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ThesisItem Open Access Strain improvement of oyster mushrooms- pleurotus cystidiosus O.K. Mill and pleurotus opuntiae (Durieu and LEV.) SACC.(Department of Plant Pathology, College of Agriculture, Vellayani, 2018) Krishnapriya, P J; KAU; Geetha, DThe present study entitled “Strain improvement of oyster mushrooms: Pleurotus cystidiosus O.K.Mill and Pleurotus opuntiae (Durieu and Lev.) Sacc.” was carried out in College of Agriculture, Vellayani during 2015-2018, with the objective to standardize the techniques for production of oyster mushrooms: P. cystidiosus and P. opuntiae; and to study their morphological, physiological and cultural characteristics as well as nutritional and organoleptic qualities; and to undertake genetic improvement by protoplast fusion. The mushrooms were collected from two locations of Thiruvananthapuram and three fast growing isolates of Pleurotus spp. viz., PC2 (Vellayani), PNC1 (Chirayinkeezhu) and PO1 (Vellayani) were selected for the study. These isolates were identified as P. cystidiosus subsp. abalonus, P. cystidiosus and P. opuntiae using internal transcribed spacer (ITS) primers and subsequent sequencing; and registered at Genbank database with accession numbers KY214254, KY887023 and KY214255 respectively. The fast growing isolates of P. cystidiosus (coremial), P. cystidiosus (non-coremial) and P. opuntiae recorded maximum growth on PDPA amended with one per cent yeast under dark condition. The optimum temperatures for the growth were 30 0C, 25 to 30 0C and 25 0C respectively whereas, the optimum pH were 8, 8 and 7 to 8 respectively. Studies with different substrates and amendments for spawn production revealed that sorghum with one per cent yeast was the best for P. cystidiosus (coremial) and P. opuntiae whereas, paddy grains with one per cent yeast for P. cystidiosus (non-coremial). Experiments with different substrates and amendments for mushroom production revealed that rubber wood sawdust sprayed with 2.5 per cent of 1 M potassium dihydrogen phosphate recorded the maximum BE for P. cystidiosus (non-coremial) (192.76 per cent). P. opuntiae recorded the maximum BE in rubber wood sawdust amended either with 4 per cent neem cake (91.38 per cent) or wheat bran (91.37 per cent). Major insect pests observed in the beds of Pleurotus spp. were phorid flies, spring tails, black ants and staphylinid beetles. The competitor moulds observed were different species of Coprinus, Aspergillus, Penicillium and Trichoderma. Sporocarps soaked in one per cent CA for 15 minutes followed by mechanical drying and powdering was the best post harvest treatment for both P. cystidiosus (non-coremial) and P. opuntiae. Mycelium of P. cystidiosus (coremial) showed black coremial structures, representing its asexual stage (Antromycopsis broussonetiae Pat. & Trab.). The coremia comprised of elliptical (16.31 µm x 7.48 µm) and round conidia (8.06 to 8.49 µm). The black colour of coremia was due to melanin which was extracted (255.56 mg l-1) and characterized. The performance of long duration P. cystidiosus (non-coremial) and short duration P. opuntiae was compared with two ruling mushrooms of Kerala viz., long duration P. florida (Mont.) Singer and short duration P. eous (Berk.) Sacc. The study revealed that P. cystidiosus (non-coremial) and P. opuntiae showed higher BE compared to P. florida and P. eous, respectively. P. cystidiosus (non-coremial) recorded maximum moisture (94.05 per cent), starch (200.55 mg g-1), protein (30.2 mg g-1), fat (4.25 per cent), antioxidants (485.45 μg equivalent gram of ascorbic acid-1), beta-carotene (25.69 µg 100 mg-1), polyphenols (7.55 mg g-1) and energy (359.45 Kcal) compared to other Pleurotus spp. Sensory evaluation of mushroom products made from the species of Pleurotus was done and masala curry prepared from P. cystidiosus (non-coremial) scored the maximum value for overall acceptability. Shelf life of P. cystidiosus (non-coremial) was higher (5 days) compared to P. opuntiae, P. florida and P. eous (3 days each) in perforated poly propylene covers stored under refrigeration. Vanillin (0.05 per cent) and carbendazim (1 mM) were selected as dual biochemical markers for the PEG mediated protoplast fusion. Three days old P. cystidiosus (non-coremial) and four days old P. opuntiae recorded the maximum protoplast yield at five and four hours after incubation respectively with 0.6 M KCl and 30 mg ml-1 of enzyme consortium. Eight fusant lines with varied mycelial characters were obtained. Among fusants, F6 and F8 did not segregate in the second generation whereas, F4 segregated. F6 and F8 recorded higher BE of 168.05 and 99.95 per cent respectively compared to the parental lines and other fusants. Sporocarp of F6 and F8 was morphologically similar to P. cystidiosus (non-coremial) and P. opuntiae respectively; and F8 also exhibited low temperature adaptability. The present investigation indicated the exploitability of two promising isolates viz. P. opuntiae for tropical areas and P. cystidiosus (non-coremial) for cooler regions of Kerala using locally available materials and the standardized cultivation practices. The present study also standardized the protoplast fusion technique between P. cystidiosus (non-coremial) and P. opuntiae; and two fusant lines (F6 and F8) recorded higher BE which can be used for future breeding programmes.ThesisItem Open Access Immunomolecular detection and characterisation of potyviruses infecting cowpea (Vigna unguiculata (L>) Walp.) and papaya (Carica papaya L.)(Department of Plant Pathology, College of Agriculture, Vellayani, 2015) Krishnapriya, P J; KAU; Umamaheswaran, KThe study entitled “Immunomolecular detection and characterisation of Potyviruses infecting cowpea (Vigna unguiculata (L.) Walp) and papaya (Carica papaya L.)” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during 2013-2015 with the objective to study the symptomatology and transmission of Potyviruses infecting cowpea and papaya, along with their biological, immunological, molecular detection and characterisation. Symptoms caused by Blackeye cowpea mosaic virus (BlCMV) in cowpea include mosaic and vein banding and that of Papaya ring spot virus (PRSV) in papaya showed mosaic and shoe string on leaves and ring spot on fruits and leaves. BlCMV and PRSV were mechanically transmitted (80 and 100 per cent respectively). Seed transmission (48.5 per cent) was recorded for BlCMV in cowpea. PRSV was not transmitted through seeds. BlCMV was transmitted by Aphis craccivora Koch and Aphis gossypii Glover (30 per cent respectively). Papaya mealy bug (Paracoccus marginatus Williams and Garnara de Willink) (80 per cent) was the most efficient vector of PRSV followed by Aphis gosssypii Glover (40 per cent) and Aphis craccivora Koch (30 per cent). Host pathogen interaction studies in resistant and susceptible genotypes of the cowpea (CO6 and Vellayani Jyothika) and papaya (Pusa Nanha and local variety) indicated a significant reduction in total carbohydrates and chlorophyll contents in susceptible genotypes on inoculation with Potyviruses, whereas resistant genotypes showed no significant difference. However, phenol, protein and defense related enzymes showed a significant increase in resistant genotypes on inoculation, compared to the susceptible genotypes. Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE were conducted for the comparison of proteins and isozymes respectively, in resistant and susceptible genotypes. Eleven novel proteins were induced in CO6 whereas ten new proteins were observed in Vellayani Jyothika. Nine and eight induced proteins were identified in Pusa Nanha and local variety of papaya respectively. Three isoperoxidases with Relative mobility (Rm) values of 0.13, 0.20 and 0.27 were unique for CO6. There was no unique isozyme on inoculation in Vellayani Jyothika. Isoperoxidases with Rm values of 0.08 and 0.23 were unique for Pusa Nanha. However no specific isozyme was observed in local variety of papaya. Chenopodium amaranticolor and Chenopodium quinoa were identified as indicator plants for the biological detection of BlCMV and PRSV. Immunological studies using Direct antigen coating-Enzyme linked immunosorbent assay (DAC-ELISA) and Dot immunobinding assay (DIBA) and molecular detection viz. Reverse transcription-Polymerase chain reaction (RT-PCR) identified the Potyviruses as Bean common mosaic virus (BCMV) strain Blackeye and PRSV respectively. Comparative amino acid sequence alignment revealed a 95 and 100 per cent homology with coat proteins of BCMV and PRSV respectively. The transmission studies revealed that Potyviruses were transmitted mechanically and by insects. Present study also revealed that BlCMV was transmitted through seeds while, PRSV was not seed transmitted. The host pathogen interaction studies identified phenol, protein and defense related enzymes along with induced proteins and isoforms of peroxidase could be used as biochemical markers to identify resistance and susceptibility in plants. The present molecular study confirmed that BlCMV infecting cowpea was closely related to BCMV, a strain of Blackeye. PRSV in the present investigation was found related to PRSV isolates reported from other parts of India. Comparative sequence analysis of BlCMV and PRSV Potyviruses showed that the sequences were entirely different and showed only a 51.12 per cent similarity at nucleotide levels. Serological and sequence studies thus suggest that BlCMV caused the mosaic disease in cowpea and PRSV, the ring spot disease in papaya.