1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)

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Expression profiling of genes associated with nitrogen use efficiency in rice varieties under organic and conventional management
(Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2024-05-02) Elizabeth Jose; KAU; Soni, K B
The study entitled “Expression profiling of genes associated with nitrogen use efficiency in rice varieties under organic and conventional management” was conducted in the Department of Molecular Biology and Biotechnology during 2019-2023. The objectives of the study were to identify differentially expressed genes associated with nitrogen use efficiency (NUE) in rice varieties under organic and conventional management and to find out the polymorphism in the gene sequences. A pot culture experiment was conducted in a completely randomized design with five replications. Four rice varieties namely Jaiva, Ezhome 2, Uma and Jyothi were compared under two nutrient management practices (KAU PoP, Adhoc Organic PoP of KAU) along with an absolute control. Under organic management, nutrients were applied on N equivalent basis. The observations on morphological, physiological, biochemical and yield parameters and expression of genes associated with NUE were taken at seedling, panicle initiation and grain filling stages. All rice varieties showed significantly taller plants, greater number of tillers per hill, higher grain and straw yields per hill with KAU PoP. Jaiva and Ezhome 2 showed a significantly greater number of productive tillers per hill (P=0.001 and P=0.02) under KAU PoP, and Ezhome 2 produced higher filled grains per panicle under organic PoP. Jyothi showed significantly higher root depth and root biomass per hill (46.34 cm, 18.6 g) under organic PoP. At the vegetative stage, all varieties except Jaiva and Uma, exhibited significantly higher photosynthetic rates under KAU PoP. The varieties Ezhome 2 and Uma showed higher leaf protein content under organic PoP. All rice varieties, except Ezhome 2, showed significantly higher amino acid content under KAU PoP. The nutrient management practices did not significantly affect the reducing sugar content in Jyothi, compared to other varieties which showed higher values under KAU PoP. All the varieties showed higher NUE with KAU PoP than organic PoP. Maximum NUE was seen in Jyothi (0.28 kg yield increase/g N applied) under KAU PoP. Variation in NUE under both nutrient management was found to be lesser in the variety Jaiva (23.8%). 195 Expression profiles of NUE-associated genes such as OsNRT1.1a, OsNRT1.1b, OsAMT1.1, OsAMT1.2, OsAMT1.3, OsNRT2.1, OsNAR2.1, OsGS1.1, OsNADH-GOGAT1 were analyzed at different stages of growth by performing RT-qPCR using SYBR Green dye and β-actin as the reference gene. At the three-week seedling stage, ammonium uptake and transporter genes OsAMT1.1, OsAMT1.2, and nitrate uptake and transporter gene OsNRT2.1 which are root-specific in nature, exhibited significantly higher expression under organic PoP in Jaiva and Jyothi. Jyothi also showed a significantly high expression of glutamine synthase gene, OsGS1.1 in the leaf and root tissues (0.72 and 8.28 folds respectively). OsNADH-GOGAT1, a gene associated with nitrogen assimilation exhibited significantly higher expression in the root tissues of all rice varieties, except Jaiva under organic PoP. At the panicle initiation stage, Uma and Jyothi showed comparatively higher expression of OsNRT1.1a, OsNRT1.1b, OsAMT1.1, OsAMT1.2, OSAMT1.3, OsNRT2.1, OsNAR2.1 and OsNADH-GOGAT1 under organic PoP. At the grain-filling stage, Uma showed significantly higher expression of OsAMT1.1 (3.19 folds), OsAMT1.2 (5.24 folds), OsAMT1.3 (7.65 folds), OsNAR2.1 (11.02 folds), OsNADH-GOGAT1 (2.2 folds) in root tissues under organic PoP. Jyothi, under organic management, showed significantly higher expression of the OsNRT2.1 gene (6.95 folds) in root tissues. In Jaiva, nitrogen assimilatory genes like OsGS1.1 and OsNADH-GOGAT1 showed higher expression in leaf and root tissues under PoP and for Jyothi, they were found elevated (P<0.005) under organic PoP. The full-length sequencing of differently expressed OsAMT1.1 of Jaiva yielded a sequence comprising 2671 bp. This is the initial comprehensive gene sequencing report for the OsAMT1.1 gene of an organic rice variety. It showed a similarity (99.7%) to that of NERICA L19 (O. sativa × O. glaberrima). Uma exhibited an insertion of 41 bp in the promoter region compared to the other three varieties. Furthermore, some single nucleotide polymorphisms were also identified in the promoter region of Jaiva, Ezhome, Uma and Jyothi. In conclusion, the present study explored the response of the key genes involved in the uptake, transport and assimilation of nitrate and ammonium under different nutrient management strategies in determining nitrogen use efficiency in rice varieties. 196 Jaiva and Jyothi exhibited superior efficiency in both nitrate and ammonium uptake, correlating with higher NUE and narrower yield gaps between organic and fertilizer-based management. In the initial stages of crop growth, an elevated expression of OsAMT1.1 and OsAMT1.2 was seen in Jaiva and Jyothi under organic management, whereas OsAMT1.3, which has a N-derepressible role was highly expressed in Uma under organic management. The variety, Uma was found to be less nitrogen-efficient and was highly influenced by ammoniacal fertilizer. Even though OsAMT1.1 and OsAMT1.2 were less expressed in the initial stages of Uma, a significant increase was observed in its later stages. OsNRT2.1 and its partner protein OsNAR2.1, which are important in regulating nitrate transport and signaling, were upregulated in Jaiva and Jyothi under organic PoP, influencing root development and contributing to superior agronomic use efficiency. The higher expression of OsGS1.1 in the seedling and grain filling stages of Jyothi under organic PoP contributed to grain development and filling, demonstrating better NUE and a reduced yield gap. Ezhome 2, by exhibiting higher OsGS1.1 expression at the seedling stage, displayed increased filled grains, emphasizing the significance of early gene expression in yield determination. Furthermore, OsNADH-GOGAT1's role in ammonia assimilation, productive tiller formation, and grain development was evident, with varieties like Ezhome 2 showcasing its positive impact on grain number and tiller productivity under organic PoP. The findings highlight the complexity of nitrogen response and metabolism in rice and emphasize the importance of nutrient management strategies in achieving optimal NUE and crop yield. The observed variations in the gene expression across different varieties and growth stages provide a foundation for further research and potential molecular interventions to enhance NUE in rice.
Assessment, mapping and modelling of soil carbon pools and stock in selected agro-ecological units of south Kerala
(Department of Soil Science and Agricultural Chemistry, College of Agriculture ,Vellayani, 2024-04-22) Bincy, B; KAU
A study entitled “Assessment, mapping and modelling of soil carbon pools and stock in selected agro-ecological units of south Kerala” was carried out to examine the impact of various agricultural land use systems on soil carbon fractions, pools and stock, soil aggregation, glomalin and polysaccharide contents, and to generate soil carbon maps using GIS and predict soil carbon changes in the future climate change scenario using modelling technique. The agro-ecological units (AEUs) of south Kerala namely, southern coastal plain (AEU 1), Onattukara sandy plain (AEU 3), southern laterites (AEU 8), south central laterites (AEU 9) and southern and central foothills (AEU 12) were selected for the study. In each AEU, different agricultural land use categories as described by IPCC for the carbon inventory such as, garden land (coconut), wet land (rice), fallow land (uncultivated) and plantation (rubber) were also selected. Soil profiles upto a depth of one meter was taken from the selected sites and samples were collected from various depth intervals of 0 to 25, 25 to 50, 50 to 75 and 75 to 100 cm. The surface soil samples (0 – 25 cm) were analyzed for soil properties such as pH, EC, CEC, texture, soil carbon fractions (organic, inorganic, water soluble, permanganate oxidizable, microbial biomass carbon and mineralizable carbon) and organic matter fractions (humic acid, fulvic acid and humin). The depth wise distribution of soil carbon pools (active, passive and slow carbon pools), bulk density and total organic carbon (TOC) were analyzed. Soil organic carbon stock, density, carbon indices (CPI, CLI, CMI, LQI, GWP) and carbon proportion/ turnover were computed. Soil aggregation was evaluated by analyzing water stable aggregates, mean weight diameter, macro-micro aggregate size distribution, aggregate associated organic carbon, glomalin and total polysaccharides in soil. GIS based thematic maps of soil organic carbon, stock, density and land quality were generated in ArcGIS 10.5.1 software. The soil organic carbon changes in the future climate change scenario were predicted using DNDC (Denitrification Decomposition) model. The results revealed that soil texture was loamy sand to sandy clay loam in AEU 1, sandy loam to sandy clay loam in AEU 3 and 9, sandy loam to sandy clay in AEU 8 and sandy clay loam to sandy clay in AEU 12. The sand fraction (45.32-80.34 %) was found to be more than silt (9.60 – 21.14%) and clay (8.87-39.15 %). The pH and EC of soils ranged 254 from 4.77 to 5.73 and 0.06 to 0.44 dS m⁻1 respectively. The CEC of soil varied between 2.60 and 6.69 c mol(p+) kg-1 with the highest value in rubber land use and the lowest in uncultivated land. Soil organic carbon (SOC) ranged from 0.34 to 0.73, 0.44 to 0.99, 0.40 to 1.09, 0.25 to 0.97 and 0.26 to 1.26 per cent in AEU 1, 3, 8, 9 and 12 respectively. Among the land uses, rubber land use recorded the highest SOC (1.03 %) followed by coconut (0.75 %) and rice (0.60 %) and the lowest SOC was observed in uncultivated land (0.34 %). The water soluble, permanganate oxidizable and the particulate organic carbon varied from 34.01 to 162.81 mg kg-1, 1.21 to 7.37 mg g-1 and 0.37 to 2.75 per cent respectively. The different AEUs and land uses followed the order AEU 12 > AEU 9 >AEU 8 > AEU 3>AEU 1 and rubber > coconut > rice >uncultivated land respectively. Similar trend was observed for microbial biomass carbon which ranged from 121.2 to 424.4 mg kg-1 with the highest value in AEU 12 and rubber land use. Percentage contribution of water soluble, particulate and permanganate oxidizable C to TOC was 0.31 to 0.43, 40.12 to 69.72 and 12.62 to 19.73 per cent respectively. The organic matter fractions viz. humic acid, fulvic acid and humin varied from 0.57 to 2.06, 0.73 to 2.33, 0.62 to 1.59 per cent respectively in different AEUs. The rubber land use showed significantly higher humic acid (1.72 %), fulvic acid (2.01 %) and humin (1.44 %) than coconut, rice and uncultivated land. Percentage contribution of humic acid, fulvic acid and humin to total organic matter ranged from 29.40 to 32.51, 32.30 to 36.42 and 26 to 29.25 per cent respectively. The active, slow and passive pools of carbon in soil ranged between 0.09 and 1.03, 0.17 and 0.78, 0.13 and 1.26 per cent respectively with the highest value in AEU 12 and the lowest in AEU 1. In different land uses it followed the order rubber >coconut> rice> uncultivated land. The soil carbon pool showed a gradual decline from 0 - 25 cm to 75 - 100 cm depth with a decrease of 0.82 to 0.14, 0.63 to 0.18 and 0.97 to 0.24 per cent for active, slow and passive carbon pools respectively. The passive pool of carbon (31.95 – 38.08 %) contributed more towards total organic carbon than active (23.64-37.66 %) and slow (22.73-32.12 %) pools. 255 With depth (0-25 cm to 75-100 cm) bulk density of soil increased from 1.38 to 1.66, 1.33 to 1.68, 1.35 to 1.64, 1.33 to 1.60 and 1.29 to 1.55 Mg m-3 in AEU 1, 3, 8, 9 and 12 respectively. Higher bulk density was observed in uncultivated land than rice, coconut and rubber land uses. The TOC in soil ranged from 0.72 to 2.80, 0.69 to 2.89, 0.79 to 3.46, 0.88 to 3.18 and 0.75 to 3.56 per cent in AEU 1, 3, 8, 9 and 12 respectively. In all the AEUs the highest TOC was registered from rubber land use followed by coconut, rice and the lowest from uncultivated land. The TOC content decreased with depth (0 - 25 cm to 75 - 100 cm) and the values were 1.99 to 0.95, 2.24 to 0.92, 2.45 to 1.05, 2.53 to 1.05 and 2.38 to 1.01 per cent for AEU 1, 3, 8, 9 and 12 respectively. Total soil organic carbon stock varied from 162.71 to 293.22, 175.80 to 275.79, 188.63 to 322.36, 199.73 to 331.48, 144.54 to 355.63 Mg ha-1 in AEU 1, 3, 8, 9 and 12 respectively with the highest stock in AEU 12. Among the land uses it followed the order rubber>coconut>rice>uncultivated land. SOC stock decreased from 0 to 25 cm to 75 to 100 cm depths and the values were 76.28 to 36.59, 79.97 to 35.88, 86.70 to 39.61, 88.10 to 39.48 and 79.90 to 35.80 Mg C ha-1 for AEU 1,3, 8, 9 and 12 respectively. The soil organic carbon density of the surface (0-25 cm) soil varied from 3.07 to 6.01, 3.68 to 5.76, 3.84 to 7.81, 4.19 to 7.19 and 3.27 to 6.96 kg m-2 in AEU 1, 3, 8, 9 and 12 respectively with the highest density observed for AEU 12. With respect to different land uses it followed the order rubber (6.96 kg m⁻2 )> coconut (5.86 kg m⁻2) > rice (5.10 kg m⁻2) > uncultivated land (3.61 kg m⁻2). The carbon indices such as lability, pool and management index ranged from 0.30 to 0.93, 0.19 to 0.51 and 5.55 to 38.42 respectively in different AEUs and were found to be the highest in AEU 12 and rubber land use. The land quality index based on SOC stock in kg m-2 was rated as medium (6 - 9 kg m-2 ) in all AEUs. The mineralizable C content in soil varied from 1.40 to 3.45, 1.18 to 3.41, 1.04 to 3.04, 1.23 to 3.35 and 1.01 to 3.02 mg g⁻¹ in AEU 1, 3, 8, 9 and 12 respectively. The highest value was observed from AEU 1 and rice land use. Similar trend was obtained for global warming potential of soils based on CO2 evolution which varied from 31.82 to 78.41, 26.89 to 79.02, 22.89 to 69.02, 28.03 to 76.06 and 22.96 to 68.64 in AEU 1, 3, 8, 9 and 12 respectively. The C proportion and turnover rates were in the range of 0.25 to 0.77 256 and 0.04 to 0.17 respectively. The C proportion was the highest in AEU 12 and rubber land use whereas the C turnover was the highest in AEU 1 and rice land use. With respect to aggregate distribution, the larger size fractions (5-8, 2-5, 1-2 & 0.5-1 mm) were found to be higher in rubber land use and the smaller size fractions (0.25-0.5 & 0.1-0.25 mm) were higher in rice land use. The macro aggregate fraction (> 250 μm) ranged from 22.60 to 75.70 per cent with the highest value in rubber land use while the micro aggregate fraction (53 – 250 μm) ranged from 10.78 to 56.46 per cent with the highest value in rice land use. The water stable aggregates and mean weight diameter were ranged from 22.60 to 34.31 per cent and 0.56 to 2.90 mm respectively and were the highest in rubber land use. The aggregate associated organic carbon was the highest in the size fraction of 1 to 2 and 0.5 to 1 mm (1.03-1.27 g kg-1) compared to other fractions. The organic carbon associated with macro (> 250 μm) and micro (53-250 μm) aggregates are in the range of 2.16 to 5.70 and 0.30 to 0.95 g kg-1 respectively with the highest value in rubber land use. The glomalin related soil protein ranged from 1.22 to 6.35 mg g-1 and polysaccharides from 2.12 to 9.24 mg g-1 . Irrespective of the AEUs the highest glomalin and polysaccharides were observed in rubber land use. The DNDC model predicted organic carbon changes in coconut land use systems in Thuravoor (AEU 1), Bharanikkavu (AEU 3), Ookkod (AEU 8), Karavaram (AEU 9) and Vellarada (AEU 12) under RCP 4.5 and RCP 8.5 future climate change scenario. The DNDC model predicted an increase in soil organic carbon status from 2020 to 2050 under RCP 4.5 and RCP 8.5 scenario but the rate of increase in soil carbon were more pronounced under 4.5 scenario where relatively lower CO2 emission was observed. The predicted SOC for 2050 under RCP 4.5 scenario were 1.26, 1.04, 1.44, 1.23 and 1.27 for Thuravoor (AEU 1), Bharanikkavu (AEU 3), Ookkod (AEU 8), Karavaram (AEU 9) and Vellarada (AEU 12) respectively. The organic carbon fractions, pools and stock were the highest in agro-ecological unit 12. Among the land uses, rubber contributed more to the SOC stock and pools indicating the prevalence of conducive environment for the buildup of carbon. The macro 257 and micro aggregates, aggregate associated carbon, glomalin, polysaccharides and C proportion were also higher in rubber land use indicating it as a potential carbon sink. Among the carbon fractions particulate organic carbon contributed more to total organic carbon. Among the carbon pools passive pool contributed more towards the total organic carbon. The carbon associated with larger fractions (1-2 mm and 0.5-1 mm) and macro aggregates (> 250 μm) were high in rubber land use indicating physical protection and sequestration in soil. The DNDC model predicted an increase in the SOC status from 2020 to 2050 under RCP 4.5 and RCP 8.5 scenario of future climate change.
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, R
The 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.
Molecular diagnosis and management of Papaya ringspot virus causing papaya ringspot diseases
(Department of Plant Pathology, College of Agriculture,Vellayani, 2024-04-17) Josiya Joy; KAU; Radhika, N S
The research work entitled “Molecular diagnosis and management of Papaya ringspot virus causing papaya ringspot disease” was undertaken in the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram, during 2019-24, with the objectives; molecular diagnosis and recombinant coat protein production of Papaya ringspot virus (PRSV), and evaluation of the efficacy of beneficial microorganisms and botanical inthe management of papaya ringspot disease (PRSD). Roving survey was carried out across five Agro-ecological units (AEUs) of Kerala. The disease incidence (DI) ranged from 50.25 per cent (Kayyur-Cheemeni) to 100 per cent (Kalliyoor, Venganoor, Balaramapuram, Pallichal, Kayamkulam, Mavelikkara, Velukkara, Irinjalakuda and Shoranur). Vulnerability index (VI) of the plants to PRSV in the surveyed locations ranged from 33.54 (Badiyadkka) to 98.22 (Kalliyoor). Serological and molecular detection confirmed the presence of PRSV in all the 20 symptomatic samples collected during survey. Phylogenetic tree constructed with the deduced amino acid sequences of CP gene of 11 Kerala PRSV isolates, revealed that Thiruvananthapuram isolates clustered together, indicating their relatedness during evolution. Mechanical inoculation on two months old Red Lady papaya seedlings under insect proof conditions was carried out to identify the most virulent PRSV isolate collected from different AEUs. At three months after inoculation (MAI), Kalliyoor isolate exhibited highest VI (96.63) followed by Alur (95.48) and Venganoor (95.22) isolates. The lowest VI was observed in Kayyur-Cheemeni isolate with 57.95 VI, followed by Cherpulassery (60.58). The recombinant coat protein of PRSV was induced in pLATE 31 expression vector with C terminal histidine tag, within BL21(DE3)pLysS expression host. PRSV recombinant coat protein was purified using Ni-NTA column chromatography. A single band was observed at 35 kDa in SDS-PAGE analysis and western blotting with PRSV antiserum, confirmed the presence of purified recombinant coat protein in the soluble fraction. Pot culture experiment was conducted to evaluate the management of PRSD using Piriformospora indica. The initial colonization of P. indica inside the papaya roots was observed five days after germination of the seeds grown in P. indica massmultiplied medium. Prophylactic colonization of P. indica exhibited lowest VI (23.10) and 68.27 per cent reduction in VI over control diseased plants at five months after PRSV inoculation (MAI). Double antibody sandwich - Enzyme linked immunosorbent assay (DAS-ELISA) at 5 MAI revealed lowest absorbance (0.23) indicating lowest virus titre in P. indica pre-colonized plants upon PRSV inoculation, compared to control diseased plants (1.23). The accumulation of reactive oxygen species (ROS) i.e., H2O2 and superoxides in the leaves were analyzed using DAB (diaminobenzidine) and NBT (nitro blue tetrazolium chloride) staining respectively. P. indica-colonized plants upon PRSV inoculation indicated a higher initial accumulation of ROS at three weeks after inoculation (3 WAI) and further reduction at 6, 9 and 12 WAI of PRSV, compared to control diseased plants. P. indica-colonized plants also exhibited enhanced antioxidant defence enzyme activity viz., catalase, peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, glutamate synthase and superoxide dismutase compared to control diseased plants. Amongst PRSV inoculated treatments, P. indica pre-colonized plants exhibited highest number of leaves (20.86), leaf area (365.14 cm2), plant height (108.29 cm), stem girth (7.26 cm), shoot biomass (423.43 g), root biomass (133.96 g) and chlorophyll content (2.84 mg g-1 of fw) at five months after transplanting (5 MAT). Effect of P. indica, Bougainvillea spectabilis leaf extract (10 %) and Pseudomonas fluorescens (2 %) were evaluated against natural incidence of PRSD under field conditions at Instructional farm, College of Agriculture, Vellayani and Coconut Research Station, Balaramapuram, Thiruvananthapuram. P. indica-colonized plants exhibited lowest VI (37.63), with highest reduction in VI over control (53.85 %) followed by B. spectabilis treated plants (37.49 %) and P. fluorescens treated plants (33.31 %) at 12 months after planting (12 MAP). In DAS-ELISA, lowest virus titre with absorbance of 0.438 was observed in P. indica-colonized plants at 12 MAP, compared to the highest virus titre in control plants (1.267). B. spectabilis treated plants (0.596) also exhibited reduction in virus titre followed by P. fluorescens treated plants (0.625) at 12 MAP. P. indica-colonized plants exhibited enhancement in growth parameters viz., number of leaves (24.00), leaf area (1127 cm2), stem girth (42.74 cm) and plant height (207.39 cm) at 12 MAP. P. indica-colonized plants also enhanced the yield by 44.68 per cent followed by B. spectabilis treated plants (24.13 %) and P. fluorescens treated plants (17.99 %). Moreover, fruits from P. indica-colonized plants expressed significantly superior quality parameters. Thus, findings from the present study could aid in the preliminary detection and management of the virus, thus mitigating the widespread infection caused by PRSV. The recombinant PRSV coat protein produced in this study could be used for the development of PRSV antiserum. Additionally, our research highlights the efficacy of eco-friendly management strategies for papaya ringspot disease, with P. indica-colonization @ 106 cfu g-1 and also with four foliar sprayings as well as soil drenching of B. spectabilis leaf extract (10 %) applied at fortnightly intervals, starting from one month after planting. More field trials are to be conducted to integrate this strategy in integrated disease management (IDM) package for the effective and sustainable management of PRSD in papaya.
Piriformospora indica and its water diffusible exudates for the management of chilli anthracnose incited by colletotrichum capsici (Syd.) butler and bisby
(Department of Plant Pathology, College of Agriculture, Vellayani, 2024-07-05) Elizabeth, T Jojy; KAU
The research work entitled “Piriformospora indica and its water diffusible exudates for the management of chilli anthracnose incited by Colletotrichum capsici (Syd.) Butler and Bisby” was carried out in the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during 2017- 2023. The study was undertaken with the objective to evaluate P. indica- and its water diffusible exudates- primed chilli seedlings and plants against foliar infection of C. capsici; and study the biochemical and molecular mechanisms involved in this tripartite interaction. A survey conducted in the five agroclimatic zones (ACZs) of Kerala showed that the highest disease incidence (DI) and Percent Disease Index (PDI) were recorded at RARS, Pilicode (DI-90 & PDI-52.60) of northern zone, while the lowest DI was observed at farmer’s field, Kottarakkara (20%) and ORARS, Kayamkulam recorded the lowest PDI (23.63). Chilli anthracnose symptoms namely leaf spot, leaf blight, die-back, fruit rot and mummified fruits were observed in different survey locations of the five agroclimatic zones (ACZ) of Kerala. Nine C. capsici isolates and one isolate of C. gloeosporioides were obtained from the surveyed locations. All the C. capsici isolates produced sparse mycelial growth with concentric zonations of acervuli on potato dextrose agar (PDA) medium. The upper side of culture plates appeared in different shades of white and off-white to grey with regular or irregular margins, while the reverse side looked yellowish brown to black. Isolate Cc4 produced maximum mycelial growth diameter of 8.6 cm compared to 7.2 cm (minimum) in Cc2 on 7th day after inoculation (DAI). Further, microscopic characters such as mycelial width, size of conidia, acervuli, appressoria, number and length of setae were significantly different in the C. capsici isolates. The most virulent isolate of C. capsici was screened based on the lesion size produced on the artificially inoculated leaves and fruits of chilli var. Vellayani Athulya. On 7th DAI, Cc3 isolate of C. capsici produced maximum lesion size of 2.52 cm while isolate Cc4 produced minimum lesions (0.74 cm) on 292 inoculated chilli leaves. Similarly, tender, mature and ripe fruits of var. Vellayani Athulya inoculated with Cc3 isolate produced the highest lesion size (2.48, 2.34 and 2.56 cm respectively) and isolate Cc4 recorded the lowest lesions (1.62, 1.16 and 1.38 cm respectively) on 7th DAI. Thus, isolate Cc3 from Thrissur, was selected as the most virulent isolate of C. capsici. Ten selected KAU/ICAR released varieties of chilli were screened with Cc3 isolate by detached leaf and fruit inoculation method to identify the most susceptible variety. Leaves of Vellayani Athulya recorded the highest lesion size of 2.80 cm on 5 DAI compared to 1.66 cm (lowest) in Manjari. Maximum lesion size of 2.96 cm was observed on the tender fruits of Vellayani Athulya and minimum was recorded on Vellayani Thejus (1.52 cm). Lesions of 3.50 cm (highest) were observed on mature Vellayani Athulya fruits as against 2.00 cm on Manjari fruits. Similarly, ripe fruits of Vellayani Athulya recorded maximum lesion size of 3.46 cm and minimum in Vellayani Samrudhi (1.30 cm) at 7 DAI. Also, in the in vivo study, higher lesion size (3.00 cm) was observed in Vellayani Athulya fruits and lowest (1.62 cm) in fruits of Vellayani Samrudhi at 7th DAI. Standardization of P. indica-colonization in chilli var. Vellayani Athulya was done on plant nutrient medium (PNM) and chilli roots were sampled at different intervals viz., 3rd, 5th, 7th, 10th, and 15th days after colonization (DAC). Young, double-walled chlamydospores were observed within the root cells at 5 DAC. P. indica colonization gradually increased and reached a maximum of 100 per cent at 15 DAC. Dual culture of P. indica and C. capsici showed the appearance of inhibition zone (8.33 DAI) and antibiosis (12.67 DAI) at the point of interaction of the two fungi. Further microscopic observations revealed thickening and lysis of pathogen mycelium by P. indica. The growth of C. capsici was suppressed (3.85 cm) in dual culture compared to normal (6.28 cm) in control at 7th DAI. 57.22 per cent mycelial growth inhibition due to P. indica was observed at 15 DAI. Antagonistic property of P. indica-water diffusible exudates (Pi-WDE) against C. capsici was studied by in vitro poison food technique. Significant 293 differences were observed in the colour and nature of C. capsici growth on PDA media, added with different day-old Pi-WDE in comparison with control. Among them, ten day-old Pi-WDE recorded the highest inhibition (67.30 %) of pathogen mycelial growth whereas the lowest in one day-old Pi-WDE (9.29 %). Mass multiplication of P. indica in the portray medium was carried out. Rapid growth of the endophyte was recorded in a combination of farmyard manure (FYM) and coir pith (1:1), added with 2 per cent gram flour. P. indica completely covered the inoculated trays within 7 DAI. This medium was used to colonize chilli plants with P. indica for further experiments and thus, four treatments viz., P. indica alone (T1), C. capsici alone (T2), P. indica-primed seedlings + C. capsici (T3) and control (T4) were systematized for the in vitro and in vivo evaluation experiments. In vitro study of P. indica-primed chilli seedlings against C. capsici significantly reduced anthracnose lesion size and disease severity to 1.30 cm and 30.63 per cent respectively compared to 2.06 cm and 77.00 per cent in C. capsici alone at 7 DAI. Maximum root and shoot weight were recorded in the P. indicacolonized plants, irrespective of the pathogen. Pot culture experiment was also carried out in the chilli var. Vellayani Athulya. The development of anthracnose symptoms on leaves was delayed by 4 days in P. indica-colonized chilli plants as against 2 days in control. The lesion size and disease severity due to anthracnose was lower in P. indica-colonized chilli leaves (1.58 cm & 54.90 %) compared to C. capsici alone (2.86 cm & 85.20 %) at 10 DAI. Similarly, minimum lesion size and disease severity of 1.17 cm and 27.92 per cent was observed in fruits from pathogen inoculated P. indicacolonized chilli plants than in C. capsici alone (3.56 cm & 91.67 %) at 10 DAI. Field studies were conducted in rabi and summer seasons with two treatments viz., P. indica-colonized and non-colonized plants. P. indica-colonized chilli plants recorded lower anthracnose severity of 27.00 per cent in Rabi and 15.50 per cent in summer compared to 56.50 and 47.00 per cent respectively in control. P. indica colonization improved the biometric characters such as plant height, number of leaves, leaf area, number of secondary and tertiary roots, shoot 294 weight, root weight in primed chilli plants. In addition, yield of chilli was enhanced in endophyte-colonized plants compared to control. Biochemical studies on the mechanism of disease management in P. indica-colonized chilli plants at 0, 12, 24 and 72 hours after inoculation (HAI) showed increased activity of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (PO). In contrast, the activities of 4-coumaryl CoA ligase (4-CL) and polyphenol oxidase (PPO) were enhanced in chilli plants inoculated with C. capsici alone. Isoenzyme analysis revealed higher induction of isoenzymes of PO and PPO in the P. indica-colonized plants compared to control. Enhanced induction of pathogenesis related (PR) proteins was noticed in the P. indica-colonized plants inoculated with the pathogen at 72 HAI. The capsaicin content was drastically reduced in C. capsici inoculated chilli fruits. However, fruits from P. indica-colonized plants had high capsaicin content (>30% over control) irrespective of infection. Molecular studies of the defense related genes revealed the positive role of P. indica in the management of anthracnose disease in chilli. P. indica reduced anthracnose symptoms in colonized chilli plants by the upregulation of genes involved in phenyl propanoid pathway (PAL1, 4-CL, CAD and CHS), jasmonic acid signaling pathway (PDF1.2 and AOS) and salicylic acid signaling pathway (PR1, EDS1 and PAL3); and downregulating the LOX gene involved in jasmonic acid signaling. In summary, P. indica and its WDE inhibited C. capsici under in vitro conditions. Anthracnose incidence and severity were considerably reduced in the treated chilli seedlings and plants compared to control. In addition, P. indica enhanced growth as well as yield in colonized chilli plants, thereby enhancing the fruit quality. Further, the endophyte improved disease resistance in chilli plants by increasing the defense-related enzyme activities and expression of defense genes. Thus, the root fungal endophyte, P. indica can be considered as an efficient biocontrol agent in the management of chilli anthracnose.
Technology assessment and refinement in coastal homegardens of Kerala: linkages with biodiversity and household livelihoods
(Department of Agricultural Extension Education, College of Agriculture , Vellayani, 2024-01-15) Roshni Thampi; KAU; Allan Thomas; Joy, M
The study entitled “Technology assessment and refinement in coastal homegardens of Kerala: Linkages with biodiversity and household livelihood” conducted from 2019 to 2023 focused on the primary objectives to assess crop diversity, identify the dominance of crops, delineate technology needs, sorter dimensions of technologies and analyze the adoption and technology gaps through action research for sustainable coastal home garden systems. The study also examined the social anthropogenic variables of coastal home garden farmers and explored the linkages between biodiversity and household livelihoods in terms of perceptions and practices by farmers. Constraints in the coastal home garden farming system as perceived by farmers and extension personnel were also studied. The study was conducted in homegardens of three coastal Agro Ecological Units (AEU 1- Southern coastal plains, AEU 3- Onattukara Sandy Plains and AEU 4- Kuttanad) of southern Kerala. 35 farmer respondents were chosen from the home gardens of selected panchayats, comprising 15 participants and 20 non-participants who had at least 25 cents. The total numbers of respondents for the study were 165 comprising 105 coastal home garden farmers, 30 Agricultural Officers and 30 Scientists. Within the framework of Participatory Technology Assessment and Refinement, a collaborative effort was made to select appropriate technology for the primary crops within specific AEUs. This encompassed the evaluation of technology for bittergourd in AEU 1, chilli in AEU 3, and cowpea in AEU 4. Furthermore, Front Line Demonstrations (FLDs) were conducted to showcase and validate KAU-proven technologies that were deemed suitable for these specific crops. The structural configuration in terms of dominance-biodiversity profile of crops in coastal home gardens of southern Kerala was studied under two aspects viz., numerical and economic dominance and the diversity profile of coastal homegardens in Kerala. The study revealed that in AEU 1, out of the 82 identified crops, the most dominant one was coconut (5.90) followed by bittergourd (5.81) whereas in AEU 3 a total of 78 crops were identified, of which the maximum dominance was noted with black pepper (5.65) followed by chilli (5.26). In AEU 4 a total of 85 crops were identified of which the maximum dominance was noted with cowpea (5.24). The study compared the mean diversity index across different AEUs and found that AEU 4 exhibited the highest diversity index at 1.547, followed by AEU 3 at 1.159, and AEU 1 at 1.058. When examining the crop-wise diversity profile of coastal AEUs, fruits had the highest diversity index at 0.998, followed by vegetables at 0.878, and spice crops at 0.850. Conversely, the lowest biodiversity index was recorded for tubers at 0.360 and ornamental plants at 0.357. In AEU 1, the maximum diversity index was observed for spices at 0.801, followed by vegetables at 0.770, and medicinal crops at 0.741, with tubers having the lowest diversity index at 0.350. In AEU 3 and AEU 4, the highest diversity index was noted for fruit crops at 1.181 and 1.164, respectively, while the lowest diversity index was recorded for medicinal plants at 0.225 and 0.226 respectively. One-way ANOVA analysis revealed no significant difference in crop diversity among selected AEUs, but there was a significant difference in crop-wise diversity for plantation crops, multipurpose trees, medicinal plants, tubers, and ornamental plants. In terms of region-wise biodiversity, the mid-regions had the highest diversity index at 1.433, while the courtyard of coastal home gardens had the lowest diversity at 1.117. The study on vertical diversification of crops in different coastal AEUs revealed that in AEU 1 maximum diversification was seen for banana with six levels of diversification whereas in AEU 3 maximum diversification was seen for banana and coconut with five levels of diversification. In AEU 4 the maximum diversification was seen for coconut and banana with four levels followed by cowpea and black pepper with two levels of diversification. The extent of horizontal diversification was also recorded and found that 40 % of the homegardens in AEU 1 had 5 tier diversification whereas in AEU 3, 60 % of the home gardens had 6 tier or more diversification. In case of AEU 4, 73.33% of the homegardens were having 6 and more tiers of diversification. The study revealed that a significant percentage (53.33%) of homegardens in all AEUs had six or more tiers of diversification. This study involved assessing the technology requirements for the top dominant crops in the selected AEUs. The study identified that in AEU 1, the maximum technology need was recorded with production practices of bittergourd (6.83), whereas in AEU 3, the highest technology need was noted with protection practices (leaf curl disease management) in chilli (6.67). In AEU 4, highest technology need was noted for production practices in cowpea (6.50). Based upon the technology needs of the dominant crops in different AEUs, FLDs were conducted in selected three homegardens on production aspects of bittergourd (AEU 1) and cowpea (AEU 4) and protection aspects for chilly (AEU 3). The FLD in all the AEUs of study clearly highlights that scientific approach in farming and correct use of technology in accordance to KAU POP will help the farmer to derive more profit through improved production. The B:C ratio for FLD studies on bittergourd cowpea was found to be higher (1.96 and 1.78) compared to farmers practices (1.37 and 1.42). Research showed that using scientific methods like KAU practices reduced leaf curl disease in chilli by 48.19%, emphasizing the importance of adopting these techniques. The study also examined how farmers adopted KAU practices. It was found that 77.14% of AEU 1 farmers had low adoption, while 74.29% of AEU 4 farmers had high adoption. In AEU 3, 51.43% had low adoption. The study also categorized farmers as innovators (0.95%), early adopters (16.19%), early majority (34.29%), late majority (32.38%), and laggards (16.19%). In the study, a reliable scale was developed to assess technology-related aspects in Kerala's coastal home gardens. The Single ANOVA tool helped choose indicators with a mean value below 4.49, resulting in 26 variables across 8 dimensions. For reliability, the odd-even split-half method was used, yielding a strong correlation of 0.846 for the half-test and 0.917 for the full test. Pearson's correlation coefficients also confirmed the scale's validity by exceeding 0.25 at a 0.05 significance level. The scale was administered to respondents and the results showed that participating farmers perceived economic dimensions (4.62), environmental dimensions (4.30), and psychological dimensions (4.31) as the most important. Non-participating farmers, on the other hand, considered economic dimensions (4.61), psychological dimensions (4.14), and human resource dimensions (4.12) to be significant. Extension personnel, in their evaluation, rated economic dimensions (4.28), human resource dimensions (4.04), and political dimensions (4.03) as the most important factors. Cluster analysis was done for different dimensions for different categories of respondents and it invariably shows interaction effects of different dimensions of technology. The analysis was done using Ward linkage with 26 indicators, representing 8 dimensions of technology in coastal homegardens. For participant farmers 3 clusters were identified. The first cluster comprised of 10 indicators viz., profit, sustainability, technical efficiency, interpersonal relationship, initial cost, bureaucratic support, perception of technology, coordination, supply chain and acquisition of information that showed strong association. The second cluster encompassed 12 indicators that demonstrated proficiency in areas such as credit access, effective decision-making, keen observability, viability, adept resource management, resource recycling capacity, reasonable price expectations, local resource utilization, ready availability, utilization of family labour, dependable delivery reliability, and skilled labour requirements. The third cluster, comprised of four dimensions, exhibited characteristics related to flexibility, strong social networking, effective budgeting abilities, and openness to utilizing open-source technology. Analysis of livelihood assets revealed that social assets scored the highest mean value (239.3) followed by human assets (227.3), physical assets (185.6), natural assets (172.3) and financial assets (176). ANOVA confirmed that there is significant difference between AEUs in case of all assets except human assets and Pearson correlation analysis was conducted to examine the strength and significance of the relationship between these variables. The results showed a moderate positive correlation (r = 0.311, p < 0.05) between total assets and biodiversity. This suggests that as the total assets of the communities increase, the level of biodiversity also tends to increase. The study delved into gender roles within various coastal home gardens, with a focus on dominant crops. In AEU 1, particularly in bittergourd cultivation, men were predominantly engaged in labour-intensive tasks like land preparation (54.29%) and drain preparation (85.71%). Women, conversely, played more active roles in activities such as weeding (51.43%), kitchen gardening (57.14%), and harvesting (51.43%). Both genders actively participated in operations like irrigation (62.86%), plant protection activities (57.14%), and post-harvest tasks (51.43%). In AEU 3, where chilli cultivation was highlighted, men took on tasks like drain preparation (62.86%) and fencing (74.29%), while women were prominently involved in sowing seeds (68.57%), planting seedlings (57.14%), plant protection (57.14%), kitchen gardening (54.29%), harvesting (74.29%), and post-harvest operations (62.86%). Both men and women contributed to activities like applying manure (62.86%) and fertilizers, as well as weeding (54.29%). In AEU 4, centered on cowpea cultivation, men primarily handled land preparation (60%). Interestingly, women took on most responsibilities related to cowpea cultivation including, applying manure (57.14%), sowing seeds (51.43%), planting seedlings (51.43%), applying fertilizer (57.14%), weeding (51.43%), kitchen gardening (57.14%), and harvesting (60%). Both genders were involved in applying irrigation (54.29%). A chi-square test assessed the significance of gender differences in home garden activities, revealing a strong association between gender and agricultural tasks. The calculated chi-square statistic was 192.78, surpassing the table value of 33.92 at a 0.05% significance level, affirming the substantial gender differences in home garden activities. The study identified a total of 22 constraints within coastal home gardens, which were rated by respondents. The most significant challenges reported by coastal home garden farmers were, lack of scientific knowledge (1031) followed by salinity issues (rated at 1010), flooding concerns (1008), ineffectiveness of plant protection methods at recommended dosages (rated at 969), insufficient knowledge about plant protection chemicals (960), the impact of climate change (948), and the absence of proper drainage systems (916), listed in descending order of importance. Key suggestions to improve coastal home gardens as perceived by farmers and extension personnel were, the creation of customized packages of practices tailored to the coastal home garden system, enhanced on-going support and guidance from extension agencies for the adoption of recommended scientific practices and the promotion of Farmer Producer Organizations (FPOs) to enhance post-harvest handling and marketing Social anthropogenic variables of coastal home garden farmers like age, education, gender, occupation, economic motivation, extension contribution, irrigation potential, information source utilization, credit orientation, training need, market orientation and adoption were also studied. In essence, the research revealed a multifaceted profile of dominance and diversity within the coastal home garden community. Furthermore, it outlined specific technology requirements, adoption patterns, and dimensions conducive to the development of coastal home gardens. Additionally, the study delved into aspects related to livelihood assets, gender roles, constraints, and provided valuable suggestions to promote the sustainability of coastal home gardens in the Kerala region.
Bio-elicitation of Ashwagandha (Withania Somnifera (L.) Dunal) for improved growth, yield and secondary metabolite production
(Department of Plantation, Spices, Medicinal and Aromatic Crops, College of Agriculture,Vellayani, 2024-10-10) Manisha Elza Jacob; KAU; Deepa, S Nair
The study titled “Bio-elicitation of Ashwagandha (Withania somnifera (L.) Dunal) for improved growth, yield and secondary metabolite production” was carried out in the Department of Plantation, Spices, Medicinal and Aromatic Crops, College of Agriculture, Vellayani, during December 2019 to September 2023 with the objective to study the effect of individual application of chitosan and the root endophytic fungus, Piriformospora indica and their combined application on plant growth, yield and secondary metabolite production in Withania somnifera. The study was carried out in W. somnifera as four experiments (i) Biopriming of seeds using chitosan, (ii) Bio-elicitation by foliar application of chitosan, (iii) Bio-elicitation using P. indica, and (iv) Bio-elicitation by combined application of chitosan and P. indica. The second experiment consisted of two parts, preparation of chitosan nanoparticles and foliar application of chitosan. In the first experiment, seeds of Ashwagandha were primed with fourteen treatments, comprising of four different concentrations of chitosan (0.5 mg mL-1, 1 mg mL-1, 2.5 mg mL-1, and 5 mg mL-1) for three different durations (2 h, 4 h and overnight), along with overnight hydropriming and control (without priming). Different germination and seedling growth parameters of the seeds and seedlings were recorded. The highest germination per cent (76.00) and survival per cent (73.33) were observed in the priming treatment chitosan 5 mg mL-1 for 4 h. This was on par with the treatment chitosan 5 mg mL-1 for 2 h (69.33) and hydropriming treatment (70.67) with respect to germination per cent. Hydropriming recorded the highest germination index (1.79) and the treatment chitosan 2.5 mg mL-1 for 2h recorded the highest allometric index (0.91). Respective results were on par with the priming treatment chitosan 5 mg mL-1 for 4 h. Hydropriming recorded the lowest mean germination time (8.83 days). The highest values of seedling vigour indices I (8890.00) and II (45.15) were also recorded in the treatment chitosan 5 mg mL-1 for 4 h. The duration of priming had a significant effect on seedling growth parameters. Overnight priming treatments with chitosan were found to have an inhibitory effect on germination and seedling growth parameters with values on par with or lower than that of the control. Based on the results, seed priming with chitosan 5 mg mL-1 for 4 h, was selected as the best priming treatment for further experiments in the study. The preparation of chitosan nanoparticles was carried out by ionotrophic gelation method by dropwise addition of tripolyphoshate (TPP) solution to chitosan solution, under stirring at room temperature. Eighteen treatments formed by three levels of chitosan (1 mg mL-1, 2 mg mL-1 and 3 mg mL-1), three levels of TPP (0.5 mg mL-1, 0.75 mg mL-1 and 1 mg mL-1) and two speeds of rotation (700 rpm and 800 rpm) were tried for preparing the nanoparticles. The absorption spectra of the resultant solutions were observed at eight different wavelengths, i.e., 200 nm, 220 nm, 240 nm, 260 nm, 280 nm, 300 nm, 320 nm and 340 nm. The factor combinations with chitosan 1 mg mL-1 and TPP 0.5 mg mL-1 at 700 rpm, chitosan 2 mg mL-1 and TPP 0.75 mg mL-1 at 700 rpm, and chitosan 3 mg mL-1 and TPP 1 mg mL-1 at 700 rpm had an absorption peak at 220 nm. These were further characterized for particle size and were observed to have mean particle sizes of 222.9 nm, 333.4 nm and 366.9 nm, respectively. As the particles formed by various chitosan - TPP combinations did not fall in the nanoscale range of less than 200 nm, the further experiments involving chitosan nanoparticles were carried out using commercially available chitosan nano particles of size 80 to 100 nm. In the experiment involving foliar application of chitosan, seeds primed with the best priming treatment selected (chitosan 5 mg mL-1 for 4 h) were sown in protrays and thirty-day old seedlings were transplanted to growbags. The eight treatments in this experiment consisted of chitosan and chitosan nanoparticles spray, each at three different concentrations (1 mg mL-1, 2.5 mg mL-1 and 5 mg mL-1). The primed and non-primed seeds (absolute control) without any foliar spray application served as the control treatments. The treatments were imposed as foliar spray at transplanting, 15, and 45 days after that. The treatment with chitosan 2.5 mg mL-1 was found to be the best in terms of parameters like plant height, number of leaves and leaf area, recording the values 123.67 cm, 178.83 leaves and 119.99 cm2 respectively, at harvest. The treatment with a higher concentration of chitosan nanoparticles (5 mg mL-1) recorded the lowest number of days to flowering from transplanting (30.67 days), and also a lesser number of days to fruitset from flowering (9.17 days). The highest number of branches (9.97) and flowering branches (8.15) at harvest were observed in the treatment, chitosan 1 mg mL-1. The treatment with the lowest concentration of chitosan nanoparticles (1 mg mL-1) recorded the lowest values in various growth parameters, viz., plant height, number of branches, number of flowering branches, leaf area and collar girth. With respect to biochemical parameters, the highest content of total proteins (7.12 mg g-1) and carbohydrates (22.41 mg 100 g-1) were observed in the treatment with chitosan nanoparticles 2.5 mg mL-1 at harvest. The treatment with chitosan 1 mg mL-1 recorded the highest peroxidase (32.35 U mg-1 protein), catalase (17.57 U mg-1 protein) and superoxide dismutase (1.68 U mg-1 protein) activities. Similar to growth parameters, chitosan 2.5 mg mL-1 recorded the highest leaf area index (25.75) and leaf area duration (1020.92 days). All the treatments involving chitosan as well as chitosan nanoparticles were observed to have significantly higher photosynthetic rate at harvest. The highest stomatal conductance (162.97 mmoles m-2 s-1) was observed in the treatment chitosan 1 mg mL-1, the highest proline content (44.03 μM g-1 tissue) was observed in the treatment chitosan nanoparticles 2.5 mg mL-1, and the highest cell membrane integrity (91.80 per cent) was observed in chitosan nanoparticles 5 mg mL-1 at harvest. This treatment also had the highest total chlorophyll content at harvest. The shoot weight was observed to be significantly higher in chitosan treatments over chitosan nanoparticles as well as the control treatments. However, with respect to the dry weight of shoot, the lowest concentration of chitosan nanoparticles (1 mg mL-1) was observed to be on par with chitosan foliar spray treatments. The treatments with chitosan nanoparticles were observed to give significantly higher berry weight and seed yield per plant over chitosan and control treatments. The root parameters, viz., root length, root diameter, root volume and root yield (fresh and dry) were also observed to be significantly higher in treatments with chitosan nanoparticles over that of chitosan and control treatments. Root being the officinal part of the plant, the secondary metabolite, withanolide content in it was recorded. The highest withanolide content was observed in the treatment chitosan 2.5 mg mL-1 (0.45 per cent), and was on par with the treatment chitosan 5 mg mL-1 (0.43 per cent). Withanolide yield per plant was the highest in the treatment chitosan nanoparticles 2.5 mg mL-1 (5.46 mg plant-1) which had 4.4 times increase in withanolide yield over control. This was on par with the treatment chitosan 5 mg mL-1 (4.99 mg plant-1) which had 3.9 times increase in withanolide yield over control. Among the foliar spray treatments, chitosan 5 mg mL-1 and chitosan nanoparticles 2.5 mg mL-1 were selected as the best two treatments in terms of higher withanolide yield, for proceeding to the combination trials. In the experiment involving application of P. indica, P. indica mycelium, culture filtrate and cell wall extract were applied to the plants raised from primed and non-primed seeds. The seedlings raised from the primed and non-primed seeds were transplanted at 30 days after sowing to growbags. P. indica mycelium was applied at transplanting; and P. indica culture filtrate and cell wall extract were applied as foliar spray at transplanting, 15 and 45 days after transplanting. With respect to growth parameters, the highest plant height (120.19 cm), number of flowering branches (7.25), leaf area (111.84 cm2) and collar girth (2.67 cm) were observed in the plants derived from primed seeds treated with P. indica mycelium. Significantly higher protein content and defense enzymes were observed in the plants raised from non-primed seeds treated with P. indica cell wall extract. Physiological parameters like leaf area index and leaf area duration were found to be the highest in the plants raised from primed seeds treated with P. indica cell wall extract (1.40 and 21.65 days) at harvest. The highest stomatal conductance (129.30 mmoles m-2 s-1), photosynthetic rate (26.60 μCO2 moles m-2 s-1), proline content (45.37 μM g-1 tissue) cell membrane integrity (70.06 per cent) and total chlorophyll content (2.44 mg g-1 fresh weight) were recorded in the plants raised from primed seeds treated with P. indica mycelium. Shoot weight per plant was found to be significantly higher in plants raised from both primed and non-primed seeds treated with P. indica mycelium. Number of berries per plant, berry weight and seed yield per plant were found to be significantly higher in plants raised from primed seeds treated with P. indica mycelium and culture filtrate. All the root parameters, root length, root diameter, root volume, root yield per plant (fresh and dry) were observed to be the highest in plants raised from primed seeds treated with P. indica mycelium. The withanolide content was observed to be significantly higher in the roots of plants raised from primed seeds treated with P. indica mycelium (0.35 per cent) and culture filtrate (0.37 per cent). The same treatments showed significantly higher withanolide yield of 4.05 mg (3.82 times increase over control) and 3.60 mg per plant (3.28 times increase over control), respectively. Thus, the plants raised from primed seeds treated with P. indica mycelium and culture filtrate were selected as the best two treatments in terms of higher withanolide yield, for proceeding to the combination trials. For the combined application of chitosan and P. indica, the best two treatments selected from the individual application of chitosan and P. indica were used for studying their combined effect on growth, yield and secondary metabolite production in W. somnifera. The highest plant height (91.86 cm), number of leaves (143.70), leaf length (17.01 cm), leaf breadth (9.48 cm), and leaf area (113.28 cm2) were observed in the combination treatment of chitosan 5 mg mL-1 and P. indica mycelium. The highest number of branches (8.25), flowering branches (7.60) and collar girth (2.42 cm) were observed in the combination treatment involving chitosan 5 mg mL-1 and P. indica culture filtrate. The same treatment recorded the highest protein content (8.45 mg g-1). The highest peroxidase activity (34.74 U mg-1 protein), catalase activity (14.67 U mg-1 protein) and superoxide dismutase activity (1.48 U mg-1) were observed in the treatment involving chitosan of 5 mg mL-1 and P. indica mycelium. The same treatment recorded the highest value with respect to physiological parameters, viz., leaf area index (19.53), leaf area duration (803.07 days), stomatal conductance (137.11 mmoles m-2 s-1), photosynthetic rate (33.73 μCO2 moles m-2 s-1) and total chlorophyll content (3.76 mg g-1 fresh weight). The highest proline content was recorded in the treatment involving chitosan nanoparticles 2.5 mg mL-1 and P. indica mycelium (34.22 μM g-1 tissue) and highest cell membrane integrity in the combination treatment of chitosan nanoparticles 2.5 mg mL-1 and P. indica culture filtrate (83.69 per cent). The shoot weight and all the root growth parameters, viz., root length, root diameter, root volume and root yield were found to be the highest in the combination treatment of chitosan 5 mg mL-1 and P. indica mycelium. The combination treatments involving chitosan nanoparticles 2.5 mg mL-1 and P. indica mycelium/culture filtrate had the highest number of berries, berry weight and seed yield per plant. Significantly higher withanolide content was observed in roots of the plants treated with chitosan 5 mg mL-1 and P. indica culture filtrate. Withanolide yield per plant was the highest in the treatment comprising chitosan nanoparticles 2.5 mg mL-1 and P. indica culture filtrate and was on par with the combination treatment of chitosan 5 mg mL-1 and P. indica mycelium/culture filtrate. In the study, it was observed that all the combination treatments gave better performance than the control in terms of root yield, withanolide content and withanolide yield per plant. The concentration of chitosan and exposure time of priming had a profound influence on seed germination and seedling growth parameters in W. somnifera. The bio-elicitors, chitosan/chitosan nanoparticles and P. indica when applied individually and in combination to chitosan primed seeds have been observed to enhance growth, yield and metabolite production in a dose dependent manner in W. somnifera.
Characterisation of Exacum bicolor Roxb.(KANNANTHALI) for aesthetic ,medicinal value and refinement of domestication practices
(Department of Floriculture and Landscaping, College of Agriculture,Vellanikkara, 2024-07-01) Aswathi, S Vasu; KAU; Sreelatha,U
India is blessed with a wide variety of endemic plant species due to the presence of four out of 34 biodiversity hotspots in the world. Endemic species with narrow geographical range of area are most likely to become extinct due to fragmentation of habitat, human interferences, deforestation, and expansion of agricultural land. Special attention must be given to such small natural populations before they get vanished. Many of such native plant species may also form an important source for sustainable landscaping and xeriscaping. Exacum bicolor which belongs to Gentianaceae family is an herbaceous perennial, endemic to peninsular India. The flowers which are a mix of colors imparted by the white petals with purple tips and bright, yellow-colored stamens, are the most beautiful part of the plant. Mature sturdy stem that ensures the non-lodging habit of Eaxcum bicolor even during heavy rainfall, makes the plant more suitable for gardening. The plant also holds enormous medicinal properties and was traditionally used by “vaidyas” for the treatment of various diseases. Though being frequently quoted by Malayalam literary writers, the plant is not that familiar to many common people due to its restricted occurrence. Exacum bicolor is an inhabitant of the dry grasslands of the lateritic hillocks and the shola grasslands in the high ranges of Kerala. The plant is enlisted as endangered due to its rapid shrinking population imposed by enmasse collection of the plants that happened decades back for herbal formulations, never ending, severe habitat destruction and also mass uprooting of the plant for its beauty. Discontinuous and isolated geographical situation of the habitats are other unique characteristics of the plant. The majority of the habitats in the lateritic hillocks are under private possession and are very likely to be exploited soon. Therefore, ex-situ conservation is of urgent need for conservation of Exacum bicolor. Evaluation of genetic variability among the available genotypes of the species and standardization of domestication practices are of utmost importance. In this context, the present study was undertaken with the objectives of morphological characterization of Exacum bicolor genotypes for aesthetic value, metabolite profiling for medicinal value, and standardization of propagation and management practices for popularization as a novel ornamental plant. The study was organized into three major experiments as morphological characterization, phytochemical analysis and standardization of domestication practices. In the first experiment, morphological characterization, eleven available genotypes of Exacum bicolor were collected and evaluated for various vegetative and floral parameters. A significant difference was noticed among the genotypes for both vegetative and floral characters. Plant characters such as stem girth (2.29 cm), number of primary branches (4.11), leaf length (8.76 cm), and floral characters such as number of flowers per plant (184.00) and duration of flowering (47.21 days) were found higher in the genotype KAU Ex 11. Qualitative characters like shape of the leaves, leaf tip as well as petal shape also differed among the genotypes. Most of the genotypes possessed linear-lanceolate leaves, whereas KAU Ex 7 and KAU Ex 14 (oblanceolate leaves), KAU Ex 9 (ovate-elliptic leaves) and KAU Ex 10 (ovate leaves) showed other leaf forms. Overall scoring of Exacum bicolor genotypes based on the number of flowers per plant and the duration of flowering resulted in the highest rank to KAU Ex 11, followed by KAU Ex 15. Wide variation in genetic parameters of various characters indicated occurrence of considerable diversity among the genotypes for these traits. The difference between phenotypic coefficient of variance (PCV) and genotypic coefficient of variance (GCV) was narrow for characters viz. number of leaves, leaf width, leaf length, number of flowers per plant and duration of flowering, indicating the impact of genetic makeup for these traits rather than influence of environment. High heritability (more than 90%) coupled with high GAM were recorded for number of leaves, leaf length, leaf width, days to bud initiation, duration of flowering which indicate the additive and dominance gene action for these characters and possibility of selection based on these traits for further crop improvement programmes. K-mean cluster analysis of eleven genotypes yielded three clusters. Four genotypes each were included in cluster 1 and cluster 2, and three genotypes in cluster 3. The clustering pattern indicated that, geographical origin had no influence on grouping of genotypes. Association among various vegetative and floral characters analyzed via genotypic and phenotypic correlation revealed that Genotypic correlation coefficients of all the attributes were greater than or equal to the phenotypic correlation coefficients which indicated that influence of environment is negligible on phenotypic variation. The second experiment was phytochemical analysis. With respect to phytochemical analysis, extractive yield in methanol, contents of total polyphenols, alkaloids, flavonoids and saponins were estimated. The extractive yield in methanol (18.98 g/100 g) and alkaloids (0.77%) were found higher in the genotype KAU Ex 7, total polyphenols were higher in KAU Ex 10 (397.03 mg GAE/100g), flavonoids in KAU Ex 16 (1541.50 mg QE/100 g) and saponins in KAU Ex 12 (3.19 %). Gas chromatography-mass spectrometry analysis of sequential extracts of two genotypes of Exacum bicolor yielded a number of possible compounds which possess various therapeutic properties. The presence of secoiridoid namely, gentiolacotne [1H,6HPyrano[ 3,4-c]pyran-1,6-dione, 5-ethyl-3,4,5,8-tetrahydro-5-hydroxy-, (.+-.)] was first reported in Exacum bicolor. The third experiment, refinement of domestication practices, was attempted on enhancement of seed germination, seedling growth, standardization of seed germination media, potting media, vegetative propagation through cuttings, spacing & pinching, elucidation of floral biology and assessment of seed dormancy. The seeds were subjected to treatments viz. priming with certain chemical/bio agents, heat scarification, and light stratification by exposing to fluorescent and ultraviolet (UV) light. Priming with GA3 at 750 mg/L for 24 h recorded higher seed germination (77.50%), seedling survival (69.25%), shoot length (23.03 mm), root length (25.63 mm), dry weight of seedlings (1.60 mg), vigour index I (3771.14) and II (124.04). Among heat scarification treatments tested, significantly highest seed germination (73.93%) and seedling survival (67.86%) was exhibited when the seeds were preheated at 120ͦC for 15 minutes. Preheating at 80ͦC for 15 minutes duration recorded significantly longest shoots (41.40 mm) and roots (21.24 mm). Exposure of seeds at 80 ͦC for 30 minutes recorded highest vigour index I (3149.54), and 100 ͦC for 15 (108.07) minutes and 120 ͦC for 15 minutes (99.90) duration of exposure yielded higher vigour index II. Light stratification using fluorescent light was found to be more beneficial for seed germination and seedling survival. Fluorescent light exposure at 12/12 h light/dark photoperiod for 6 days recorded higher seed germination (92.38%), seedling survival (92.14%), and vigour index I (7876.43) and II (296.72), whereas shoot length (41.09 mm), root length (67.01 mm), and seedling dry weight (4.80 mg) were found higher when seeds were exposed to UV light for 45 minutes. Analysing the germination media for seed germination in Exacum bicolor, seeds were sown in germination media comprised of various combinations of cocopeat, sand, FYM, perlite and vermiculite alone and along with arbuscular mycorrhizal fungus (AMF) and Phosphobacterium. Highest seed germination was observed in media composed of cocopeat and sand (1:1) (76.21%). Seedling survival, shoot length, root length and seedling dry weight were recorded higher in media containing cocopeat and sand (1:1) along with AMF (10g/pot), and cocopeat, perlite, vermiculite (3:1:1) along with AMF (10g/pot). Analysing the nutrient management for growth enhancement, seedlings were applied with nutrients viz. N: P: K as 30: 10: 10 and 19: 19: 19 and ‘Amino rich’ alone and in combination with different concentrations of plant growth regulators viz. GA3 and IAA. Foliar application of N: P: K as 30: 10: 10 alone in progressive doses (0.5 g/L in week 1 of germination; 1 g/L in week 2; 2 g/L from week 3 onwards) recorded highest seedling survival (70.12%), fresh weight (63.70 mg), and dry weight of seedlings (3.62 mg). Various combinations of growing media were tested to analyse the effect of different potting media on the growth and flowering of Exacum bicolor. Plant height was found highest in media composed of soil and AMF (10 g/ Kg medium) (39.36 cm), stem girth in soil + sand + vermicompost (1:1:1) + AMF (10g/kg medium), and number of leaves were found highest in soil + AMF (10g/kg medium) (25.44). Number of flowers per plant was found greatest in potting media comprised of soil + sand + vermicompost (1:1:1) (44.50). The potting media with soil alone (37.06 days) recorded longer duration of flowering. Larger flowers with longer petals were observed in potting media composed of cocopeat + perlite + vermiculite (3:1:1) + AMF (10g/kg medium). Based on the scoring and ranking of the twelve potting media treatments for vegetative and floral parameters, the media comprised of soil + sand + vermicompost (1:1:1) + AMF (10g/kg medium) recorded the highest rank (rank 1) followed by soil + sand + vermicompost (1:1:1) and coco peat + sand + vermicompost (3:1:1). Vegetative propagation of Exacum bicolor was performed through terminal stem cuttings treated with various concentrations of indole-3-butyric acid (IBA), charcoal powder, commercial hormone powder (Rootex). A significant difference was observed among the treatments, with 96.67 percent cuttings rooted when cuttings were treated with IBA at 5.0 mg/L. cuttings treated with IBA at 20.0 mg/L resulted in greatest number of roots (19.45) and IBA at 15.0 mg/L produced longer roots (42.86 cm). Influence of pinching and spacing on vegetative and floral parameters of Exacum bicolor was analyzed by adopting three levels of spacing viz. 20 × 20 cm, 30 × 20 cm and 30 × 30 cm and giving seven levels of pinching viz. no pinching, single pinching at 15, 30 and 45 days after transplanting (DAT), double pinching at 15 & 30, 15 & 45, and 30 & 45 DAT. Analysis of pinching and spacing on vegetative and floral parameters of Exacum bicolor showed significantly greater number of branches in all the double pinching treatments. However, plants spaced at 30 x 20 cm and double pinched on 15 & 45 days after transplanting (DAT) showed compact growth habit with stouter stem and a higher number of branches which are suitable for bedding plants. The treatment also recorded a greater number of flowers/plant (86.50) with longer duration of flowering (47.0 days). Floral biology and pollination behaviour of Exacum bicolor was studied analysing the anthesis time, pollen viability, stigma receptivity and mode of pollination. It was found that anthesis in Exacum bicolour was occurring between 4.00 am and 6.00 am with peak time of anthesis at 5.00 am (56.25%). Pollen viability gradually decreased with flower aging. Significantly higher viability of pollen grains was observed for flowers on the previous day of anthesis (D0), and flowers on the day of anthesis (D1) from 8.00 am to 1.00 pm, flowers on second day of anthesis (D2 flowers) up to 11.00 am, for third day of anthesis (D3) and fourth day of anthesis (D4 flowers) up to 9.00 am. The results of the stigma receptivity test revealed that pollinating flowers on the third day of anthesis at 9.00 am ensures successful seed set, capsule weight and seed weight per capsule. Pollination behaviour of Exacum bicolor was studied by assessing five methods of pollination such as controlled selfing, controlled out-crossing, forced open pollination, self-pollination (bagging) and open pollination. Cent percent fruit set was obtained in controlled selfing, controlled outcrossing and in open pollination indicating that the plant is both self and cross compatible. Lack of fruit set in self-pollination (bagging) is an indication of lack of the phenomenon of apomixis to promote natural self-pollination in this species. Seed dormancy was assessed by performing seed germination test immediately after seed extraction and on alternate days subsequent to seed extraction up to 35 days (D1, D3, D5,… D35) and thereafter at weekly intervals, until germination percentage reached greater than 50 percent. The germination test was performed without any seed treatment (control) and with seed priming using 750 mg/L GA3 for 24 h. Lack of seed germination in control even up to the 84th day of seed extraction indicated the occurrence of seed dormancy in this species. However, seeds treated with GA3 germinated from the first day seed extraction onwards. From the study, it could be concluded that Exacum bicolor can be effectively utilised as an ornamental potted plant, and standardisation of domestication practices ensures measures for successful cultivation of the plant for the plains in Kerala
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.
Development and evaluation of high yielding, mosaic tolerant backcross progenies in bitter gourd (Momordica charantia L.) variety Preethi using morphological, biochemical and molecular markers
(Department of Genetics and Plant Breeding, College of Agriculture ,Vellayani, 2024-05-23) Ankitha, M O; KAU; Bindu, M R
The present research work entitled ‘Development and evaluation of high yielding, mosaic tolerant backcross progenies in bitter gourd (Momordica charantia L.) variety Preethi using morphological, biochemical and molecular markers’ was conducted in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani and Farming Systems Research Station (FSRS), Sadanandapuram during the year 2020-2023, with an objective to develop high yielding mosaic tolerant backcross progenies in bitter gourd using morphological, biochemical and molecular markers. Thirty three bitter gourd genotypes, including KAU released varieties (2 No’s), NBPGR accessions (13 No’s), and local collections from all over India were used for screening mosaic tolerance. Out of the 33 genotypes, 26 genotypes were Momordica charantia var. charantia and seven were Momordica charantia var. muricata. All these genotypes were artificially inoculated with the three viruses Cucumber Mosaic Virus (CMV), Tomato Leaf Curl New Delhi Virus (ToLCNDV) and Papaya Ringspot virus (PRSV) through wedge grafting. Wedge grafting was done using the infected plant shoots as scion and the collected genotypes as root stock and regrowth from the cotyledonary axis was examined for symptom expression. Out of the 33 genotypes screened, three were highly resistant, four were resistant, five were moderately resistant, six were moderately susceptible, ten were susceptible and five were highly susceptible. The genotypes Lodhi local, Udayagiri local and Therthali local recorded a lowest Vulnerability Index of zero. Molecular markers reported in Cucurbitaceae family were validated for bitter gourd mosaic resistance gene. SSR-11-1 marker for CMV resistance and CAPS marker for Potyvirus resistance gene were used, but no amplification was obtained. Double Antibody Sandwich ELISA (DAS-ELISA) was performed to confirm the resistance reaction of three highly resistant genotypes identified in seedling screening. Optical density (OD) value of the genotypes for the three viruses, CMV, ToLCNDV and PRSV were less than twice the OD value of the un-inoculated healthy plant which confirmed highly resistant disease reaction of genotypes. Molecular confirmation was done by using coat protein primer (Deng primer) specific to the Begomovirus group. Deng primer amplifies coat protein gene of ToLCNDV (520 bp), so that band will be present in only susceptible genotypes and will be absent in resistant ones. Plant defense related enzymes such as peroxidase, polyphenol oxidase and phenyl alanine ammonialyase was estimated and there was increased rate of synthesis of these enzymes in the identified resistant genotypes. So the identified resistant genotypes, Lodhi local, Udayagiri local and Therthali local were used as the donor parent for imparting mosaic resistance into the bitter gourd variety Preethi. Lodhi local is M. charantia var. charantia genotype where as both Udayagiri local and Therthali local are M. charantia var. muricata genotypes. High yielding variety released from KAU viz., ‘Preethi’ was selected as the recurrent parent in the study. Preethi was crossed with the three donor parents and F1s were produced. The F1s were morphologically evaluated with the parents for seventeen characters and it was observed that all the characters of F1 were approximately the average of two parents. All the F1s were backcrossed with Preethi to produce BC1F1 segregants. In the backcross progeny of the cross involving Preethi and Lodhi local, a total of 176 BC1F1 lines were developed. BC1F1 lines were artificially inoculated for their disease reaction. Among the 176 BC1F1 lines, 22 were found to be highly resistant to mosaic disease, 30 were resistant, 30 were moderately resistant, 26 were moderately susceptible, 35 were susceptible and 33 were highly susceptible. Confirmation of resistance was done using DAS- ELISA, Deng primers and estimation of defense enzymes. All the 17 biometrical characters were recorded and the Euclidean distance of the highly resistant BC1F1 lines from the recurrent parent Preethi was calculated using proximity dissimilarity matrix analysis. The 14 BC1F1 lines with high phenotypic similarity to Preethi was backcrossed to develop BC2F1 lines. In the backcross progeny of the cross involving Preethi and Udayagiri local, a total of 170 BC1F1 lines were produced. Among them 15 BC1F1 lines were highly resistant. Resistant reaction of identified 15 BC1F1 was confirmed by DAS-ELISA, molecular screening and biochemical analysis. Euclidean distance of the highly resistant 15 BC1F1 lines from the recurrent parent revealed that eight lines showed similarity with Preethi and they were backcrossed to get BC2F1 lines. A total 147 BC1F1 lines of the cross involving Preethi and Therthali local were screened at seedling stage. Out of the 147 lines, 16 BC1F1 lines were highly resistant. DAS-ELISA, molecular screening using aforementioned Deng primer confirmed the resistant reaction of these lines. Euclidean distance using biometric characters found that, out of 16 highly resistant BC1F1 lines eight lines had close proximity with Preethi. These lines were used to produce BC2F1 lines. The 190 BC2F1 lines of the cross involving Preethi and Lodhi local were screened at seedling stage and in 24 BC2F1 lines, there was absence of virus coat protein band which confirmed the highly resistant disease reaction of the aforementioned lines. The 12 BC2F1 lines with the shortest Euclidean distance and high phenotypic similarity with Preethi were selfed to generate BC2F2 seeds. In the 134 BC2F1 lines of the cross involving Preethi and Udayairi local, seedling screening recorded 17 highly resistant lines. After molecular confirmation of mosaic resistance four BC2F1 lines with close proximity to Preethi were selfed to get BC2F2 seeds. Out of the 143 BC2F1 lines of the cross involving Preethi and Therthali local, 20 BC2F1 lines were highly resistant. The molecular analysis of the 20 BC2F1 lines also confirmed the highly resistant reaction. Four BC2F1 lines with the shortest Euclidean distance was selected and selfed to produce BC2F2 seeds. Although there were BC2F2 seeds of three different crosses, only the BC2F2 seeds of the cross involving Preethi and Lodhi local was carried forward for further backcrossing. This is due to the low yield potential of the backcross progenies of the crosses involving M. charantia var. muricata genotypes. So 206 BC2F2 lines of the cross involving Preethi and Lodhi local were artificially screened at seedling stage for mosaic incidence. Out of the 206 BC2F2 lines, 42 plants were highly resistant to bitter gourd mosaic viruses. The 42 mosaic tolerant backcross inbred lines developed in the study can be carried forward for the development of a mosaic tolerant essentially derived variety (EDV) in the background of high yielding variety Preethi. The backcross progenies obtained in the crosses involving muricata genotypes can be further evaluated for its nutraceutical values.

1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)

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