Browsing by Author "Radhika, N S"

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Characterization of different viruses infecting small Cardamom (Elettaria cardamomum Maton) and production of disease free plants
(Department of Plant Pathology, College of Agriculture, Vellayani, 2019) Vangala Bhavana; Radhika, N S
The study entitled “Characterization of viruses infecting small cardamom (Elettaria cardamomum Maton) and production of disease free plants” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2017-2019, with the objective to study the occurrence and distribution of viruses infecting small cardamom, molecular characterization of the viruses and elimination of viruses through meristem culture for the production of disease free planting material. Survey was conducted in Kattappana, Pampadumpara and Ambalapara panchayats of Kattappana block; and Nedumkandam, Thukkupalam and Chembalam panchayats of Nedumkandam blocks of Idukki district during November 2018 – May 2019. The incidence of katte disease caused by Cardamom mosaic virus (CdMV) was present in all the panchayats surveyed and it ranged from 3.75 to 43.0 per cent in Kattappana block and 5.0 to 31.33 per cent in Nedumkandam block. Disease incidence for chlorotic streak caused by Banana bract mosaic virus (BBrMV) was recorded from Kattappana (41%), Pampadumpara (30%) and Nedumkandam (8.33%) panchayats. The aphids infestation was absent in all the surveyed plots. Colocasia spp. and Alpinia spp. were the major plants observed in and around the cardamom fields and were not having visible symptoms of the viral infections. The virus inoculums were maintained under insect proof net house at Cardamom Research Station, Pampadumpara. Katte disease produced slender chlorotic flecks developing into pale green discontinuous stripes running parallel to veins from midrib to leaf margin of the infected leaves. Mosaic mottling and chlorotic specks were seen on the infected leaves and young pseudostems. In case of severe infection, plants produced stunted tillers. Chlorotic streak disease was characterised by continuous and discontinuous chlorotic streaks along veins and midribs of the infected leaves and green discontinuous spindle streaks on pseudostem. CdMV (a potyvirus) and BBrMV in cardamom was detected using polyclonal antibodies of Potato virus Y (PVY) and BBrMV respectively procured from DSMZ, Germany by direct antigen coating- Enzyme linked immunosorbent assay (DAC-ELISA) and Dot immunobinding assay (DIBA).The highest virus titre of CdMV and BBrMV was obtained in samples collected from Pampadumpara and Kattappana respectively. Molecular detection of the viruses was carried out using reverse transcriptase - polymerase chain reaction (RT-PCR) with specific primers for CdMV and BBrMV; and obtained amplicons of expected size of 879-905 bp for CdMV- and 625-633 bp for BBrMV- infected samples. The sequences of the isolates of CdMV from Kattappana, Pampadumpara and Nedumkandam were subjected to BLAST analysis and found to be similar to Indian cardamom mosaic virus isolates from Thalathamane and Appangala with > 96 per cent similarity. The BBrMV in cardamom from Kattappana, Pampadumpara and Nedumkandam was similar to Banana bract mosaic virus (BBrMV) CdM isolate of Karnataka (91.01%), Coimbatore (90.29%) and Thrissur (95.76%) respectively. Phylogeny tree constructed in MEGA 6.0 software differentiated CdMV and BBrMV into four clades, in which CdMV Kattappana and Nedumkandam isolates were clustered together whereas CdMV Pampadumpara isolate was in separate clade. Similarly, BBrMV isolates of Pampadumpara and Nedumkandam clustered together while BBrMV Kattappana was in separate clade. Meristem of 2 mm size separated from infected plants were grown in Murashige and Skoog medium supplemented with 3 mg benzyl amino purine (BAP), 1.5 mg indole acetic acid (IAA) and 0.8 mg kinetin expressed direct organogenesis but multiple shoots were not produced. The TC plants were subjected to DAC-ELISA with the specific polyclonal antibodies and PCR with specific primers of the viruses and confirmed that the plants produced from meristems were free of both the viruses. Thus, the present study revealed that two viral diseases viz., katte and chlorotic streak affecting small cardamom in Idukki. Serologically and molecularly it was detected that katte disease was caused by Cardamom mosaic virus (CdMV) and chlorotic streak disease was caused by Banana bract mosaic virus (BBrMV), and the viruses could be eliminated from the infected plants through meristem tip culture to produce the diseases free plants.
Disease resistance in the management of cowpea aphid-borne mosaic virus
(Department of Plant Pathology, College of Agriculture, Vellayani, 1999) Radhika, N S; Umamaheswaran, K
Investigations were undertaken on the virus causing severe mosaic on cowpea (Vigna unguiculata (L.) Walp) in Kerala. The characteristic symptoms appeared as vein clearing, light and dark green mottling, severe mosaic, dark green vein banding, blistering, distortion and reduction in leaf size. The virus was mechanically transmitted through sap extracted in 0.01 M phosphate buffer (pH 7.0). The virus was efficiently transmitted by the aphid vector, Aphis craccivora. Seed transmission of eleven per cent was recorded in the variety Sharika. Thermal inactivation point was recorded at a range of 60 - 65° C, dilution end point at a range of 10-3 - 10-4 and longevity in vitro for four hours at room temperature (28 ± 4° C) and six hours under refrigerated condition (8° C). A. craccivora could efficiently transmit the virus with an acquisition access of ten minutes and inoculation access of one minute. Pre-acquisition starvation increased the rate of transmission while post-acquisition starvation decreased the rate. A single aphid was capable of transmitting the virus. The virus causing severe mosaic was identified as blackeye cowpea mosaic virus by ELISA. The virus could also be detected by Ouchterlony immunodiffusion test. Electron microscopic studies revealed the presence of flexuous, filamentous particles of 750 nm in length. Two varieties Co-6 and Cc-Selection were grouped as no symptom producing among 65 genotypes screened for resistance. Fifty three F2 progenies of the cross Sharika and Co-6 and twenty five F2 progenies of the cross Co-Selection and Sharika were long poded and resistant. Biochemical changes indicated a lower carbohydrate content in resistant compared to susceptible. Chlorophyll content decreased in the susceptible variety due to virus infection. Increase in protein was observed in both resistant and susceptible. The phenol content did not show variation between the varieties. Peroxidase, polyphenol oxidase and phenylalanine ammonia-lyase activities increased in the resistant variety. Bioassay of chemicals and neem oil on local lesion host (c. amaranticolor) indicated a per cent inhibition of 68.92 by neem oil in pre-inoculation application and 65.45 per cent inhibition by manganese chloride in post-inoculation application. On cowpea plants, pre-inoculation application of neem oil (ten per cent) concentration was found to be effective in reducing the symptoms due to viral infection.
Etiology of bud proliferation in vegetable cowpea
(Department of Plant Pathology, College of Agriculture, Vellayani, 2022-02-11) Devika, S; Radhika, N S
The study entitled „Etiology of bud proliferation in vegetable cowpea‟ was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2019-2021 with an objective of studying the symptomatology, immuno-molecular detection and characterization of incitant/(s) of bud proliferation in vegetable cowpea. Bud proliferation disease of cowpea has been observed in different varieties in different locations. Purposive sampling was carried out in different locations of Thiruvananthapuram, Kollam, Alappuzha and Thrissur and collected symptoms were used for further studies. The characteristic symptom of the disease is the abnormal proliferation of bud which showed an increase in number of buds up to 16-35. The proliferated buds also showed pinkish- brown patches in it. Noticeable symptoms were also seen in the leaves of the diseased plant. They were smaller, crinkled and unusually dark in colour. The fasciation of stem has also been observed in the fields of Onattukara and Chavara. The infected plants were stunted and completely sterile. The highest disease incidence was found to be in the variety Bhagyalakshmy cultivated in Mannuthy (6.57 per cent). The population of hoppers was observed in the field and the insects identified were Exitanus sp., Balclutha sp., Nilaparvata lugens, Ptoleria sp., Nisia nervosa. The weeds Phyllanthus, Neer-grampu and Jack bean were found to be showing similar symptoms near the diseased cowpea fields. The graft transmission was successful from cowpea to periwinkle with 40 per cent efficiency. The leaves of the graft inoculated periwinkle plants showed severe interveinal chlorosis and later on yellowing. Graft transmission was unsuccessful from cowpea to cowpea. The transmission studies for viruses from cowpea to cowpea and cowpea to Chenopodium revealed the absence of viruses. The DAPI staining of diseased and healthy plants affirmed the presence of phytoplasma in the diseased samples. Small fluorescent-coloured bodies were seen in the stem and leaf of infected plants compared to healthy. The hormonal analysis of the symptomatic plants compared to the healthy ones showed significant difference. The GA content in diseased leaf and bud was increased by 20.88 and 17.46 per cent respectively. The IAA content in diseased leaf (older) and bud was increased by 61.55 and 46.52 per cent respectively. The serological detection for viruses using monoclonal antibodies of CABMV and BICMV and polyclonal antibodies of TSWV and WSMoV divulged the absence of viruses in the diseased samples. The graft inoculated periwinkle plants also showed no presence of viruses. The molecular detection of phytoplasma with nested PCR was carried out. The first primers P1/P7 amplified a 1.8kb fragment and the second set of primers; R16F2n/R16R2 amplified a 1.2kb fragment, giving positive results. The final amplified product was sequenced and by BLAST analysis it was found that the 16S rDNA sequence shared 99.80 per cent similarity with that of the „Candidatus Phytoplasma asteris‟ reference strain (GenBank accession: M30790). Hence the phytoplasma under study is a „Candidatus Phytoplasma asteris‟-related strain. The virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession: AP006628). The phytoplasma under study was found to be a member of 16SrI-B.
Integrated management of viral diseases of bittergourd (momordica charantia L.)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2018) Radhika, N S; Umamaheswaran, K
Management of virus disease complex in chilli using the beneficial fungal root Endophyte Piriformospora indica
(Department of Plant Pathology, College of Agriculture, Padannakkad, 2023-09-15) Meera Nair, V; Radhika, N S
The research work, “Management of virus disease complex in chilli using the beneficial fungal root endophyte Piriformospora indica” was undertaken in the Department of Plant Pathology, College of Agriculture, Padannakkad during 2021- 2023 with the objective to explore the use of the beneficial fungal root endophyte Piriformospora indica for the management of chilli leaf curl virus complex infecting chilli.Infected plant samples were collected from the chilli plots in the Instructional Farm, College of Agriculture, Padannakkad. The chilli variety Anugraha recorded higher disease incidence (D.I.-66 per cent) and vulnerability index (V.I.-15.66) than the local variety (D.I.-64 per cent and V. I. - 14.66 per cent). The major symptoms observed in the fields were leaf curling, puckering and swelling of veins. Molecular detection for the presence of associated viruses was done with begomovirus coat protein specific primers viz., Deng and AV/AC yielded amplicons of 520 bp and 575 bp respectively confirming its association with the disease. RT- PCR with Cucumber mosaic virus (CMV) coat protein specific primers could not detect the presence of CMV. Chilli seeds of variety Vellayani Athulya were sown on P. indica mass-multiplied coir pith-cow dung mixture (1:1) amended with 2 per cent gram flour. Chlamyodspores of P. indica were observed in the root cortical region five days after co-cultivation (DAC). Seeds sown on P. indica mass multiplied medium germinated early (seven days) and completed 50 per cent germination within ten days compared with untreated seeds (ten days for germination and 17 days for 50 per cent germination). Pot culture experiments were conducted with seven treatments and eight replication in a completely randomized design (CRD). The virus was transmitted through wedge grafting at intervals of 2, 5, 10, and 15 days. Pre-colonization of P. indica followed by graft transmission of the virus after 15 days took 28 days for symptom expression while non-colonized, grafted plants took 13 days. Pre-colonized plants expressed low V.I. (25) against noncolonized, grafted plants (64) at 45 DAT. Colonization of P. indica (2 days) after graft transmission of the virus recorded a V.I. (36) at 45 DAT and took 15 days for symptom expression while non-colonized grafted plants recorded a V.I. (65) and took 12 days for symptom expression. PCR amplification using virus specific primers confirmed the transmission of the virus in all grafted treatments. Field study laid out in the Instructional Farm I, College of Agriculture, Padannakkkad with P. indica colonized and non-colonized chilli seedlings of variety Vellayani Athulya recorded per cent D.I. (74) and V.I. (14.66) while the non-primed plants recorded a D.I. (86) and V.I. (32.27) at 90 days after transplanting. Amplicons in agarose gel electrophoresis confirmed that the virus titre in P. indica colonized chilli plants was significantly lower compared to those in control. P. indica colonization recorded improved growth characteristics such as the number of leaves (72.53), leaf area (30.59 cm2) and number of branches (10.78) compared to the non-colonized plants. The days taken for flowering (18.22) was early and the number of fruits per plant increased (68.23) significantly in P. indica colonized plants. P.indica colonized plants recorded a yield of 683 g while non-colonized plants yielded 480.29 g per plant. Mites and mole crickets along with diseases like powdery mildew and fruit rot were also observed. Biochemical basis for P.indica conferred tolerance was estimated based on reactive oxygen species (ROS) and hydrogen peroxide production and the activity of defense related enzymes. The presence of ROS was assessed using nitro blue tetrazolium (NBT) and H2O2 by diaminobenzidine (DAB) staining techniques. Intense colour development was obtained in non-colonized plants inoculated with the virus. Plants pre and post-colonized with P. indica exhibited reduced stain intensity compared to plants containing only the virus with the pre-colonized plants showing better stain reduction than the post-colonized ones. The ROS scavenging enzymes include catalase, peroxidase, superoxide dismutase and phosphatase activity was higher in pre-colonized plants than in post-colonized plants. Plants infected only by the virus expressed a significant reduction in the activities of catalase, peroxidase, superoxide dismutase, phosphatase, and total soluble protein compared to pre-colonized plants at all time intervals. However, at the final harvest, this treatment exhibited an increase in total protein although still lower than pre-colonized plants. The present study revealed that P. indica is effective in managing chilli leaf curl disease both in controlled and open field condition by increasing the production of ROS scavenging enzymes. P. indica was found to improve the germination, growth and development of chilli plants of variety Vellayani Athulya. Gene expression studies in future can unravel the tripartite interaction between plant, virus and the endophyte in rendering tolerance to plants against virus and enhancing plant growth.
Molecular characterization of blackeye cowpea mosaic virus causing mosaic disease in cowpea (Vigna unguiculata (L.) Wal) in Kerala
(Department of Plant Biotechnology, College of Agriculture ,Vellayani, 2022) Tania Mathew; Pampackal; Radhika, N S
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.
Nutrient based management of chilli leaf curl virus in Chilli (Capsicum annuum L.)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2019) Shilpa Sankar; Radhika, N S
The study entitled “Nutrient based management of Chilli leaf curl virus in Chilli (Capsicum annuum L.) was conducted at Department of Plant Pathology, College of Agriculture, Vellayani from 2017 to 2019. The main objectives were to serologically and molecularly characterize the virus causing leaf curl in chilli and to study the role of nutrient application in the management of the disease. The present investigation was carried out in four experiments viz., collection of Begomovirus infecting chilli from different cultivated areas, symptomatology, serological diagnosis and molecular characterization of the virus causing chilli leaf curl and nutrient based management of chilli leaf curl. The survey was undertaken from December 2018 to March 2019 to investigate the disease incidence in major chilli growing areas of Palakkad (Vadakarapathy and Kozhinjanpara) and Thiruvananthapuram districts (College of Agriculture, Vellayani) of Kerala. In Thiruvananthapuram, the disease incidence ranged from 69.33 to 80 per cent whereas Palakkad recorded maximum incidence of 73.33 per cent in Vadakarapathy village and Kozhinjanpara village recorded the disease incidence of 55.71 to 71.70 per cent. The common symptoms of the disease were upward curling and puckering of leaves, and stunting of whole plant. Other symptoms viz., reduced leaf size, yellowing, petiole elongation, crinkling and mottling of leaves with few or no fruits were also observed. Serological diagnosis of the disease was carried out using triple antibody sandwich – enzyme linked immunosorbent assay (TAS-ELISA) using polyclonal antisera specific to Begomovirus, Sri Lankan cassava mosaic virus (SLCMV). All the samples collected from College of Agriculture, Vellayani were detected with the virus whereas none of the samples from Palakkad district were positive to the virus. Molecular characterization of the virus using universal primers specific to coat protein of Begomovirus viz., AV-AC and DENG through Polymerase chain reaction (PCR) revealed that the DNA isolated from samples of Vellayani could yield an amplicon size of ̴ 550 bp (AV-AC) and ̴ 500 bp (DENG); thus confirming the presence of the virus. But no PCR amplification was observed in any of the samples from Palakkad district. Blast analysis with the sequence of coat protein of Vellayani revealed that the virus had 96.63 per cent similarity with Chilli leaf curl Vellanad virus. Nutrient status of healthy and diseased leaves revealed that the per cent of major nutrients viz., nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg); and micronutrients viz., manganese (Mn), zinc (Zn), copper (Cu) and boron (B) in the infected leaves was low compared to the healthy leaves but in sufficient level. Whereas, the per cent of sulphur (S) and iron (Fe) in infected leaves was high and in toxic level. Comparatively higher levels of all the major and micro nutrients were recorded in the soils collected from the infected fields than the soils from the healthy field. A pot culture study was conducted at Department of Plant Pathology in completely randomized design consisting of ten treatments and three replications with chilli variety Vellayani Athulya from May 2019 to August 2019. Cent per cent disease incidence was recorded in all chilli plants before imposing the treatments. Soil samples were taken and analysed for major and micro nutrients at pre-treatment stage. Urea, rajphos, murate of potash, lime and magnesium sulphate (MgSO4) were used as the source of major nutrients like N, P, K, Ca and Mg, respectively. Whereas, manganese sulphate (MnSO4), zinc sulphate (ZnSO4), copper sulphate (CuSO4), borax and potassium silicate were used as source for micronutrients such as Mn, Zn, Cu, B and Si, respectively. After imposing the treatments, the highest coefficient of infection (75.0) was recorded in untreated infected plants whereas, the plants supplemented with nutrients as per package of practices (POP) + B @ 10 kg ha-1, POP + (ZnSO4) @ 20 kg ha-1 and, basal application of 1/2 N + full P + 1/2 K followed by 0.5 per cent foliar application of NPK 19:19:19 at fortnightly recorded the lowest coefficient of infection (25.0). TAS-ELISA conducted with all the experimental plants confirmed the presence of virus. Lower virus titre value was observed in the plants supplemented with nutrients as per POP recorded maximum level of major nutrients. Among the treatments highest fruit yield of 48.85 g plant1 was obtained from plants supplemented with nutrients as per POP + B @ 10 kg ha-1 was found to be most effective in reducing the coefficient of infection with better yield. Thus, the present study revealed that the serological diagnosis of the disease carried out using TAS-ELISA with antisera SLCMV and molecular characterization of the virus using primers AV-AC and DENG through PCR confirmed the presence of virus in Vellayani. The BLAST analysis of Chilli leaf curl Vellayani isolate thus showed 96.63 per cent similarity with Chilli leaf curl Vellanad virus. It was also indicated that Chilli leaf curl virus in chilli could be managed by the application of nutrients as per package of practices recommendation along with boron as borax @ 10 kg ha-1 which need to be validated under field conditions.
Prevalence of sesamum phyllody in Onattukara tract and evaluation of fungal root endophyte Piriformospora indica for its management
(Department of Plant Pathology, College of Agriculture, Vellayani, 2022) Gifty, K J; Radhika, N S
The research work entitled ‘Prevalence of sesamum phyllody in Onattukara tract and evaluation of fungal root endophyte Piriformospora indica for its management’ was conducted during 2019-21 at Department of Plant Pathology, College of Agriculture, Vellayani and Onattukara Regional Agricultural Research Station with the objectives to study the symptomatology, molecular detection and characterization of phytoplasma inciting sesamum phyllody disease in AEU 3 (Onattukara tract); and evaluation of fungal root endophyte P. indica for its management. Phytoplasma infected sesamum samples were collected from D and F blocks of Onattukara Regional Agricultural Research Station and Karthikapally. Karthikapally recorded highest disease incidence (39.44 per cent) and vulnerability index (23.75). Chocolate weed, Melochia corchorifolia, was found to be exhibiting symptoms of shoot proliferation. Hoppers collected from the infected fields were identified as Orosius albicintus, Hishimonas phycitis and Nephotettix sp. Disease symptoms were observed at the stage of flowering of sesamum plants in all the sampled locations. The associated symptoms were reduction in internodal length of stem, axillary bud proliferation, thickening of the floral veins, phyllody and floral proliferation. Microtome sections of infected and healthy leaf, stem of sesamum stained with 4,6-diamidino-2-phenylindole (DAPI) stain, and observed under fluorescence microscope emitted diffuse fluorescence from the infected tissues, which was brighter than the one from the parenchymal cells indicating the presence of phytoplasma in the infected tissues. Studies on variations in the level of gibberellic acid (GA) and indole-3-acetic acid (IAA) in phyllody infected and healthy sesamum was undertaken. GA content was increased by 2.25 times and 10.46 times, and IAA content was decreased by 1.25 times and 1.97 times in leaves and flowers of infected samples compared to the healthy samples. Molecular characterization of sesamum phyllody was performed with leaf samples collected from ORARS lowland, ORARS upland, Vellayani and Karthikapally. Amplicons of 1.4kb was obtained by amplifying with universal primers P1/P6 for detection of phytoplasma. The sequences obtained were subjected to BLAST analysis and the 16S rDNA gene sequence showed that all the isolates shared more than 99 per cent similarity with that of the ‘Candidatus phytoplasma aurantifolia’ strains in GenBank data base. In the phylogenetic tree constructed, the sesamum phyllody phytoplasma under study clustered with the 16SrII group (Candidatus Phytoplasma aurantifolia) phytoplasmas causing sesamum phyllody in various regions. The virtual RFLP pattern generated by iPhyClassifier, derived from 16S rDNA fragment was found to be identical to the reference pattern of 16Sr group II, subgroup D (GenBank accession: Y10097). Based on the results obtained from sequence analysis and virtual RFLP pattern, the phytoplasma associated with sesamum phyllody was identified as ‘'Candidatus Phytoplasma aurantifolia”-related strain belonging to subgroup 16SrII-D. P. indica obtained from Department of Plant Pathology, College of Agriculture, Vellayani was mass multiplied in sterilized coir pith: FYM mixture (1:1) amended with 2 per cent gram flour and sesamum seeds were sown. Colonization was observed seven days after germination. Wedge grafting was standardized in sesamum at 30 days after germination. Pot culture experiment was conducted to evaluate the effect of P. indica against phytoplasma causing sesamum phyllody, by grafting the colonized and non-colonized plants with infected scion. P. indica colonization could significantly reduce the incidence and severity of infection. After 30 and 45 days of grafting, an incidence of 20 and 60 per cent, and severity of 5 and 50 were recorded in the colonized plants grafted with infected scion, whereas an incidence of 60 and 80 per cent and severity of 45 and 75 were recorded in non-colonized plants grafted with infected scion. In colonized plants, enhanced shoot and root length at 30 and 55 days after germination were recorded and also earliness in flowering compared to noncolonized plants. Hence the associated symptoms of phytoplasma infection in sesamum are virescence, thickening of floral veins, phyllody and floral proliferation. The study revealed the association of Candidatus phytoplasma aurantifolia group with sesamum phyllody prevalent in Onattukara tract. The evaluation of beneficial fungal root endophyte P. indica against phytoplasma revealed delayed expression of symptoms in the colonized plants.
Prevalence of sesamum phyllody in Onattukara tract and evaluation of fungal root endophyte Piriformospora indica for its management
(Department of Plant Pathology, College of Agriculture, Vellayani, 2022) Gifty, K J; KAU; Radhika, N S
The research work entitled ‘Prevalence of sesamum phyllody in Onattukara tract and evaluation of fungal root endophyte Piriformospora indica for its management’ was conducted during 2019-21 at Department of Plant Pathology, College of Agriculture, Vellayani and Onattukara Regional Agricultural Research Station with the objectives to study the symptomatology, molecular detection and characterization of phytoplasma inciting sesamum phyllody disease in AEU 3 (Onattukara tract); and evaluation of fungal root endophyte P. indica for its management. Phytoplasma infected sesamum samples were collected from D and F blocks of Onattukara Regional Agricultural Research Station and Karthikapally. Karthikapally recorded highest disease incidence (39.44 per cent) and vulnerability index (23.75). Chocolate weed, Melochia corchorifolia, was found to be exhibiting symptoms of shoot proliferation. Hoppers collected from the infected fields were identified as Orosius albicintus, Hishimonas phycitis and Nephotettix sp. Disease symptoms were observed at the stage of flowering of sesamum plants in all the sampled locations. The associated symptoms were reduction in internodal length of stem, axillary bud proliferation, thickening of the floral veins, phyllody and floral proliferation. Microtome sections of infected and healthy leaf, stem of sesamum stained with 4,6-diamidino-2-phenylindole (DAPI) stain, and observed under fluorescence microscope emitted diffuse fluorescence from the infected tissues, which was brighter than the one from the parenchymal cells indicating the presence of phytoplasma in the infected tissues. Studies on variations in the level of gibberellic acid (GA) and indole-3-acetic acid (IAA) in phyllody infected and healthy sesamum was undertaken. GA content was increased by 2.25 times and 10.46 times, and IAA content was decreased by 1.25 times and 1.97 times in leaves and flowers of infected samples compared to the healthy samples. Molecular characterization of sesamum phyllody was performed with leaf samples collected from ORARS lowland, ORARS upland, Vellayani and Karthikapally. Amplicons of 1.4kb was obtained by amplifying with universal primers P1/P6 for detection of phytoplasma. The sequences obtained were subjected to BLAST analysis and the 16S rDNA gene sequence showed that all the isolates shared more than 99 per cent similarity with that of the ‘Candidatus phytoplasma aurantifolia’ strains in GenBank data base. In the phylogenetic tree constructed, the sesamum phyllody phytoplasma under study clustered with the 16SrII group (Candidatus Phytoplasma aurantifolia) phytoplasmas causing sesamum phyllody in various regions. The virtual RFLP pattern generated by iPhyClassifier, derived from 16S rDNA fragment was found to be identical to the reference pattern of 16Sr group II, subgroup D (GenBank accession: Y10097). Based on the results obtained from sequence analysis and virtual RFLP pattern, the phytoplasma associated with sesamum phyllody was identified as ‘'Candidatus Phytoplasma aurantifolia”-related strain belonging to subgroup 16SrII-D. P. indica obtained from Department of Plant Pathology, College of Agriculture, Vellayani was mass multiplied in sterilized coir pith: FYM mixture (1:1) amended with 2 per cent gram flour and sesamum seeds were sown. Colonization was observed seven days after germination. Wedge grafting was standardized in sesamum at 30 days after germination. Pot culture experiment was conducted to evaluate the effect of P. indica against phytoplasma causing sesamum phyllody, by grafting the colonized and non-colonized plants with infected scion. P. indica colonization could significantly reduce the incidence and severity of infection. After 30 and 45 days of grafting, an incidence of 20 and 60 per cent, and severity of 5 and 50 were recorded in the colonized plants grafted with infected scion, whereas an incidence of 60 and 80 per cent and severity of 45 and 75 were recorded in non-colonized plants grafted with infected scion. In colonized plants, enhanced shoot and root length at 30 and 55 days after germination were recorded and also earliness in flowering compared to noncolonized plants. Hence the associated symptoms of phytoplasma infection in sesamum are virescence, thickening of floral veins, phyllody and floral proliferation. The study revealed the association of Candidatus phytoplasma aurantifolia group with sesamum phyllody prevalent in Onattukara tract. The evaluation of beneficial fungal root endophyte P. indica against phytoplasma revealed delayed expression of symptoms in the colonized plants.
Prevalence of sesamum phyllody in Onattukara tract and evaluation of fungal root endophyte Piriformospora indica for its management
(Department of Plant Pathology, College of Agriculture, Vellayani, 2022) Gifty, K J; Radhika, N S
The research work entitled ‘Prevalence of sesamum phyllody in Onattukara tract and evaluation of fungal root endophyte Piriformospora indica for its management’ was conducted during 2019-21 at Department of Plant Pathology, College of Agriculture, Vellayani and Onattukara Regional Agricultural Research Station with the objectives to study the symptomatology, molecular detection and characterization of phytoplasma inciting sesamum phyllody disease in AEU 3 (Onattukara tract); and evaluation of fungal root endophyte P. indica for its management. Phytoplasma infected sesamum samples were collected from D and F blocks of Onattukara Regional Agricultural Research Station and Karthikapally. Karthikapally recorded highest disease incidence (39.44 per cent) and vulnerability index (23.75). Chocolate weed, Melochia corchorifolia, was found to be exhibiting symptoms of shoot proliferation. Hoppers collected from the infected fields were identified as Orosius albicintus, Hishimonas phycitis and Nephotettix sp. Disease symptoms were observed at the stage of flowering of sesamum plants in all the sampled locations. The associated symptoms were reduction in internodal length of stem, axillary bud proliferation, thickening of the floral veins, phyllody and floral proliferation. Microtome sections of infected and healthy leaf, stem of sesamum stained with 4,6-diamidino-2-phenylindole (DAPI) stain, and observed under fluorescence microscope emitted diffuse fluorescence from the infected tissues, which was brighter than the one from the parenchymal cells indicating the presence of phytoplasma in the infected tissues. Studies on variations in the level of gibberellic acid (GA) and indole-3-acetic acid (IAA) in phyllody infected and healthy sesamum was undertaken. GA content was increased by 2.25 times and 10.46 times, and IAA content was decreased by 1.25 times and 1.97 times in leaves and flowers of infected samples compared to the healthy samples. Molecular characterization of sesamum phyllody was performed with leaf samples collected from ORARS lowland, ORARS upland, Vellayani and Karthikapally. Amplicons of 1.4kb was obtained by amplifying with universal primers P1/P6 for detection of phytoplasma. The sequences obtained were subjected to BLAST analysis and the 16S rDNA gene sequence showed that all the isolates shared more than 99 per cent similarity with that of the ‘Candidatus phytoplasma aurantifolia’ strains in GenBank data base. In the phylogenetic tree constructed, the sesamum phyllody phytoplasma under study clustered with the 16SrII group (Candidatus Phytoplasma aurantifolia) phytoplasmas causing sesamum phyllody in various regions. The virtual RFLP pattern generated by iPhyClassifier, derived from 16S rDNA fragment was found to be identical to the reference pattern of 16Sr group II, subgroup D (GenBank accession: Y10097). Based on the results obtained from sequence analysis and virtual RFLP pattern, the phytoplasma associated with sesamum phyllody was identified as ‘'Candidatus Phytoplasma aurantifolia”-related strain belonging to subgroup 16SrII-D. P. indica obtained from Department of Plant Pathology, College of Agriculture, Vellayani was mass multiplied in sterilized coir pith: FYM mixture (1:1) amended with 2 per cent gram flour and sesamum seeds were sown. Colonization was observed seven days after germination. Wedge grafting was standardized in sesamum at 30 days after germination. Pot culture experiment was conducted to evaluate the effect of P. indica against phytoplasma causing sesamum phyllody, by grafting the colonized and non-colonized plants with infected scion. P. indica colonization could significantly reduce the incidence and severity of infection. After 30 and 45 days of grafting, an incidence of 20 and 60 per cent, and severity of 5 and 50 were recorded in the colonized plants grafted with infected scion, whereas an incidence of 60 and 80 per cent and severity of 45 and 75 were recorded in non-colonized plants grafted with infected scion. In colonized plants, enhanced shoot and root length at 30 and 55 days after germination were recorded and also earliness in flowering compared to noncolonized plants. Hence the associated symptoms of phytoplasma infection in sesamum are virescence, thickening of floral veins, phyllody and floral proliferation. The study revealed the association of Candidatus phytoplasma aurantifolia group with sesamum phyllody prevalent in Onattukara tract. The evaluation of beneficial fungal root endophyte P. indica against phytoplasma revealed delayed expression of symptoms in the colonized plants.
Prevalence of virus diseases of ridge gourd (Luffa acutangula (L) Roxb) and characterization of a major associated virus
(Prevalence of virus diseases of ridge gourd (Luffa acutangula (L) Roxb) and characterization of a major associated virus, 2024-03-01) Sana, T; Radhika, N S
The research work entitled “Prevalence of virus diseases of ridge gourd (Luffa acutangula (L.) Roxb.) and characterization of a major associated virus” was carried out in the Department of Plant Pathology, College of Agriculture, Padannakkad during 2021-23 with the objectives of assessment of virus diseases of ridge gourd in North Kerala; transmission studies, identification of sources of resistance, molecular detection and characterization of coat protein of major virus associated with the disease. Survey for assessment of virus disease incidence (DI) and vulnerability index (VI) of ridge gourd was conducted in major vegetable growing areas of Kozhikode, Kannur, and Kasaragod (AEU 2 and 11) districts during 2021-2023. It was found that the DI ranged from5.3-100 percent and VI from26.57-84.08. Kunjathur, Thalangady, and Hosabettu areas of Kasaragod district recorded the highest incidence (100%). The highest VI(84.08) was recorded in Thalangady (Kasaragod). Kuttyeri recorded high disease incidence (90%) and VI (61.2)in Kannur district. In Kozhikode district,Cherukulathur recorded high disease incidence (68%) and Poolacode recorded high VI (35.71). Plants in cultivated fields at Avala, Akkuparamb, and Velom were asymptomatic. Whiteflies and aphids associated with infected ridge gourd were identified as Bemisia tabaci and Aphis gossypii. Weeds like Synedrella sp., Ageratum conizoides and Alternanthera sp. were observed with chlorosis and vein banding symptoms in the surveyed areas. Light chlorotic specks with yellowing, pale green intermingled with normal green tissues, vein banding, yellow vein mosaic, cupping, blistering, vein clearing, thickening, reduction in leaf size, puckering, and green island symptoms were observed. The infected scions collected from surveyed locations were grafted on healthy ridge gourd plants and maintained under greenhouse conditions for further studies. Mechanically inoculated ridge gourd plants (2-3 leaf stage) took four days to produce vein banding symptoms. Chlorotic lesions were observed on Chenopodium amaranticolor indicating the presence of Cucumber mosaic virus. When infected ridge gourd scions were wedge grafted on bitter gourd (Momordica charantia L.), pumpkin (Cucurbita moschata), ash gourd (Benincasa hispida), oriental pickling melon (Cucumis melo var. conomon) and ridge gourd (L. acutangula), expressed symptoms of infection 4-15 days after grafting (DAG). Grafted oriental pickling melon plants expressed low DI (33%). Seedlings raised from seeds collected from infected plants expressed pale banding symptoms (DI-15.38 %). Screening for disease resistance was undertaken with ten varieties/hybrids by wedge grafting with infected scion. After 20 days of grafting,the hybrid MHRG 7 recorded a high VI (65.33) and Arka Vikram recorded a low VI (39.33).After 40 days of grafting, hybrid Sorot F1 recorded high VI(78) and Arka Sujat recorded low VI(48). After 60 days of grafting, KRH-1 recorded the highest VI (91.33) while Arka Vikram recorded the lowest VI (66.67). RG7 and RG10 recorded moderately susceptible reactions to the virus. PCR based molecular detection of Tomato leaf curl New Delhi virus (ToLCNDV) using coat protein (CP) specific primers yielded amplicons of approximately 575 bp. The amplified PCR products were subjected to sequencing and blast analysis. All the isolated sequences were found to be related to ToLCNDV. Phylogenetic studies revealed that isolates from Thrikaripur, Mavichery, and Poolacode isolates were closely related to each other. DNA isolated from infected weeds like Synedrellasp., Alternanthera sp., graft transmitted different cucurbits such as bitter gourd, ash gourd, pumpkin, oriental pickling melon and ridge gourd detected the presence of begomovirus (575 bp amplicon). Cucumber mosaic virus was also detected from isolated samples using specific primers of CMV (CMV1 and CMV2) with an amplicon size of 400 bp. Thus, the study revealed that the ridge gourd cultivated in areas of Kasaragod, Kannur, and Kozhikode were infected with ToLCNDV and CMV; transmitted to other cucurbitaceous hosts andRG7 and RG10 were moderately susceptible among the varieties and hybrids screened. The presence of the begomovirus was detected in weeds indicating its role in perpetuation and spread of the virus disease in the field. Future emphasis should be the standardization of an early detection method for the multiple viruses involved and the developmentof an integrated management strategy for virus disease associated with ridge gour Survey for assessment of virus disease incidence (DI) and vulnerability index (VI) of ridge gourd was conducted in major vegetable growing areas of Kozhikode, Kannur, and Kasaragod (AEU 2 and 11) districts during 2021-2023. It was found that the DI ranged from5.3-100 percent and VI from26.57-84.08. Kunjathur, Thalangady, and Hosabettu areas of Kasaragod district recorded the highest incidence (100%). The highest VI(84.08) was recorded in Thalangady (Kasaragod). Kuttyeri recorded high disease incidence (90%) and VI (61.2)in Kannur district. In Kozhikode district,Cherukulathur recorded high disease incidence (68%) and Poolacode recorded high VI (35.71). Plants in cultivated fields at Avala, Akkuparamb, and Velom were asymptomatic. Whiteflies and aphids associated with infected ridge gourd were identified as Bemisia tabaci and Aphis gossypii. Weeds like Synedrella sp., Ageratum conizoides and Alternanthera sp. were observed with chlorosis and vein banding symptoms in the surveyed areas. Light chlorotic specks with yellowing, pale green intermingled with normal green tissues, vein banding, yellow vein mosaic, cupping, blistering, vein clearing, thickening, reduction in leaf size, puckering, and green island symptoms were observed. The infected scions collected from surveyed locations were grafted on healthy ridge gourd plants and maintained under greenhouse conditions for further studies. Mechanically inoculated ridge gourd plants (2-3 leaf stage) took four days to produce vein banding symptoms. Chlorotic lesions were observed on Chenopodium amaranticolor indicating the presence of Cucumber mosaic virus. When infected ridge gourd scions were wedge grafted on bitter gourd (Momordica charantia L.), pumpkin (Cucurbita moschata), ash gourd (Benincasa hispida), oriental pickling melon (Cucumis melo var. conomon) and ridge gourd (L. acutangula), expressed symptoms of infection 4-15 days after grafting (DAG). Grafted oriental pickling melon plants expressed low DI (33%). Seedlings raised from seeds collected from infected plants expressed pale banding symptoms (DI-15.38 %). Screening for disease resistance was undertaken with ten varieties/hybrids by wedge grafting with infected scion. After 20 days of grafting,the hybrid MHRG 7 recorded a high VI (65.33) and Arka Vikram recorded a low VI (39.33).After 40 days of grafting, hybrid Sorot F1 recorded high VI(78) and Arka Sujat recorded low VI(48). After 60 days of grafting, KRH-1 recorded the highest VI (91.33) while Arka Vikram recorded the lowest VI (66.67). RG7 and RG10 recorded moderately susceptible reactions to the virus. PCR based molecular detection of Tomato leaf curl New Delhi virus (ToLCNDV) using coat protein (CP) specific primers yielded amplicons of approximately 575 bp. The amplified PCR products were subjected to sequencing and blast analysis. All the isolated sequences were found to be related to ToLCNDV. Phylogenetic studies revealed that isolates from Thrikaripur, Mavichery, and Poolacode isolates were closely related to each other. DNA isolated from infected weeds like Synedrellasp., Alternanthera sp., graft transmitted different cucurbits such as bitter gourd, ash gourd, pumpkin, oriental pickling melon and ridge gourd detected the presence of begomovirus (575 bp amplicon). Cucumber mosaic virus was also detected from isolated samples using specific primers of CMV (CMV1 and CMV2) with an amplicon size of 400 bp. Thus, the study revealed that the ridge gourd cultivated in areas of Kasaragod, Kannur, and Kozhikode were infected with ToLCNDV and CMV; transmitted to other cucurbitaceous hosts andRG7 and RG10 were moderately susceptible among the varieties and hybrids screened. The presence of the begomovirus was detected in weeds indicating its role in perpetuation and spread of the virus disease in the field. Future emphasis should be the standardization of an early detection method for the multiple viruses involved and the developmentof an integrated management strategy for virus disease associated with ridge gour
Varietal screening of black pepper to cucumber mosaic virus and Piper yellow mottle virus and their sero molecular detection
(Department of Plant Pathology, College of Agriculture, Vellayani, 2020) Arya, M; Radhika, N S