Browsing by Author "Krishnapriya, P J"

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Etiology and immuno- molecular detection of the virus causing mosaic disease in snake gourd
(Department of Plant Pathology, College of Agriculture,Vellayani, 2024-01-23) Ammu Asok, A; Krishnapriya, P J
The study entitled “Etiology and immuno-molecular detection of the virus causing mosaic disease in snake gourd” was carried out at College of Agriculture, Vellayani during 2021-2023 with the objectives of symptomatology, maintenance of virus in systemic and local lesion host, host range studies, seed transmission, aphid transmission, immunological diagnosis, molecular diagnosis and host pathogen interaction of the viruses causing mosaic disease in snake gourd (Trichosanthes anguina). Mosaic diseased snake gourd samples were collected from three taluks of Thiruvananthapuram district (Thiruvananthapuram, Neyyattinkara and Varkala) of Kerala during January-March and July-September of 2021-23. The characteristic symptoms of snake gourd mosaic disease were mosaic, mosaic mottling, vein banding, blistering, distortion, puckering on leaves and deformed fruits. The disease incidence in majority of the fields was found to be over 25.0 per cent and disease severity progressed with age of the crop. In the surveyed locations, the highest disease incidence of 94.0 to 100 per cent and vulnerability index of 72.9 to 82.7 were observed in commercial variety Kaumudi from fields of College of Agriculture, Vellayani. In snake gourd variety Baby, disease incidence of 28.5 per cent and vulnerability index of 46.6 were observed. Similar symptoms of mosaic were observed on weeds like Hemidesmus indicus (F: Apocynaceae) and Synedrella nodiflora (F: Asteraceae) in snake gourd field. The snake gourd mosaic viruses were sap transmissible and maintained in systemic host viz., snake gourd varieties Kaumudi and Baby and also in local lesion hosts viz., Chenopodium amaranticolor and Nicotiana tabacum var. Samsun. Local lesion assay revealed that viruses associated with snake gourd mosaic disease were Potyviruses and Cucumoviruses. Based on host range studies, virus was mechanically transmissible to plants of family Cucurbitaceae viz., pumpkin, bitter gourd, cucumber, ridge gourd, bottle gourd, water melon and family Solanaceae viz., Nicotiana glutinosa and Nicotiana tabacum var. Samsun. However, no symptoms were produced in tomato, chilli, brinjal and papaya. Host range studies identified viruses associated with the snake gourd mosaic disease as Papaya ring spot virus (PRSV) Type W and Cucumber mosaic virus (CMV). No seed transmission was recorded in snake gourd plants raised from seeds produced by snake gourd mosaic viruses infected plant. Aphid transmission of the virus of 30.0 per cent was observed with Aphis craccivora and 50.0 per cent was observed with A. gossypii. The viruses causing snake gourd mosaic disease were immunologically detected using Double antibody sandwich-Enzyme linked immunosorbent assay (DAS-ELISA) and Direct antigen coating-ELISA (DAC-ELISA) with polyclonal antibodies specific to PRSV and CMV respectively. Dot immunobinding assay (DIBA) detected CMV from snake gourd mosaic diseased samples. Reverse transcriptase-Polymerase chain reaction (RT-PCR) was carried out and amplicons of sizes 1200 bp and 400 bp were obtained with primers specific to coat protein of PRSV and 2a protein of CMV respectively. Comparative nucleotide sequence alignment of the isolates revealed an 86.5 and 93.8 per cent homology with PRSV and CMV isolates from Tamil Nadu respectively. Comparative amino acid sequence alignment of the isolates revealed an 88.9 and 97.8 per cent homology with coat protein of PRSV-W and 2a protein of CMV respectively. The sequences were deposited in NCBI GenBank with accession numbers viz., OR601008 and OR601007. Phylogenetic tree was constructed and PRSV isolate grouped in the same clade along with other reported PRSV type-W isolates, thus the isolate was identified as PRSV Type-W. CMV isolate from snake gourd grouped in the same clade along with other CMV isolates from snake gourd and Musa sp. Virus inoculated snake gourd plants showed a significant reduction in total chlorophyll content (0.61 mg g-1) and increase in protein content (13.79 mg g-1) at 5 weeks after transmission. The activities of defense related enzymes (peroxidase, polyphenol oxidase, phenylalanine ammonialyase, superoxide dismutase and catalase) were higher in inoculated plants compared to healthy plants. Thus, the present study revealed that snake gourd mosaic disease is caused by the combined infection of PRSV-W and CMV in the surveyed locations of Thiruvananthapuram district. Synedrella nodiflora was identified as the suitable weed host and viruses were transmitted through Aphis gossypii and Aphis craccivora.
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; Umamaheswaran, K
The 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.
Management of snake gourd mosaic disease using endophytes
(Department of Plant Pathology, College of Agriculture, Vellayani, 2025-05-29) Farhana, A P.; Krishnapriya, P J
The study entitled "Management of snake gourd mosaic disease using endophytes" was conducted at Department of Plant Pathology, College ofAgriculture, Vellayani during 2023-2025 aimed at the utilisation ofbacterial endophyte viz., Bacillus velezensis PCSE 10 and fungal endophytes viz., Arbuscular Mycorrhizal Fungi (AMF) and Piriformospora indica for the management of snake gourd mosaic disease; and the elucidation of biochemical mechanisms involved in resistance or tolerance of snake gourd plants against viruses. Mosaic, mosaic mottling, vein banding, puckering and blistering on leaves were the symptoms ofsnake gourd mosaic disease observed from fields at Instructional farm, College ofAgriculture, Vellayani, Thiruvananthapuram. Disease incidence and severity progressively increased over different months after transplanting, with the highest observed values at 3 MAT i.e., 100 per cent and 81.6 respectively. The virus inoculum was maintained in snake gourd variety Kaumudi through mechanical transmission and typical symptoms of mosaic disease appeared after 14 days of inoculation. The diseased samples reacted positively to primers specific to coat protein of PRSV (PRSV Fl and F2) and 2a protein of CMV (CMV 1 and 2); and amplicons of sizes 850 bp and 450 bp were obtained respectively.Successful root colonization of snake gourd plants with fungal endophytes viz., AMF (presence of arbuscules and vesicles) and P indica (chlamydospores) was observed at 30 days after inoculation with them. Pot culture studies with endophyte colonized snake gourd plants revealed that, those colonized with P indica took the highest number of days for symptom expression (22 days) followed by plants colonized with B. velezensis (21 days) and AMF (20 days). P. indica colonized plants recorded a reduced disease incidence (20.30 per cent, 33.48 per cent and 46.61 per cent) and severity (9.30, 14.60 and 48.00) followed by B. velezensis (33.47 per cent, 46.43 per cent and 66.33 per cent; 10.60, 18.67 and 64.00) and AMF colonized plants (40.74 per cent, 60.38 per cent and 73.59 per cent; 13.32,29.33 and 76.33) at 40, 50 and 60 DAS respectively. The combined application of endophytes did not give any significant difference in reducing the expression of disease symptoms. Reaction of inoculated samples to polyclonal antibody of coat protein of CMV in DAC-ELISA and PRSV in DAS-ELISA was analysed. Among the endophyte colonized plants, P indica colonization resulted in the lowest virus load of CMV (0.0040, 0.0186 and 0.0202) and PRSV (0.0020, 0.0131 and 0.0149) followed by B. velezensis (0.0050, 0.0030;0.0200, 0.0143 and 0.0213, 0.0168) and AMF (0.0070,0.0048; 0.0236, 0.0161 and 0.0241, 0.0197) at 40, 50 and 60 DAS respectively. Based on the remission of symptoms and reduced virus titre values_,_ pre colonization with P. indica followed by individual pre colonizations with B. velezensis and AMF were identified as the three promising endophytes in alleviating symptoms of snake gourd mosaic disease.P. indica colonized plants gave the highest activities of peroxidase (30.46 g min' g' fw), polyphenol oxidase (20.32 g min'g' fw) and phenylalanine ammonia lyase (104.86 g min'g' fw) followed by colonization with B. velezensis (28.59, 14.41 and 99.28 g min'g' fw) and AMF (27.84, 4.64 and 96.51 g min'g' fw) at 50 DAS. Similarly, P indica colonization recorded the highest activities of catalase (292.31 mg g' fiw) and ascorbic acid oxidase (0.760 g min'g' fw) followed by B. velezensis (284.86 mg g' fw, 0.64 g min'g' fw) and AMF (278.73 mg g' fw,0.43 g min'g' fw) at 60 DAS. SDS PAGE analysis identified the induction of a novel protein (31.57 kDa) in plants in response to colonization with AMF. However, pre colonization with P. indica and B. velezensis PCSE 10 reduced the number of profiled proteins when compared to control plants.Field studies revealed that, P indica colonization increased the number of days taken for symptom development (36.8 days) followed by B. velezensis (31.4 days) and AMF (28.2 days). Similarly, reduced disease incidence was observed in P. indica colonized plants (88.36 per cent) followed by B. velezensis (92.95 per cent) and AMF (99.74 per cent) at 90 DAS. Also, P indica colonization resulted in the highest yield/snake gourd plant (7.037 kg). The present study identified pre colonization of snake gourd plants with P. indica followed by colonization with B. velezensis and AMF as the three promising treatments in alleviating snake gourd mosaic disease owing to their ability in reducing virus titre and disease incidence/severity, induction of defense related enzymes and novel proteins. Similarly, the promising endophytic treatments reduced the expression of snake gourd mosaic disease symptoms with improved vegetative characters under field conditions.
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; Geetha, D
The 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.