiling of leaves was done in all the five treatments of experiment one, using SDS-PAGE, in which, there was variation in the intensity of large subunit (55 kDa) as well as small subunit (around16 kDa) of RuBisCO, among the treatments. The intensity of those bands were found higher in WCT and Kerasree selectively fertilized hybrid .The band expressed in selectively fertilized hybrid in between 26 kDa to 36 kDa was absent in other treatments.. SSR markers CncirE2, CncirE10, CncirE12 and CncirH4 were screened in five treatments and polymorphism was observed in CncirE2, CncirE12, and CncirE10. RNA-seq tranascriptomic analysis of Kerasree selectively fertilized hybrid in reference with Kerasree was done to identify the key differentially expressed genes (DEGs) responding to water stress tolerance in Kerasree selectively fertilized hybrid. A total of 16671 genes were found expressed in Kerasree SF differing from Kerasree. 9635 genes were upregulated and remaining 7036 genes were down regulated in Kerasree SF.Abiotic stress related genes like late embryogenesis abundant protein Lea5 and HSPs were found differently expressed in Kerasree S.F hybrid in response to water stress. Gene ontology of the identified genes revealed that in terms of biological processes the following were the most represented GO terms (GO:0006749, 24 proteins, GO:0006468, 23 proteins,GO:0009415,12). In cellular component (GO: 0016020, 31 proteins) and in molecular function (GO: 0005524, 56 proteins, GO: 0009408, 21 proteins) the above shown were the most represented GO terms. In experiment three, physiological assessment of temperature stress tolerance of selectively fertilized coconut hybrids was done. Pollen grains were collected from the selectively fertilized coconut palms during summer season. The pollen grains were incubated in specific pollen germination media and exposed to different temperature ranging from 35oC to 45oC for a period of 2 hours. The temperature at which only 20-30 % of pollen germinated was identified as the critical temperature stress for pollen germination. From the results obtained, both the selectively fertilized hybrid of Kerasree and Keraganga were noted with maximum critical temperature for pollen germination (42oC) followed by WCT (40oC).This indicated 200 the temperature stress tolerance of selectively fertilized hybrids over their normal hybrids. Pollen viability was found maximum in WCT followed by Kerasree selectively fertilized hybrid. Accumulation of starch was found significantly higher in Kerasree selectively fertilized hybrid and the protein in WCT. The fourth experiment was conducted to screen temperature stress tolerance of 30 coconut genotypes in Regional Agricultural Research Station, Pilicode for recognizing the temperature stress tolerance through critical temperature for pollen germination. Significant genetic variation in critical temperature was discerned among the treatments. Andaman ordinary and Tanjore coconut genotypes were observed with higher critical temperature (42oC) for pollen germination. Hence they can be used as a pollen parent in hybridization programs for developing temperature stress tolerance. The present study revealed that Kerasree selectively fertilized and Keraganga selectively fertilized hybrids of coconut were more tolerant to water stress than normal hybrids in terms physiological, biochemical and molecular parameters. Selectively fertilized hybrids developed through pollen selection at critical water potential also have temperature stress tolerance. By selective fertilization techniques it may be possible to add water stress and temperature stress tolerance traits to high yielding coconut hybrids.Item 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 BEnvironmental 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.