1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Gene expression and bioactivity estimation of Piriformospora Indica co-cultivated red amaranth (Amaranthus tricolor) plants under temperature stress conditions(Department of molecular biology and biotechnology,college of agriculture, Vellayani, 2023-11-22) Sneha Stephen; Sabu, K KAmaranthus tricolor is one of the major leafy vegetables. Red amaranth is most vulnerable to climatic changes, especially a rise in temperature. Piriformospora indica symbiosis with red amaranth reduces the negative effects of temperature stress. . This study was aimed to gene expression and bioactivity estimation of P. indica co-cultivated red amaranth plants under temperature stress conditions. One week old red amaranth seedlings were is transferred to a suitable medium containing soil with P. indica for co-culture. P. indica co-cultured red amaranth was maintained in a growth chamber with various temperature treatments for a period of three hours at 25οC, 30C, 35C and 40C. Uninoculated plants maintained at 25οC, 30C, 35C, and 40C was be served as control. The changes in plant phenotypes and biochemical parameters were measured. To identify temperature stress tolerant genes conferred by P. indica, colonized and uncolonized plants were analyzed after 3 hours of temperature stress induction. Results obtained showed that colonization of red amaranth roots by P. indica revealed significant variation in leaf number, leaf length, leaf area and plant height of P. indica plants compared to untreated plants for a period of 2 months at 15 days intervals. In addition, chlorophyll a, b, a + b, carotenoid and anthocyanin contents were increased in the leaves of the P. indica colonized plants under temperature stress conditions. Mineral estimation revealed that plants treated with P. indica increased the accumulation of Ca and Mg elements and slightly decreased the oxalate content. Furthermore, the expression of the various genes catalase, ascorbate peroxidase, superoxide dismutase, dihydroflavonol 4- reductase, sHSP 17.8 and 17.9 genes were significantly up- regulated in P. indica treated plants in comparison with untreated plants. At 25°C the expression of CAT, APX, SOD, DFR, sHSP 17.8 and sHSP 17.9 were up-regulated by 1.5, 2, 2.5, 2, 2.9, and 1.4 folds. At 30°C the expression of CAT, APX, SOD, DFR, sHSP 17.8 and sHSP 17.9 were up regulated by 2.5, 2.45, 2.8, 2.5, 3.1 and 1.9 folds. At 35°C the expression of CAT, APX, SOD, DFR, sHSP 17.8 and sHSP 17.9 were up-regulated by 3.35, 2.8, 3.5, 3.56, 3.35 and 112 2.6 folds. At 40°C the expression of CAT, APX, SOD, DFR, sHSP 17.8 and sHSP 17.9 were up-regulated by 3.5, 3, 3.7, 3, 3.68 and 3.2. In conclusion, P. indica has the potential to enhance the growth of red amaranth under temperature stress and increased phenotypic and physiological parameters of the plant which would help them to grow in higher temperature zonesItem Role of microbes in the management of cassava mosaic disease(Department of molecular biology and biotechnology, college of agriculture , Vellayani, 2023-09-18) Athulya, V AThe study entitled “Role of microbes in the management of Cassava Mosaic Disease” was carried out at the Division of Crop Protection, ICAR- Central Tuber Crops Research Institute and the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani in the year 2022-2023 to analyse the role of microbial biocontrol agents in reducing the incidence of Cassava Mosaic Disease. Infected cuttings of two susceptible cassava varieties H226 and Sree Prakash were collected from the fields at ICAR-CTCRI. They were identified to be infected with Cassava Mosaic Virus by visual observation of disease symptoms. DNA was isolated from the leaves of these plants by CTAB method and PCR was performed to confirm infection using the coat protein primer pair. Ten isolates of Trichoderma asperellum and Bacillus sp. were used. They were cultured in Potato Dextrose Broth and Nutrient Broth respectively. The infected cuttings were immersed in the microbial suspensions and treated using a sett treating device for 15 min at 15 lbs pressure. They were then planted in trays in a mixture of coir pith and soil, along with untreated control. Six replicates were used for all. Biometric observations like number of leaves, branch length and symptom scores were recorded for three months from the date of planting. DNA was isolated at one and three months after planting and PCR was performed to confirm infection. qPCR was performed on this DNA to check the viral load in the plants. Three isolates of Trichoderma asperellum (T4, T7 and T8) showed reduction in viral load as compared to control in H226 plants. After the first month, plants treated with T4 had a viral load of 1.36x108 copies/10ng and T7 had 1.26x105 copies/10ng while control had 105 copies/10ng. After three months, plants treated with T4 had 53.2 copies/10ng, T7 had 58.5 copies/10ng and T8 had 68.5 copies/10ng while control had 3.9x109 11 2 copies/10ng. Two isolates (B3 and B8) of Bacillus sp. showed reduction in viral load in Sree Prakash variety. One month after planting, plants treated with B3 (Bacillus subtilis) had a viral load of 1.27x109 copies/10ng while untreated control had 7.4x105 copies/10ng. After three months, plants treated with B3 had a viral load of 1.74x104 copies/10ng and those treated with B8 had 8.1x103 copies/10ng while control had 8.7x105 copies/10ng. The biometric observation did not always show correlating trends. In Trichoderma treated H226 plants, the percentage changes in disease severity were 9.1% reduction for T7 and 10.9% reduction for T8 over control after one month. After three months, there was a 25% increase for T4, 8.3% increase for T7 and 4.2% increase for T8 in disease severity over control. In Sree Prakash plants treated with Bacillus sp., the percentage change in disease severity was 24% increase for both treatments B3 and B8 after one month, over control. After three months, the percentage change in disease severity was 26.75% increase with B3 over control. Primers were designed for two plant defense genes ETR1 and PAL1 using Primer3 online tool for future study of the expression of these genes. This may help understand the mechanism of action of these biocontrol agents in reducing viral load. Thus, certain isolates of Trichoderma asperellum and Bacillus sp. were found to have effective biocontrol activity against Cassava Mosaic Disease in this preliminary study. Further studies with a larger number of replicates and longer growth period is necessary for proper validation of the results.Item Role of microbes in the management of cassava mosaic disease(Department of molecular biology and biotechnology, college of agriculture, Vellayani, 2023-09-18) Athulya, V A.; Makeshkumar, TThe study entitled “Role of microbes in the management of Cassava Mosaic Disease” was carried out at the Division of Crop Protection, ICAR- Central Tuber Crops Research Institute and the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani in the year 2022-2023 to analyse the role of microbial biocontrol agents in reducing the incidence of Cassava Mosaic Disease. Infected cuttings of two susceptible cassava varieties H226 and Sree Prakash were collected from the fields at ICAR-CTCRI. They were identified to be infected with Cassava Mosaic Virus by visual observation of disease symptoms. DNA was isolated from the leaves of these plants by CTAB method and PCR was performed to confirm infection using the coat protein primer pair. Ten isolates of Trichoderma asperellum and Bacillus sp. were used. They were cultured in Potato Dextrose Broth and Nutrient Broth respectively. The infected cuttings were immersed in the microbial suspensions and treated using a sett treating device for 15 min at 15 lbs pressure. They were then planted in trays in a mixture of coir pith and soil, along with untreated control. Six replicates were used for all. Biometric observations like number of leaves, branch length and symptom scores were recorded for three months from the date of planting. DNA was isolated at one and three months after planting and PCR was performed to confirm infection. qPCR was performed on this DNA to check the viral load in the plants. Three isolates of Trichoderma asperellum (T4, T7 and T8) showed reduction in viral load as compared to control in H226 plants. After the first month, plants treated with T4 had a viral load of 1.36x108 copies/10ng and T7 had 1.26x105 copies/10ng while control had 105 copies/10ng. After three months, plants treated with T4 had 53.2 copies/10ng, T7 had 58.5 copies/10ng and T8 had 68.5 copies/10ng while control had 3.9x109 11 2 copies/10ng. Two isolates (B3 and B8) of Bacillus sp. showed reduction in viral load in Sree Prakash variety. One month after planting, plants treated with B3 (Bacillus subtilis) had a viral load of 1.27x109 copies/10ng while untreated control had 7.4x105 copies/10ng. After three months, plants treated with B3 had a viral load of 1.74x104 copies/10ng and those treated with B8 had 8.1x103 copies/10ng while control had 8.7x105 copies/10ng. The biometric observation did not always show correlating trends. In Trichoderma treated H226 plants, the percentage changes in disease severity were 9.1% reduction for T7 and 10.9% reduction for T8 over control after one month. After three months, there was a 25% increase for T4, 8.3% increase for T7 and 4.2% increase for T8 in disease severity over control. In Sree Prakash plants treated with Bacillus sp., the percentage change in disease severity was 24% increase for both treatments B3 and B8 after one month, over control. After three months, the percentage change in disease severity was 26.75% increase with B3 over control. Primers were designed for two plant defense genes ETR1 and PAL1 using Primer3 online tool for future study of the expression of these genes. This may help understand the mechanism of action of these biocontrol agents in reducing viral load. Thus, certain isolates of Trichoderma asperellum and Bacillus sp. were found to have effective biocontrol activity against Cassava Mosaic Disease in this preliminary study. Further studies with a larger number of replicates and longer growth period is necessary for proper validation of the results.