Browsing by Author "Radhika, N S"
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ThesisItem Open Access 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,; KAU; Radhika, N SThe 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.ThesisItem Open Access Disease resistance in the management of cowpea aphid-borne mosaic virus(Department of Plant Pathology, College of Agriculture, Vellayani, 1999) Radhika, N S; KAU; Umamaheswaran, KInvestigations 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.ThesisItem Open Access Etiology of bud proliferation in vegetable cowpea(Department of Plant Pathology, College of Agriculture, Vellayani, 2022) Devika, S; KAU; Radhika, N SThe 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.ThesisItem Open Access Integrated management of viral diseases of bittergourd (momordica charantia L.)(Department of Plant Pathology, College of Agriculture, Vellayani, 2018) Radhika, N S; KAU; Umamaheswaran, KThe present research work entitled ‘Integrated management of viral diseases of bitter gourd (Momordica charantia L.) was carried out in the College of Agriculture, Vellayani during 2014-2017, with the objectives to study the occurrence and distribution of viruses in bitter gourd in Thiruvananthapuram, Idukki and Palakkad, immunomolecular characterization of the viruses, and screening of antiviral chemicals, antiviral principles of animal, plant and microbial origin for the management of the disease. In the suvey conducted at five locations in Thiruvanaanthapuram district, Pappanchani area recorded highest incidence of viral disease (60%) while highest Vulnerability Index (V.I) was recorded from Vellayani (56.00). In Idukki district, six major bitter gourd cultivating areas were surveyed among which Rajakumary area recorded the highest disease incidence (100%) and V.I (82.00). In Palakkad district, five locations were surveyed, among which panackatri and Thekkepotta recorded highest disease incidence of 88% and highest V.I (69.00). The major insects associated with the crop were whitefly (Bemisia tabaci (Genadius) with an incidence of 10-25%, aphids (Aphis gossypii glover) with an incidence of 10-40%, Jassids (Empoasca (Empoasca) motti Pruthi) with an incidence of 10-30% and mites with an incidence of 10-50%. Phyllody and little leaf symtoms (20% incidence) were also recorded in bittetgourd form Rajakumary and Rajakkad areas in Idukki. Flat limb and multiple proliferation of shoot tip were observed at many fields in Idukki. Symptoms associated with the disease include yellow mottle, mosaic,blistering, leaf curl and reduction in leaf size. Yellow mosaic and blistering is seen in severe infection finally leading to stunting of the plant, reduced flowering an fruiting and hairyness on stem. Mechanical transmission of the virus on Datura stramonium produced yellow lacal lesions indicating the presenceof Bean Golden mosaic virus (Begomo) in the infected leaf extract. This leaf extract also produced local lesions on othe indicator hosts like Chenopodium amaranticolor and Gomphrena globosa indicating the presence of Cucumber mosaic virus (CMV) or Potato virus Y (PVY). The viruses were transmitted by whiteflies (20%) and aphids (30%) from infected bittetgourd plants to healthy seedlings. Whiteflies (Bemisia tabaci Gennadius)) and aphids (Aphis gossypii Glover) are the vectors of the respective viruses Wedge grafting diseases scion on to 3-5 leaf stage healthy seedling of bittergourd produced symptoms of infection within ten days. KAU varieties Preethi and Priyanka were found to be susceptible to infection with preethi expressing a V.I of 70.80 and Priyanka expressing a V.I of 62.50 respectively. Ensyme linked immunosorbent assay (ELISA) and Dot immunobinding assay (DIBA) revealed the presence of three viruses belonging to Begomo, CMV and PVY group causing an mixed infection in bittergourd. The presence of all the three viruses were also confirmed in electron micrograph, Begomovirus as twin particles of size 18-20 X 30nm,CMVas single particles of 18nm and PVY as lonog flexuous rod of size 750nm. PCR amplification of coat protein gene (cp gene) of virus isolates from all the three districts yielded an amplicon of size approximately equal to 570 bp. Idukki and Palakkad isolates showed 94% identity to Tomato leaf Curl Virus isolate TNUDU BGI Coat Protein (AVI) gene while Trivandrum isolate showed 95% identity to Tomato leaf Curl Virus isolate TNPDU BG4 Coat Protein (AV1) gene . Phylogenetic tree constructed using multiple sequence alignment programme showed close relation between Begomo viruses identified in bittergourd from different districts. Studies on defense related enzymes such as peroxidase (PO), polyphenol oxidase (PPO) and phenyl alanine ammonialyase PAL) showed significant activity of PO and PPO in diseased plants than in healthy plants and the activity was on par in healthy and diseased for PAL. Protien profile of healthy and diseased at different days after virus inoculation through grafting indicated the production of novel proteins in diseased. There was no difference in the native profile of peroxidase in healthy and diseased at 15 days after virus inoculation. An additional isozyme band with a Rm value of 0.5 was observed in diseased at 45 days after virus inoculation. Management of the disease with antiviral chemicals and antiviral principles of plant, animal and microbial origin was undertaken as pot culture studies with pre and post inoculation of treatments. Twelve treatments with three replications each were laid out in completely randomized design for the evaluation. The treatments included Aspirin at two levels of 100 and 150 ppm, Salicylic acid (SA) at two levels of 100 and 150 ppm and Acibenzolar S methyl (ASM) at 50 and 75 ppm concentration, and two commercial formulations viz., Perfect and virus –Ex at 0.5 and 1.0 ml concentrations. The treatments were applied three times at 10 days interval. Pre application of thrice sprapying of Acibenzolar S methyl (ASM), 75 ppm concentration (V.I-35.00) at ten days interval was statistically significant over other treatments followed by ASM-50 ppm (V.I-41.33). Post application of antiviral chemicals also showed a statistically significant effect of three times spraying ASM-50 ppm(V.I-25.00) at ten days interval followed by spraying of Virus Ex 1ml L-1 (VThe best eight treatments with control was laid out as Randomised Block Design at the Instructional Farm, College of Agriculture, Vellayani during February to May 2017 as a field trial to study the effect of treatments on natural incidence of the viruses in the susceptible variety Preethi. The treatment, three sprays of ASM-50 ppm (V.I-28.33) at ten days interval ws on par with buttermilk (Three times dilution of curd) (V.I-39.16). Yield was also significantly high in ASM-50 ppm (437g plant-1) followed by Pseudomonas fluorescens talc based formulation (2%) (233 g plant-1)among the treatments.ThesisItem Open Access 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, Vellanikkara, 2022) Tania Mathew, Pampackal; Radhika, N SThesisItem Open Access Nutrient based management of Chilli leaf curi virus in Chilli (Capsicum annuum L.)(Department of Plant Pathology, College of Agriculture, Vellayani, 2019) Shilpa Sankar; KAU; Radhika, N SThe 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.ThesisItem Open Access 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 SThe 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.ThesisItem Open Access 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; KAU; Radhika, N SVarietal screening of black pepper to Cucumber mosaic virus and Piper yellow mottle virus and their sero-molecular detection The study entitled “Varietal screening of black pepper to Cucumber mosaic virus and Piper yellow mottle virus and their sero-molecular detection’ was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2018-2020 with the objectives to study the occurrence and sero-molecular detection of Cucumber mosaic virus (CMV) and Piper yellow mottle virus (PYMoV) infecting black pepper and to identify the varietal response to infection by the viruses. As a part of the study, survey was conducted in four panchayats of Idukki (Erattiyar, Kattappana, Pampadumpara and Karunapuram) and Wayanad (Ambalavayal, Meenangadi, Poothadi and Panamaram) districts of Kerala. Plant samples showing the symptoms of viral infections were collected and severity of viral infections were assessed from farmer’s field at the survey locations. Highest disease incidence (DI) was observed in Poothadi (45.50 %) of Wayanad and lowest in Karunapuram (21.62 %) of Idukki district. Highest vulnerability index (V.I.) was recorded in Panamaram (61) of Wayanad and lowest in Pampadumpara (32) of Idukki district. Mealy bugs were found associated with the virus infected plants in survey locations. Different symptoms of viral infections observed in the field were general chlorosis, initial chlorotic specks progressing to severe mottling and curling, vein banding, vein clearing, leaf distortion and reduction in leaf size. The virus inoculum was maintained in insect proof greenhouse condition. In the field, high viral disease incidence was observed in Karimunda plants while Thekkan pepper grafted on Piper colubrinum did not express any symptom. Varietal screening with 15 varieties (KAU, IISR and local) was undertaken by wedge grafting the infected scions showing the symptoms of both viruses (CMV and PYMoV) on to healthy black pepper plants. Among the varieties screened, Karimunda was identified as the most susceptible variety with highest V.I. (48). Serological detection of CMV and PYMoV were confirmed using polyclonal antisera of Cucumber mosaic virus (CMV) and two monoclonal antibodies of Banana streak virus (BSV 1 and BSV 2; as PYMoV belongs to the same group of BSV) through DAS (Double Antibody Sandwich)-ELISA. Infection due to either of the two viruses and mixed infections by both viruses were detected in the leaf samples. Leaf curling was found associated with infection by PYMoV while leaf reduction and distortion in leaf size were found associated with CMV infection. The results were confirmed using polyclonal antibodies of CMV and Sugarcane bacilliform virus (SCBV; as PYMoV belongs to the same group of SCBV) through DIBA (Dot Immuno Binding Assay) in infected leaf samples. The quality and quantity of isolated DNA by CTAB method was also assessed. PCR based molecular detection of PYMoV using ORF III specific primers yielded amplicons of approximately 400 bp. The amplified PCR products were subjected to sequencing and blast analysis. All the isolated sequences were found to be related to PYMoV. Phylogenetic studies revealed that isolates from Idukki and Wayanad grouped in separate clades. The present study on viruses infecting black pepper indicated that infection of CMV and PYMoV either in pure form or as mixed infections was prevalent in cultivated areas of Idukki and Wayanad districts of Kerala. Serological techniques like DASELISA and DIBA could be used for detection of these viruses along with molecular detection using coat protein specific primers. Varietal screening undertaken revealed that Karimunda was the most susceptible variety. The isolates of PYMoV from different locations of Idukki and Wayanad showed similarity in ORF III to other reported PYMoV sequences. സംഗ്രഹം കുരുമുളകിലെ വൈറസ് രരോരങ്ങൾലെതിലര ഇനങ്ങളുലെ ഗ്രതികരണൈും കുരുമുളകിലെ വൈറസ് രഹതുെളുലെ സീരറോ രമോളിെുെോർ രഠനൈും എന്ന ൈിഷയത്തിൽ രഠനം ലൈള്ളോയണി കോർഷിക രകോളജിലെ സസൃരരോര ൈിഭോരത്തിൽ 2018-20 കോെയളൈിൽ നെത്തുകയുണ്ടോയി. രഠനത്തിൻ്്ലറ ഭോരമോയി കുരുമുളക് കൃഷി ൈൃരകമോയി നെത്തുന്ന ഇെുെി, ൈയനോെ് ജില്ലകളിലെ 4 രഞ്ചോയത്തുകളിൽ സർരൈ നെത്തുകയുണ്ടോയി.അതിനോയി ഇെുെിയിലെ ഇരട്ടിയോർ, കട്ടപ്പന, രോമ്പോെുംരോറ, കരുണോരുരം എന്നീ രഞ്ചോയത്തുകളും ൈയനോെ് ജില്ലയിൽ നിന്നും അമ്പെൈയൽ, മീനങ്ങോെി, രൂതോെി, രനമരം എന്നീ രഞ്ചോയത്തുകളും സന്ദർശിച്ചു.വൈറസ് രരോര െക്ഷണം ഗ്രകെിപ്പിച്ച കുരുമുളക് ൈള്ളികൾ രശഖരിെുകയും അൈയുലെ രരോര ൈൃരനൈും രരോര തീഗ്ൈതയും ൈിെയിരുത്തുകയുണ്ടോയി.ഏറ്റൈും കൂെുതൽ രരോരത്തിന്ലറ ൈൃരനം റിരപ്പോർട്ട് ലെയ്തത് ൈയനോട്ടിലെ രൂതോെി രഞ്ചോയത്തിെും (45.5 %) ഏറ്റൈും കുറൈ് ഇെുെിയിലെ കരുണോരുരം രഞ്ചോയത്തിെും ആണ്.രരോര ൈൃരനത്തിന് രഹതുെളോയ മുഞ്ഞ, മീെിമൂട്ട തുെങ്ങിയൈയുലെ സോന്നിധ്യൈും ൈള്ളികളിൽ നിന്നും രശഖരിച്ചു. ലകോെികളിൽ കണ്ട രെതരം രരോര െക്ഷണങ്ങളും രഠിെുയുണ്ടോയി.മഞ്ഞളിപ്പ്, കുരുെിപ്പ് തുെങ്ങിയൈ ആണ് ഗ്രധ്ോന രരോര െക്ഷണങ്ങൾ. ൈിൈിധ്ഇനങ്ങളുലെ രരോര ഗ്രതിരരോധ് രശഷി അളെുന്നതിനോയി രരോരമുള്ള ൈള്ളി രരോര രഹിതമോയ ൈള്ളികളിൽ ഗ്രോഫ്റ്റ്റ് ലെയ്തു. കരിമുണ്ട എന്ന ലകോെിയിനത്തിനോണ് ഗ്രതിരരോധ് രശഷി ഏറ്റൈും കുറൈ് എന്ന് കലണ്ടത്തി. രുതിയ ഇെകളിൽ രരോരെക്ഷണങ്ങൾ ഗ്രകെമോെുന്ന ദിൈസൈും അൈയുലെ തീഗ്ൈതയും രഠിച്ചു.രരോരം നിജലപ്പെുത്തുന്നതിനോയി സീരറോ രമോളിെുെോർ രീതികളോയ എെിസ, ഡിബ, രി. സി. ആർ എന്നീ ലെസ്റ്റ്കൾെ് ൈിരധ്യമോെി ലകോണ്ട് വൈറസ് സോന്നിധ്യം സ്ഥിരികരിച്ചു. രരോരകോരിയോയ ലരപ്പർ ലയരല്ലോ രമോട്ടൽ വൈറസിന്ലറ ഒ.ആർ.എഫ്റ് 3 ജനിതക മോഗ്തകളും ഇൈയ്െ് മറ്റ് വൈറസുകളുമോയി തോരതമൃ രഠനൈും (വഫ്റരെോജനി ഗ്െീ) നെത്തി.ThesisItem Open Access 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; KAU; Radhika, N SVarietal screening of black pepper to Cucumber mosaic virus and Piper yellow mottle virus and their sero-molecular detection The study entitled “Varietal screening of black pepper to Cucumber mosaic virus and Piper yellow mottle virus and their sero-molecular detection’ was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2018-2020 with the objectives to study the occurrence and sero-molecular detection of Cucumber mosaic virus (CMV) and Piper yellow mottle virus (PYMoV) infecting black pepper and to identify the varietal response to infection by the viruses. As a part of the study, survey was conducted in four panchayats of Idukki (Erattiyar, Kattappana, Pampadumpara and Karunapuram) and Wayanad (Ambalavayal, Meenangadi, Poothadi and Panamaram) districts of Kerala. Plant samples showing the symptoms of viral infections were collected and severity of viral infections were assessed from farmer’s field at the survey locations. Highest disease incidence (DI) was observed in Poothadi (45.50 %) of Wayanad and lowest in Karunapuram (21.62 %) of Idukki district. Highest vulnerability index (V.I.) was recorded in Panamaram (61) of Wayanad and lowest in Pampadumpara (32) of Idukki district. Mealy bugs were found associated with the virus infected plants in survey locations. Different symptoms of viral infections observed in the field were general chlorosis, initial chlorotic specks progressing to severe mottling and curling, vein banding, vein clearing, leaf distortion and reduction in leaf size. The virus inoculum was maintained in insect proof greenhouse condition. In the field, high viral disease incidence was observed in Karimunda plants while Thekkan pepper grafted on Piper colubrinum did not express any symptom. Varietal screening with 15 varieties (KAU, IISR and local) was undertaken by wedge grafting the infected scions showing the symptoms of both viruses (CMV and PYMoV) on to healthy black pepper plants. Among the varieties screened, Karimunda was identified as the most susceptible variety with highest V.I. (48). Serological detection of CMV and PYMoV were confirmed using polyclonal antisera of Cucumber mosaic virus (CMV) and two monoclonal antibodies of Banana streak virus (BSV 1 and BSV 2; as PYMoV belongs to the same group of BSV) through DAS (Double Antibody Sandwich)-ELISA. Infection due to either of the two viruses and mixed infections by both viruses were detected in the leaf samples. Leaf curling was found associated with infection by PYMoV while leaf reduction and distortion in leaf size were found associated with CMV infection. The results were confirmed using polyclonal antibodies of CMV and Sugarcane bacilliform virus (SCBV; as PYMoV belongs to the same group of SCBV) through DIBA (Dot Immuno Binding Assay) in infected leaf samples. The quality and quantity of isolated DNA by CTAB method was also assessed. PCR based molecular detection of PYMoV using ORF III specific primers yielded amplicons of approximately 400 bp. The amplified PCR products were subjected to sequencing and blast analysis. All the isolated sequences were found to be related to PYMoV. Phylogenetic studies revealed that isolates from Idukki and Wayanad grouped in separate clades. The present study on viruses infecting black pepper indicated that infection of CMV and PYMoV either in pure form or as mixed infections was prevalent in cultivated areas of Idukki and Wayanad districts of Kerala. Serological techniques like DASELISA and DIBA could be used for detection of these viruses along with molecular detection using coat protein specific primers. Varietal screening undertaken revealed that Karimunda was the most susceptible variety. The isolates of PYMoV from different locations of Idukki and Wayanad showed similarity in ORF III to other reported PYMoV sequences. സംഗ്രഹം കുരുമുളകിലെ വൈറസ് രരോരങ്ങൾലെതിലര ഇനങ്ങളുലെ ഗ്രതികരണൈും കുരുമുളകിലെ വൈറസ് രഹതുെളുലെ സീരറോ രമോളിെുെോർ രഠനൈും എന്ന ൈിഷയത്തിൽ രഠനം ലൈള്ളോയണി കോർഷിക രകോളജിലെ സസൃരരോര ൈിഭോരത്തിൽ 2018-20 കോെയളൈിൽ നെത്തുകയുണ്ടോയി. രഠനത്തിൻ്്ലറ ഭോരമോയി കുരുമുളക് കൃഷി ൈൃരകമോയി നെത്തുന്ന ഇെുെി, ൈയനോെ് ജില്ലകളിലെ 4 രഞ്ചോയത്തുകളിൽ സർരൈ നെത്തുകയുണ്ടോയി.അതിനോയി ഇെുെിയിലെ ഇരട്ടിയോർ, കട്ടപ്പന, രോമ്പോെുംരോറ, കരുണോരുരം എന്നീ രഞ്ചോയത്തുകളും ൈയനോെ് ജില്ലയിൽ നിന്നും അമ്പെൈയൽ, മീനങ്ങോെി, രൂതോെി, രനമരം എന്നീ രഞ്ചോയത്തുകളും സന്ദർശിച്ചു.വൈറസ് രരോര െക്ഷണം ഗ്രകെിപ്പിച്ച കുരുമുളക് ൈള്ളികൾ രശഖരിെുകയും അൈയുലെ രരോര ൈൃരനൈും രരോര തീഗ്ൈതയും ൈിെയിരുത്തുകയുണ്ടോയി.ഏറ്റൈും കൂെുതൽ രരോരത്തിന്ലറ ൈൃരനം റിരപ്പോർട്ട് ലെയ്തത് ൈയനോട്ടിലെ രൂതോെി രഞ്ചോയത്തിെും (45.5 %) ഏറ്റൈും കുറൈ് ഇെുെിയിലെ കരുണോരുരം രഞ്ചോയത്തിെും ആണ്.രരോര ൈൃരനത്തിന് രഹതുെളോയ മുഞ്ഞ, മീെിമൂട്ട തുെങ്ങിയൈയുലെ സോന്നിധ്യൈും ൈള്ളികളിൽ നിന്നും രശഖരിച്ചു. ലകോെികളിൽ കണ്ട രെതരം രരോര െക്ഷണങ്ങളും രഠിെുയുണ്ടോയി.മഞ്ഞളിപ്പ്, കുരുെിപ്പ് തുെങ്ങിയൈ ആണ് ഗ്രധ്ോന രരോര െക്ഷണങ്ങൾ. ൈിൈിധ്ഇനങ്ങളുലെ രരോര ഗ്രതിരരോധ് രശഷി അളെുന്നതിനോയി രരോരമുള്ള ൈള്ളി രരോര രഹിതമോയ ൈള്ളികളിൽ ഗ്രോഫ്റ്റ്റ് ലെയ്തു. കരിമുണ്ട എന്ന ലകോെിയിനത്തിനോണ് ഗ്രതിരരോധ് രശഷി ഏറ്റൈും കുറൈ് എന്ന് കലണ്ടത്തി. രുതിയ ഇെകളിൽ രരോരെക്ഷണങ്ങൾ ഗ്രകെമോെുന്ന ദിൈസൈും അൈയുലെ തീഗ്ൈതയും രഠിച്ചു.രരോരം നിജലപ്പെുത്തുന്നതിനോയി സീരറോ രമോളിെുെോർ രീതികളോയ എെിസ, ഡിബ, രി. സി. ആർ എന്നീ ലെസ്റ്റ്കൾെ് ൈിരധ്യമോെി ലകോണ്ട് വൈറസ് സോന്നിധ്യം സ്ഥിരികരിച്ചു. രരോരകോരിയോയ ലരപ്പർ ലയരല്ലോ രമോട്ടൽ വൈറസിന്ലറ ഒ.ആർ.എഫ്റ് 3 ജനിതക മോഗ്തകളും ഇൈയ്െ് മറ്റ് വൈറസുകളുമോയി തോരതമൃ രഠനൈും (വഫ്റരെോജനി ഗ്െീ) നെത്തി.