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ThesisItem Open Access Management of foot rot of black pepper (Piper nigrum L.) with mycoinoculant enriched vermicompost(Department of Plant Pathology, College of Agriculture, Vellayani, 2002) Divya, S; Sasikumar NairThe study, "Management of foot rot of black pepper (Piper nigrum L.) with mycoinoculant enriched vermicompost" was done at the Department of Plant Pathology, College of Agriculture, Veliayani to explore the possibility of using vermicornpost as a carrier material for mass production of biocontrol agents and to test its efficacy in controlling the disease. The talc based inoculum of Trichoderma harzianum and soil based granular inoculum of Glomus Jasciculatum were mass produced in vermicompost either alone or in combination with farmyard manure or neem cake in the ratio 1 : 1 and 5 : 1 respectively and the efficacy was compared with the standard carrier material of FYM + neem cake (10 : 1). The population of T. harzianum 15 DAI was maximum in the treatment combination of VC + NC (5 : 1). However, the influence of these carrier materials on percentage of mycorrhizal infection 50 DAI was not statistically significant. Vermicompost as a carrier material for biocontrol agents was further tested in pepper var. Karimunda inoculated with P. capsici. Carrier materials as such had no significant influence on disease control. Reduction in foliar infection, disease index, stem infection and percentage mortality was observed in treatments with biocontrol agents. Disease control was maximum with the treatments involving T. harzianum while plant growth promotion was maximum with G. Jasciculatum. The physiological changes in pepper plants after inoculation with P. capsici and biocontrol agents were studied. The phenol and OD phenol content was more in pathogen inoculated plants. The defence related enzymes peroxidase, polyphenol oxidase and phenylalanine ammonia lyase were enhanced with pathogen inoculation as well as with the application of biocontrol agents. SDS-PAGE analysis of proteins with samples extracted from plants one day after inoculation of pathogen showed the presence of two novel proteins with molecular . weights of 78 kDa and 28 kDa in diseased samples which were absent in treatments without pathogen and also in plants treated with COC.ThesisItem Open Access Studies on the burrowing nematode radopholus similis (cobb,1893) thorne 1949 on pepper(Piper nigrum L.) and its role in slow wilt disease(Department of plant pathology, University of agricultural sciences, Bangalore, 1976) Venkitesan, T S; Setty, K G HThesisItem 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 Embargo 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; 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 Evaluation of beneficial fungal root endophytes against fusarium rot in small cardamom(Department of Plant Pathology, College of Agriculture,Vellayani, 2022-04-02) Aishwarya, Manoharan.; KAU; Dhanya, M KThe study entitled “Evaluation of beneficial fungal root endophytes against Fusarium rot in small cardamom” was conducted at College of Agriculture, Vellayani and Cardamom Research Station, Pampadumpara during 2020-21 to evaluate the colonisation and interaction of two beneficial fungi, Piriformospora indica and Glomus fasciculatum, in cardamom and their potential to manage Fusarium rot disease of cardamom and elucidation of the role of gibberellic acid in the endophytic fungimediated disease tolerance. Fusarium infection in small cardamom is characterised by root rotting, eye shaped lesion on pseudostem and panicle rot. Fusarium oxysporum Schledt was isolated from the infected roots of the farmer’s field of Pampadumpara in Idukki district. Koch’s postulate was proved by inoculation of the pseudostem of one year plants using pinprick method where the isolate produced the typical eye shaped lesion within two weeks. A pot culture experiment was conducted at CRS, Pampadumpara to assess the interactive effect of the fungal root endophytes P. indica and G. fasciculatum on the vegetative growth of small cardamom seedlings and suckers using four treatments and five replications. Both individual and combinatorial effects of the endophytes on the vegetative characters like plants height, number of leaves, leaf length, leaf width, root parameters, biomass etc. were evaluated. Successful root colonization was observed in P. indica and G. fasciculatum treated roots at six and seven days after their inoculation respectively. This is the first report of colonization of P. indica in small cardamom. The study revealed that the endophytes inoculated plants were superior to control plants in which maximum shoot growth was shown by the dually colonized plants followed by the P. indica-colonized which was then followed by G. fasciculatum colonised plants. The root parameters were higher for P. indica-colonized plants compared to the combined or G. fasciculatum colonized plants in cardamom seedlings whereas dually colonized plants had better root growth in cardamom suckers. P and K analysis of the whole plant clearly indicated the enhanced nutrient uptake to root, shoot and leaf in the endophytes-colonized plants. The IAA analysis of root samples also revealed a higher IAA content in the endophytes colonized seedlings compared to the control seedlings with P. indica colonized plants recording the maximum value of 83.80 µg g-1 in seedlings and 94.30 µg g-1 in suckers; and least value being observed in control plants recording 44.80 µg g-1 in seedlings and 94.30 µg g-1 in suckers. This study stipulate a scope of better establishment of cardamom in field especially in the case of seedlings during transplantation due to well established root system. There was also a reduction in nursery period by two months in the P. indica and G. fasciculatum dually colonized seedlings compared to the non-colonized seedlings. In vitro evaluation of P. indica against F. oxysporum and in vivo evaluation of both the bioagents against F. oxysporum in small cardamom was carried out to appraise the effect of bioagents in the management of Fusarium rot disease of cardamom. In in vitro evaluation, dual culture assay of P. indica with F. oxysporum was performed in PDA in which a percent inhibition of 64.4 (%), antagonism index of 20.53 and an obscure lysis zone was observed. There wasn’t any presence of inhibition zone, antibiosis, coiling, overgrowth, sporulation etc. In in vivo evaluation, a pot culture experiment was laid out at CRS, Pampadumpara with seven treatments and three replications including a bio agent check of 2% Pseudomonas fluorescens and chemical check of 2 % carbendazim as basal drench. The study revealed that the beneficial interaction with bioagent successively decreased the disease severity and lesion size, among which P. indica treated plants showed the best result having the least disease severity of 38.07 (%) and 31.48 (%) in seedlings and suckers respectively. The disease severity of the control plants were the highest, recording 86.62 (%) and 76.77 (%) in seedlings and suckers respectively. Combinatorial and P. fluorescens treatments were the other treatments showing promising results in disease management. Total Gibberellic acid was estimated from leaf samples of challenge inoculated experimental plants to elucidate the role of GA in endophyte mediated disease tolerance and the authors couldn’t interpret any role of GA in disease development. Moreover, the GA content was higher in P. indica colonised recording 18.60 µg g-1 and 10.65 in µg g-1 in seedlings and suckers respectively; and dually colonised plants recording 18.00 µg g-1 and 9.71 µg g-1 in seedlings and suckers respectively. Thus, the present study proclaims a very pronounced plant growth promotion in cardamom seedlings and suckers by the beneficial fungal root endophytes viz., P. indica and G. fasciculatum just before transplanting to the field and therein advocating better development and establishment in the field condition. Furthermore, the results also indicates an effective management of the destructive Fusarium rot disease in small cardamom using endophyte colonization especially in P. indica colonized and dually colonized plants.ThesisItem Open Access Management of a major fungal disease in black pepper (Piper nigrum L.)(Department of Plant Pathology, College of Agriculture,Padanakkad, 2022-04-11) Pranavya, A P; KAU; Sajeesh, P KBlack pepper (Piper nigrum L.) is one of the major spice crops and plantation crops that grow as the sole crop or intercrop in Kerala. In nurseries of black pepper major biotic stress are caused by bacteria, fungus, virus and nematode, out of this fungal infection are the leading one. The seedling blight, anthracnose and collar rot were found to be more severe in the nursery. With this background, the present study was conducted to identify the major fungal pathogen associated with black pepper nursery and formulate disease management strategies against that pathogen. A survey was carried out in black pepper growing nurseries to record the different diseases. Seedling blight, anthracnose and collar rot were observed. The fungal isolates associated with seedling blight in nurseries at Padannakkad and Kanakappalli were designated as Pc1 and Pc2 respectively. Anthracnose affected seedlings were collected from Padannkkad, Balal, Panniyur and Periya black pepper nurseries and the fungal isolates associated were designated as Cg1, Cg2, Cg3 and Cg4 respectively. The collar rot samples were collected from Padannakkad, Periya and Panniyur and isolates were designated as Sr1, Sr2 and Sr3. In the case of seedling blight infection, highest disease incidence (DI) (74%) was recorded in Pc1. The highest percent disease index (PDI) in case seedling blight (32%) was recorded in Pc1 as compared to Pc2 isolate. In the case of anthracnose, higher DI (30%) and PDI (25%) were recorded in Cg1 in comparison with Cg2, Cg3 and Cg4. In case of collar rot, higher DI (20%) was recorded in case of Sr1 isolate. All the isolates were tested for its pathogenicity; in case of seedling blight, the isolate Pc1 recorded higher radial growth (7.90 cm) and recorded lesser time (2days) for the symptom development as compared to Pc2 isolate. In case of anthracnose, isolate Cg1 recorded higher radial growth (8.0 cm) and took lesser time (5days) for the symptom development in comparison with Cg2, Cg3 and Cg4. In case of collar rot isolate Sr1 recorded higher radial growth (8.96 cm) and recorded lesser time (2days) for the symptom development as compared to Sr2 and Sr3. A trial was laid out to identify the most susceptible variety of black pepper as well as the most virulent strain of the pathogen among different isolates on pepper varieties viz., Panniyur (P) 1-6, P8, P9, Vijay and Karimunda. Panniyur 2 was identified as most susceptible variety and Pc1 was the virulent isolate. Based on cultural and morphological characters and molecular characterization, Pc1 was identified as Phytophthora capsici. In vitro evaluation of biocontrol agents, chemical fungicides and organic preparations were carried out to study the efficacy against P. capsici. Under in vitro conditions biocontrol agent T. harzianum (2%) exhibited maximum inhibition (55.56%) followed by P. fluorescens (53.33%). Among the chemical fungicides copper oxychloride (0.3%) and metalaxyl (8%) + mancozeb (64%) at 0.3 percent recorded 100 per cent inhibition. Among the organic preparations, ready to use neem oil garlic soap (3%) and garlic extract at 0.3 percent recorded maximum inhibition 31.11% percent and 28.88% percent respectively in the mycelial growth of the pathogen. The best-performing treatments from the in vitro evaluation were selected for pot culture experiment on variety Panniyur 2. After 7 days of inoculation, the least PDI (0.00 %) as well as the least lesion development (0.00%) was recorded in case of metalaxyl (8%) + mancozeb (64%) at 0.6 per cent. The higher PDI (86.66%), as well as maximum lesion development (46.66%), was recorded in ready to use neem oil garlic soap (3%). The study reveals that, the seedling blight caused by Phytophthora capsici can be effectively managed with the foliar application of metalaxyl (8%) + mancozeb (64%) at 0.6 per cent.ThesisItem Open Access Comparative evaluation of different species of oyster mushroom suitable to Kerala(Department of Plant Pathology, College of Agriculture, Padannakkad, 2022-03-11) Akhil, G L; KAU; Susha S, Tharahe present study entitiled “Comparative evaluation of different species of Oyster mushroom suitable to Kerala” was carried out in the College of Agriculture, Padannakkad during 2019-2021 with the objective to identify the oyster mushroom species best suitable for cultivation under five agro-ecological zones of Kerala. Five species of Oyster mushrooms were selected for the study viz., Pleurotus florida, P. djamor, Hypsizygus ulmarius, P. sajor-caju and P. citrinopileatus. To evaluate the yield performance, five species were cultivated in farmer’s field of five agroecological zones of Kerala. The study also aims to evaluate the proximate constituents and medicinal components present in oyster mushrooms. Sensory evaluation and shelf life studies were also carried out. The sporocarps of five species of oyster mushrooms were studied for their morphological characters. P. florida produced white, delicate and fleshy sporocarp with an average weight of 11.71 g. P. djamor produced pinkish white leathery sporocarps with smaller pileus having very short stipe (0.8 cm). H. ulmarius produced creamy white fleshy sporocarps with significantly large pileus having average size of 7.0 cm x 7.52 cm. P. sajor-caju produced greyish white leathery sporocarp. P. citrinopileatus produced golden yellow delicate sporocarp with comparatively short stipe. The average size of pileus was 5.45 cm x 6.17 cm. Microscopic observations of hyphae and spores of five species of oyster mushrooms shows that hyphae of all the five species were septate, branched and hyaline with clamp connections. The width of the hyphae of all the five species ranged from 1.5- 4.5 μm. Basidiospores of P. florida and P. djamor recorded 7-12 μm in length and 2-5 μm in width whereas, H. ulmarius and P. sajor-caju produced spores with dimension of 8-12 μm× 3-6 μm. P. citrinopileatus produced spores with dimension of 7-10 μm× 2-3 μm. 141 Two substrates namely paddy straw and rubber sawdust were evaluated for the cultivation of five species of oyster mushrooms. Total yield from three harvests were significantly less in paddy straw, (954.66 g) compared to rubber sawdust which has given a yield of 1306.40 g. The yield recorded was highest for H. ulmarius cultivated on both paddy straw and rubber sawdust (1233 g kg-1 and 1611 g kg-1 respectively) which was followed by P. florida (1148.30 g kg-1 and 1582.66 g kg-1 respectively). It has been evident that selection of substrate significantly affected time taken for complete spawn run, time taken for pin-head formation, time taken for first harvest, total crop period and total yield from three harvests. The present study revealed that both the substrates viz., paddy straw and rubber sawdust were suitable for the cultivation of oyster mushrooms. So, both the substrates can be selected for cultivation based on the ease of availability and cost. In the present study, the suitability of five species of oyster mushrooms in five agro-ecological zones of Kerala were evaluated by cultivating the mushroom species at coastal plains, midland laterites, foothills, high hills and Palakkad plains. H. ulmarius was found to be the best suited oyster mushroom species for cultivation in coastal plains (102.99 % BE), foot hills (92.73 % BE) and high hills (123.30 % BE). In midland laterites and Palakkad plains, highest BE of 82.70 (%) and 103.80 (%) respectively were obtained from P. djamor. P. djamor, although reported to be performing well under warm conditions, yielded well under midland laterites and Palakkad plains. This may be due to low relative humidity and comparatively higher temperature in these zones. Earliness in primordial initiation (14-19 days) and extremely fast growth also make this species promising for cultivation in Kerala especially during dry months. The proximate analysis of five species of oyster mushrooms was carried out and it revealed that mushrooms are rich source of proteins, carbohydrates, fibres, amino acids, vitamins and minerals with less fat content. The protein content was highest in P. sajorcaju (26.02 %) and the fibre content was highest in P. florida (26.20 %). Fat content was the least in H. ulmarius with 1.70 per cent on dry weight basis. H. ulmarius (31.27 %) recorded the highest carbohydrate content among the five species of oyster mushrooms. 142 P. florida recorded the maximum moisture content (90.97 %). Highest amino acid content of 19.40 per cent was recorded from P. sajor-caju Analysis of mineral components revealed that highest content of sodium (965 ppm) and potassium (7175 ppm) was present in P. djamor. P. florida recorded highest content of calcium (1000 ppm) and magnesium (4025 ppm) which supports healthy immune system and keep bones strong. Medicinal analysis confirmed that these mushrooms were rich in bioactive components. P. djamor recorded highest content of β-glucan (30.25 g per 100g) having antitumour and immunomodulation activities, β-carotene (3.70 μg per 100g) having antioxidant properties and lovastatin (370.75 μg per 100g) which reduce blood cholesterol. P. sajor-caju recorded highest glycoprotein (435.00 μg per 100g) and terpenoid (1.26 %) content. Sensory evaluation of five species of oyster mushrooms was done using sauteed mushroom recipe to evaluate the quality attributes and consumability of the species. H. ulmarius recorded highest scores for appearance (8.83), colour (8.75), texture (8.66), flavour (8.58) and taste (8.83) on a maximum score of 9. H. ulmarius scored 8.80 in terms of overall acceptability followed by P. florida (7.60). Overall, from this study it was found that oyster mushrooms, especially H. ulmarius and P. florida are highly favoured for consumption. Shelf life of five species of oyster mushrooms were undertaken both in room temperature (26±20C) and in refrigerated conditions (150C). In the present study, comparatively higher shelf life was observed on P. djamor and P. sajor-caju which could be stored for two days after harvesting in room temperature and three days in refrigerated conditions. The present study demonstrated the exploitability of five species of oyster mushrooms in Kerala conditions. All the five species can be cultivated under the five agro-ecological zones of Kerala but H. ulmarius, P. florida and P. djamor yielded well compared to P. citrinopileatus and P. sajor-caju. H. ulmarius was found to be the best 143 suited oyster mushroom species for cultivation in coastal plains, foot hills and high hills. In midland laterites and Palakkad plains, highest yields were obtained from P. djamor can be economically cultivated regarding the yield and earliness in fruiting.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 Evaluation of Piriformospora indica against Piper yellow mottle virus in Black pepper(Department of Plant Pathology, College of Agriculture, Vellayani, 2021) Lekshmi, R Krishnan; KAU; Joy, MThe study entitled “Evaluation of Piriformospora indica against Piper yellow mottle virus (PYMoV) in black pepper” was conducted in Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during 2019-2021, to evaluate the efficiency of the root colonizing endophytic fungus P. indica in managing PYMoV in black pepper, and to elucidate the role of Reactive Oxygen Species and antioxidant enzymes in the tripartite interaction. A survey conducted in the instructional farm of College of Agriculture, Vellayani, to identify and collect PYMoV-infected black pepper plants showed that the surveyed varieties viz., Panniyur 1 to 7 and Karimunda were moderately to highly susceptible to PYMoV infection, with the disease incidence ranging from 10 to 91 per cent and disease severity as vulnerability index from 30 to 58. PYMoV-infected black pepper plants showed symptoms like chlorotic flecking, mottling, light necrosis, crinkling and distortion of leaves, reduction in leaf size and stunted growth. Presence of the virus in the samples was confirmed by polymerase chain reaction (PCR) with oligonucleotide primers specific to open reading frame III (ORF III) of PYMoV. P. indica culture obtained from Department of Plant Pathology was maintained in potato dextrose agar (PDA) and potato dextrose broth (PDB) media. P. indica was mass multiplied in coirpith-dried farm yard manure (FYM) mixture (1:1) amended with two per cent gram flour and was allowed to colonize on rooted cuttings of black pepper. P. indica colonization enhanced different biometric characters at different intervals, compared to the non-colonized control plants. P. indica colonization resulted in 25 per cent more shoot length, 23 per cent more root length, 35 per cent more number of leaves, 23 per cent more leaf area and 27 per cent more number of primary roots than the control plants at 150 days after co-cultivation (DAC). A pot culture experiment was laid out to evaluate the efficiency of prophylactic as well as curative application of P. indica, in managing PYMoV of black pepper. P. indica colonization significantly reduced the incidence and severity of PYMoD, upon pre- as well as post- inoculation of the virus, compared to the control plants. Plants colonized with P. indica and later inoculated with PYMoV as well as plants infected by PYMoV and later colonized by P. indica, both recovered the loss in biomass caused by the virus infection, compared to the non-colonized plants challenged by the virus. However, prophylactic treatment with P. indica resulted in better control of the disease compared to the curative treatment. Amplification of the virus by PCR also revealed that the virus titer was the least in P. indica-colonized plants that were post-inoculated with the virus after 30 DAC, followed by pre-inoculated plants that were later colonized with P. indica. The virus titer was the highest in the non-colonized plants inoculated with the virus. In order to evaluate the performance of P. indica in modulating the biochemical responses of black pepper plants against PYMoV infection, the activity of various enzymes which are known to play a role in the antioxidant defense systems of plants viz. catalase, peroxidase, glutathione reducatse, glutamate synthase, phosphatase and superoxide dismutase were assayed. A significant increase in the activity of these antioxidant enzymes was observed in the P. indica-colonized plants compared to the control plants, upon pre- as well as post- inoculation of PYMoV. Thus, the better performance of P. indica-colonized plants upon PYMoV infection can be correlated to the increased activities of antioxidant enzymes. PYMoV-infected plants showed increased accumulation of reactive oxygen species like superoxide anions and hydrogen peroxide, which cause oxidative stress in the plants. P. indica-colonization was able to mitigate the increase in ROS concentration caused due to PYMoV infection, thus reducing the symptoms. Thus the study reveals that, co-cultivation of rooted cuttings of black pepper (obtained from disease-free mother plants) with P. indica and keeping them under insectproof conditions for around 30 days (for successful establishment of the fungus inside the root cortex) and then planting them in the main field is a reliable and chemical-free method to control PYMoD in black pepper. Bio-protective action of P. indica might be attributed to the increased activity of antioxidant enzymes, which in turn leads to suppression in ROS accumulation in the colonized plants, upon being challenged by the virus.
