Browsing by Author "Ayisha, R"

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Biochemical response of okra to bhendi yellow vein mosaic virus and production of virus free plants
(Department of plant pathology, Vellayani, 2023-11-14) Swetha, B Nair; Ayisha, R
The research entitled ‘Biochemical response of okra to Bhendi Yellow Vein Mosaic Virus and production of virus free plants’ was undertaken at the College of Agriculture, Vellayani, Thiruvananthapuram during the years 2021 to 2023 with the objective of host pathogen interaction of Bhendi yellow vein mosaic virus (BYVMV)infecting okra, screening of different genotypes of okra cultivars for disease resistance and production of virus free plants using meristem culture. Purposive sampling survey was done and samples were collected from AEU8 and AEU 10. The disease incidence of virus infected okra plants in surveyed locations ranged from 37 to 100 %. And vulnerability index varied from 23.40 to 85.20. Highest V. I. was observed in Arka Anamika (85.2) followed by varieties, Varsha Uphar(80) and Anakomban (83.53). The okra variety, Anjitha, recorded lowest disease incidence of 37.20 per cent and vulnerability index of 23.40. Disease incidence was observed to be high at the flowering and fruiting stage. The virus causing BYVMD was serologically detected using ELISA and DIBA and found that BYVMV isolate have close relationship with tomato leaf curl New Delhi virus. The molecular detection of virus was done using PCR and an expected amplicon of size 520 bp was obtained using Deng primer. The BLAST analysis of the sequence of amplicon from okra with vein clearing symptom and enation symptom showed 96.27 % and 96.67 % similarity with DNA-A segment of bhendi yellow vein mosaic virus isolate. Out of 286seeds collected from infected plants seeds sown, only 57.14 % germination was observed. None of the seedlings established symptoms till flowering stage. Molecular analysis of random samples taken from asymptomatic leaves of grow out plants also showed negative reaction to the virus. ELISA also showed negative results confirming absence of seed transmission. But, presence of virus was detected molecularly on whole seeds taken randomly from the seeds collected from infected plants. Out of 20 seeds examined only four seeds showed positive reaction. The presence of virus was also not detected on any of the seed parts examined. Hence it can be inferred that the virus is present on seed samples but there is no seed transmission. On screening of 15varieties, Phule Vimukta variety (V.I.-13.34) was found to be moderately resistant to BYVMD and Anjitha variety (V. I.- 38.30) was moderately susceptible to BYVMD. Arka Anamika, Anakomban, Arka Nikitha and ten NBPGR accessions studied were found to be highly susceptible to BYVMD.(IC 052303, IC 00780, IC 588166, IC 002134, IC 006101,IC 002024, IC 043279, IC 093771, IC 093688, IC 045820)Defense-related enzymes activities varied with genotypes and also with the growth stages of the crop. Tolerant varieties were found to possess the highest content of defence enzymes compared to susceptible varieties. PO activity of moderately resistant variety Phule Vimukta was observed with 270.29 % and 35.02 % more enzyme activity than the susceptible variety, Anakomban at 90 days after sowing and 45 days after graft inoculation respectively. PO and PPO activity showed an increasing trend on inoculation with virus while PAL showed a decreasing trend. Standardisation of production of virus free plants using meristem culture was done. Meristem culture can be successfully done using MS media with BAP (0.5µM), NAA(0.1 µM) and GA3(0.1µM). Virus indexing of meristem cultured plants by PCR confirmed the absence of virus in regenerated plants. Based on the present study, the virus causing BYMD showing symptoms of vein clearing and enation was found to be BYVMV. Presence of virus was detected on seed (20 %) but seed transmission was not observed in grow out test. Breakdown of resistance was observed in Varsha Uphar and Arka Anamika which were earlier reported as resistant. The variety Phule Vimukta, with disease resistance and high defense related enzyme activity can be used for breeding purposes for the development of disease resistant varieties. Meristem culture can be successfully used for the production of disease-free planting materials and production of quality seeds.
Biochemical response of okra to bhendi yellow vein mosaic virus and production of virus free plants
(Department of plant pathology, Vellayani, 2023-11-04) Swetha, B Nair; Ayisha, R
Characterisation of a tospovirus causing necrosis disease of cowpea (vigna unguiculata (L.) walp.)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2005) Ayisha, R; Umamaheswaran, K
Characterization and management of viral diseases of black pepper(Piper nigrum L.)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2010) Ayisha, R; Joseph P J
A detailed survey was undertaken to study the occurrence and distribution of viral diseases in black pepper in Thiruvananthapuram and Kollam districts of Kerala. Disease incidence (DI) and per cent disease index (PDI) were determined during survey which showed that per cent DI varied between 0-57 and PDI between 0-18. The disease was prevalent in both the districts. Most of the local cultivars and improved varieties were susceptible to the disease. The characteristics symptoms of disease were chlorotic spots on emerging younger leaves, vein clearing, scattered chlorotic flecks followed by chlorotic mottling along veins leading to interveinal chlorosis and characteristic twisting and curling of leaves. The infected leaves were also observed to be small, crinkled, and brittle with reduced internodal length, leading to typical stunting of plants. Most of the diseased plants were found to be infected with both Cucumber mosaic virus (CMV) and Pepper yellow mottle virus (PYMo V). The presence of these viruses was confirmed through conducting enzyme linked immunosorbent assay (ELISA) on representative samples collected from different locations. The virus was not mechanically transmitted to healthy pepper seedlings. However the virus was found to be transmitted through grafting, insect vectors and also through seeds. The mealy bug, Ferrisia I virgata was found to be the efficient vector although aphid, Toxoptera aurantii, was also found to be transmitting the virus. Thermal inactivation point was recorded at a range of 40-450C and dilution end point between 10-3 and 10-4 for CMV. Host range studies revealed that virus could be readily transmitted to other species in Piperaceae family as well to some of the weed hosts. The virus was partially purified and antiserum was produced with a titre of 1:128. Identification and serological characterization of the virus was done using ELISA and DIBA. Molecular detection of the virus was also performed using PCR and a PCR product of amplicon size 500 bp and 300 bp were obtained for primers specific to CMV and banana streak virus (BSV) respectively. The pathophysiological studies revealed that virus infected plants showed increased phenol, carbohydrate and protein content. The chlorophyll content was found to be less in infected samples. The activity of defence related enzymes like peroxidase, polyphenol oxidase and phenylalanine ammonialyase were found to be more in infected plants. Electrophoretic analysis of virus infected samples through SOS-PAGE revealed the presence of two novel proteins in diseased samples. Analysis of isozymes through native gel revealed the production of an additional isoform of peroxidase and over expression of polyphenol oxidase in infected plants. In screening of varieties for the source of resistance Panniyur, 2, 3 and 4 were found moderately resistant and Karimunda was highly susceptible. Piper colubrinum showed resistance to the virus. Meristem culure attempted was unsuccessful and could not be used as a viable strategy for eliminating the virus infecting black pepper as the meristems were seen contaminated with the pepper badnavirus.
Endospore-forming endophytic bacteria for the management of tospovirus infection in COWPEA(Vigna unguiculata ssp. sesquipedalis)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2023-09-25) Ramseena, S R; Ayisha, R
The study entitled “Endospore-forming endophytic bacteria for the management of Tospovirus infection in cowpea (Vigna unguiculata ssp. sesquipedalis)” was carried out at College of Agriculture, Vellayani during 2021-2023 with an objective of evaluating the efficacy of Endospore-forming Endophytic Bacteria (EEB) for the management of Tospovirus causing bud necrosis in cowpea (Vigna unguiculata ssp. sesquipedalis). The virus was sap transmissible and was maintained in local lesion host, Chenopodium amaranticolor and susceptible cowpea variety, Pusa Komal by mechanical inoculation using potassium phosphate buffer. On mechanical inoculation to chenopodium plants, cowpea Tospovirus isolate took 4-5 days for symptom development and produced chlorotic and necrotic lesions. In cowpea variety Pusa Komal it took 8 days for symptom development and produced both local and systemic infections. The virus that causes cowpea bud necrosis disease was identified serologically using ELISA and DIBA, and it was discovered that Tospovirus isolation from several cowpea samples demonstrated a close relationship with Watermelon silver mottle virus (WSMoV). Molecular detection was done by RT-PCR and an expected amplicon of size 477–500 bp was obtained using the primer specific to the coat protein gene of Groundnut bud necrosis virus (GBNV). Serological and molecular detection confirmed that the virus associated with bud necrosis of cowpea belong to the serogroup IV which is Watermelon silver mottle orthotospovirus. Screening of five different Bacillus strains was done for evaluating its efficacy in managing cowpea bud necrosis disease. Bacillus pumilus VLY17, Bacillus amyloliquefaciens VLY24, Bacillus velezensis PCSE10, Bacillus amyloliquefaciens CBSE5 and Bacillus velezensis CBRE5 were used for screening. Pre and post inoculation with EEBs in C. amaranticolor and susceptible cowpea variety Pusa Komal were carried out and B. pumilus VLY17 treated plants demonstrated reduced number of local lesions (4) in C. amaranticolor, when compared to that of virus inoculated control with a significantly higher number of local lesions (11). In cowpea, the plants pre colonised with B. pumilus VLY17 were observed with low vulnerability index of (V I) (20) compared to untreated plants with a V I of 66.6. Hence, B. pumilus VLY17, with good disease-suppressing ability, was selected and further evaluated against the virus in tolerant cowpea variety, Githika, to standardise the effective method of application. The most efficient strategy of treating EEB, according to the analysis of different application techniques, was seed priming followed by foliar spray and drenching with a suspension of B. pumilus at 108 CFU per ml at cotyledonary leaf stage of cowpea plants compared to the untreated ones. The number of branches (19), fresh weight (163.6 g) and number of pods (19) obtained from B. pumilus VLY17 treated plants showed a significant increase than that of the virus control plants. The treated plants also flowered 3-4 days earlier than the virus control and exhibited considerable resistance towards further pest and disease occurrence. When analysed the enzyme activity over 3 weeks in different treatments, enzyme activity of polyphenol oxidase and phenylalanine ammonia-lyase was found to be higher at second week and peroxidase activity was higher at the third week in treated plants. On analysis of the average of enzyme activity over 3 weeks, peroxidase (74.1 μg/g of leaf tissue min -1), polyphenol oxidase (6.23 μg/g of leaf tissue min -1) and phenylalanine ammonialyase (72.7 μg of cinnamic acid per gram of leaf tissue) were found to be high in treatment, where seed priming followed by soil drenching and foliar spray at cotyledonary leaf stage prior to the virus inoculation was applied. Total protein content (49.9 μg of BSA per gram of leaf tissue) was also found to be higher in treated ones when compared to the untreated ones at second week. Expression of defense genes such as NPR1, Coi1, PAL and BGL were analysed in treated and virus control plants by RT-PCR and ImageJ software. The analysis showed that seed treatment followed by foliar spray and drenching with B. pumilus VLY17 at the cotyledonary leaf stage of cowpea plants prior to virus inoculation, gave the highest expression of defense genes such as NPR1 (46.18), Coi1 (31.56) and PAL (25.59) in terms of band peak percentage compared to the virus inoculated and absolute control plants. The BGL gene expression (33.55) was found to be less in B. pumilus VLY17 seed primed plants than the virus control plants. There are reports establishing that BGL gene expression is controlled by the virus in infected plants as it helps in the movement of the virus by hydrolysis of callose, deposited in plasmodesmata, formed as a part of defense due to virus infection. Hence, less activity of BGL gene is indicating more defense as it can hinder the movement of virus. The research highlights the biocontrol potential of B. pumilus VLY17 to reduce the disease severity by enhancing the expression of defense genes and by promoting growth and yield parameters of cowpea plants. Seed priming with B. pumilus VLY17 for 4 hours followed by soil drenching and foliar spray at cotyledonary stage of cowpea is the best method of application of B. pumilus VLY17 for the management of bud necrosis disease in cowpea.
Viricidal activity of nanoparticles against Cowpea aphid borne mosaic virus in cowpea
(Department of Plant Pathology, College of Agriculture,Vellanikkara, 2025-03-12) Anirudh, S.; Ayisha, R
The study entitled “Viricidal activity of nanoparticles against Cowpea aphid borne mosaic virus in cowpea” was carried out at the College of Agriculture, Vellanikkara during 2022-2024 to evaluate the antiviral activity of nanoparticles against Cowpea aphid-borne mosaic virus (CABMV) in cowpea. Cowpea leaf samples exhibiting symptoms such as vein-banding and mosaic patterns were collected from different locations in Vellanikkara (AEU10) and Palakkad (AEU23). The virus was maintained through mechanical inoculation for further studies. The virus is sap- transmissible and was mechanically inoculated using 0.01 M phosphate buffer. Successful transmission of virus by mechanical inoculation (98%) to the local lesion host, Chenopodium amaranticolor, and the propagative host, cowpea, was possible. The virus maintained in Chenopodium and cowpea was further isolated and characterized molecularly using RT-PCR. RT-PCR yielded an expected amplicon size of 812 bp with specific primers targeting the coat protein gene of Potyvirus. Sequence analysis through BLAST revealed 95.81% similarity with Cowpea aphid-borne mosaic virus (CABMV) and the sequence was deposited in NCBI with accession number PQ511232. Screening of nanoparticle (NP) formulations using silver nanopowder (SRL Catalog No: 44416) and silicon dioxide nanopowder (SRL Catalog No: 69294), was done to evaluate their antiviral activity against CABMV. Simultaneous inoculation of virus and nanoparticles (NPs), as well as pre-and post-inoculation of virus followed by NP foliar spray, was done to evaluate the efficacy of NPs at different concentrations. Direct inoculation/simultaneous inoculation of virus and NPs was done to evaluate the virucidal activity of NPs on the virus. In these studies, viral sap was mixed with NPs and incubated for 10 minutes before application to Chenopodium. A reduced number of diffused local lesions were observed on leaves inoculated with viral sap and NPs. A comparatively higher number of intact local lesions (34) were observed on plants treated with viral sap without NPs. Transmission electron microscopic (TEM) analysis of viruses from diffused and intact local lesions revealed aggregated viral particles in diffused local lesions. To further characterize viral particle aggregation, dynamic light scattering (DLS) analysis was performed on viral sap treated with NPs and untreated viral sap. The hydrodynamic radius of the particles in the NP- treated viral sap was found to be larger, with a mean size of 956.46 nm, compared to viral sap (512.56 nm) and nanosuspension alone (421 nm). The increase in size is likely due to the aggregation of viral particles on the NPs, facilitated by electrostatic attraction between the virus and the NPs. Pre- and post-inoculation studies using various concentrations of silver and silicon NPs on Chenopodium revealed that treatments T8 and T16, pre- and post- inoculation of the virus combined with a foliar spray of silicon NPs at 1000 ppm were themost effective, showing no local lesions compared to 34 local lesions observed in the viral control. This was followed by T14 (silicon NPs at 200 ppm), which also demonstrated significant efficacy. Pre- and post-inoculation studies on cowpea confirmed that foliar application of silicon NPs at 1000 ppm after challenge inoculation with the virus significantly reduced disease severity, with a zero-vulnerability index (VI), compared to 94.44 in the viral control. The reduction in disease severity was further validated by measuring the viral titre using DAS-ELISA with CABMV-specific antiserum (DSMZ PV RT–0417, DAS-ELISA set). Treatments T14 (Si 200 ppm) and T16 (Si 1000 ppm) showed 80% and 86% reduction in viral titre with less absorbance of 0.023 and 0.031 respectively. Based on the VI and viral titre, silicon 1000ppm was used for further studies. Various application methods for silicon NPs were standardized, and their efficacy was assessed in combination with the promising endophyte Bacillus pumilus VLY17. The applications include seed treatment, foliar application, and their combinations for silicon NPs at 1000 ppm. For Bacillus pumilus, seed treatment and soil drenching were done. A total of 19 treatments involving NP applications, both individually and in combination with Bacillus pumilus, were developed and tested. Based on the VI and reduced viral titre, treatment T9 (Bacillus seed treatment followed by Si NP foliar 1000ppm) was identified as the most effective. Evaluation of yield and plant growth parameters also highlighted T9 as the best performing treatment. T9 resulted in a 2.5-fold increase in pod yield per plant (138.7 g/plant) compared to the viral control (52.3 g/plant). Additionally, root length (56.5cm), fresh weight, and dry weight of both root (14.9g and 6.63g) and shoot (91.8g and 43.7g) were significantly higher than those in the viral control. Furthermore, T9 exhibited a substantially reduced viral titre (OD at 405nm -0.126) compared to the viral control (OD at 405nm-1.248). Analysis of defense related enzymes was conducted across all treatments to investigate the underlying mechanisms of reduced disease severity in treated plants. Treatment T9 showed a 21% increase in peroxidase activity (0.418 μmol/min/ml), a 46% increase in polyphenol oxidase activity (0.57 change in absorbance per minute/g), and a 2.36% increase in PAL activity (1.30 mg of cinnamic acid g-1 fresh weight) compared to viral control. Additionally, the activity of antioxidant enzymes, superoxide dismutase (SOD) and catalase, was analyzed. T9 exhibited enhanced SOD activity, with 358 units/ml at 20 days after treatment (DAT), and a 25% increase in catalase activity (121.42 enzyme units/g) compared to the viral control at 20 DAT. The research highlights the potential of silicon NPs in reducing disease severity caused by CABMV, promoting growth and yield parameters, and enhancing the activity of antioxidant and defense related enzymes. Seed treatment with Bacillus pumilus, combined with foliar application of silicon NPs at 1000 ppm, emerged as an effective management strategy for controlling CABMV in cowpea.