1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item 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 SThe 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.Item Piriformospora indica and its water diffusible exudates for the management of chilli anthracnose incited by colletotrichum capsici (Syd.) butler and bisby(Department of Plant Pathology, College of Agriculture, Vellayani, 2024-07-05) Elizabeth, T Jojy; KAUThe research work entitled “Piriformospora indica and its water diffusible exudates for the management of chilli anthracnose incited by Colletotrichum capsici (Syd.) Butler and Bisby” was carried out in the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during 2017- 2023. The study was undertaken with the objective to evaluate P. indica- and its water diffusible exudates- primed chilli seedlings and plants against foliar infection of C. capsici; and study the biochemical and molecular mechanisms involved in this tripartite interaction. A survey conducted in the five agroclimatic zones (ACZs) of Kerala showed that the highest disease incidence (DI) and Percent Disease Index (PDI) were recorded at RARS, Pilicode (DI-90 & PDI-52.60) of northern zone, while the lowest DI was observed at farmer’s field, Kottarakkara (20%) and ORARS, Kayamkulam recorded the lowest PDI (23.63). Chilli anthracnose symptoms namely leaf spot, leaf blight, die-back, fruit rot and mummified fruits were observed in different survey locations of the five agroclimatic zones (ACZ) of Kerala. Nine C. capsici isolates and one isolate of C. gloeosporioides were obtained from the surveyed locations. All the C. capsici isolates produced sparse mycelial growth with concentric zonations of acervuli on potato dextrose agar (PDA) medium. The upper side of culture plates appeared in different shades of white and off-white to grey with regular or irregular margins, while the reverse side looked yellowish brown to black. Isolate Cc4 produced maximum mycelial growth diameter of 8.6 cm compared to 7.2 cm (minimum) in Cc2 on 7th day after inoculation (DAI). Further, microscopic characters such as mycelial width, size of conidia, acervuli, appressoria, number and length of setae were significantly different in the C. capsici isolates. The most virulent isolate of C. capsici was screened based on the lesion size produced on the artificially inoculated leaves and fruits of chilli var. Vellayani Athulya. On 7th DAI, Cc3 isolate of C. capsici produced maximum lesion size of 2.52 cm while isolate Cc4 produced minimum lesions (0.74 cm) on 292 inoculated chilli leaves. Similarly, tender, mature and ripe fruits of var. Vellayani Athulya inoculated with Cc3 isolate produced the highest lesion size (2.48, 2.34 and 2.56 cm respectively) and isolate Cc4 recorded the lowest lesions (1.62, 1.16 and 1.38 cm respectively) on 7th DAI. Thus, isolate Cc3 from Thrissur, was selected as the most virulent isolate of C. capsici. Ten selected KAU/ICAR released varieties of chilli were screened with Cc3 isolate by detached leaf and fruit inoculation method to identify the most susceptible variety. Leaves of Vellayani Athulya recorded the highest lesion size of 2.80 cm on 5 DAI compared to 1.66 cm (lowest) in Manjari. Maximum lesion size of 2.96 cm was observed on the tender fruits of Vellayani Athulya and minimum was recorded on Vellayani Thejus (1.52 cm). Lesions of 3.50 cm (highest) were observed on mature Vellayani Athulya fruits as against 2.00 cm on Manjari fruits. Similarly, ripe fruits of Vellayani Athulya recorded maximum lesion size of 3.46 cm and minimum in Vellayani Samrudhi (1.30 cm) at 7 DAI. Also, in the in vivo study, higher lesion size (3.00 cm) was observed in Vellayani Athulya fruits and lowest (1.62 cm) in fruits of Vellayani Samrudhi at 7th DAI. Standardization of P. indica-colonization in chilli var. Vellayani Athulya was done on plant nutrient medium (PNM) and chilli roots were sampled at different intervals viz., 3rd, 5th, 7th, 10th, and 15th days after colonization (DAC). Young, double-walled chlamydospores were observed within the root cells at 5 DAC. P. indica colonization gradually increased and reached a maximum of 100 per cent at 15 DAC. Dual culture of P. indica and C. capsici showed the appearance of inhibition zone (8.33 DAI) and antibiosis (12.67 DAI) at the point of interaction of the two fungi. Further microscopic observations revealed thickening and lysis of pathogen mycelium by P. indica. The growth of C. capsici was suppressed (3.85 cm) in dual culture compared to normal (6.28 cm) in control at 7th DAI. 57.22 per cent mycelial growth inhibition due to P. indica was observed at 15 DAI. Antagonistic property of P. indica-water diffusible exudates (Pi-WDE) against C. capsici was studied by in vitro poison food technique. Significant 293 differences were observed in the colour and nature of C. capsici growth on PDA media, added with different day-old Pi-WDE in comparison with control. Among them, ten day-old Pi-WDE recorded the highest inhibition (67.30 %) of pathogen mycelial growth whereas the lowest in one day-old Pi-WDE (9.29 %). Mass multiplication of P. indica in the portray medium was carried out. Rapid growth of the endophyte was recorded in a combination of farmyard manure (FYM) and coir pith (1:1), added with 2 per cent gram flour. P. indica completely covered the inoculated trays within 7 DAI. This medium was used to colonize chilli plants with P. indica for further experiments and thus, four treatments viz., P. indica alone (T1), C. capsici alone (T2), P. indica-primed seedlings + C. capsici (T3) and control (T4) were systematized for the in vitro and in vivo evaluation experiments. In vitro study of P. indica-primed chilli seedlings against C. capsici significantly reduced anthracnose lesion size and disease severity to 1.30 cm and 30.63 per cent respectively compared to 2.06 cm and 77.00 per cent in C. capsici alone at 7 DAI. Maximum root and shoot weight were recorded in the P. indicacolonized plants, irrespective of the pathogen. Pot culture experiment was also carried out in the chilli var. Vellayani Athulya. The development of anthracnose symptoms on leaves was delayed by 4 days in P. indica-colonized chilli plants as against 2 days in control. The lesion size and disease severity due to anthracnose was lower in P. indica-colonized chilli leaves (1.58 cm & 54.90 %) compared to C. capsici alone (2.86 cm & 85.20 %) at 10 DAI. Similarly, minimum lesion size and disease severity of 1.17 cm and 27.92 per cent was observed in fruits from pathogen inoculated P. indicacolonized chilli plants than in C. capsici alone (3.56 cm & 91.67 %) at 10 DAI. Field studies were conducted in rabi and summer seasons with two treatments viz., P. indica-colonized and non-colonized plants. P. indica-colonized chilli plants recorded lower anthracnose severity of 27.00 per cent in Rabi and 15.50 per cent in summer compared to 56.50 and 47.00 per cent respectively in control. P. indica colonization improved the biometric characters such as plant height, number of leaves, leaf area, number of secondary and tertiary roots, shoot 294 weight, root weight in primed chilli plants. In addition, yield of chilli was enhanced in endophyte-colonized plants compared to control. Biochemical studies on the mechanism of disease management in P. indica-colonized chilli plants at 0, 12, 24 and 72 hours after inoculation (HAI) showed increased activity of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (PO). In contrast, the activities of 4-coumaryl CoA ligase (4-CL) and polyphenol oxidase (PPO) were enhanced in chilli plants inoculated with C. capsici alone. Isoenzyme analysis revealed higher induction of isoenzymes of PO and PPO in the P. indica-colonized plants compared to control. Enhanced induction of pathogenesis related (PR) proteins was noticed in the P. indica-colonized plants inoculated with the pathogen at 72 HAI. The capsaicin content was drastically reduced in C. capsici inoculated chilli fruits. However, fruits from P. indica-colonized plants had high capsaicin content (>30% over control) irrespective of infection. Molecular studies of the defense related genes revealed the positive role of P. indica in the management of anthracnose disease in chilli. P. indica reduced anthracnose symptoms in colonized chilli plants by the upregulation of genes involved in phenyl propanoid pathway (PAL1, 4-CL, CAD and CHS), jasmonic acid signaling pathway (PDF1.2 and AOS) and salicylic acid signaling pathway (PR1, EDS1 and PAL3); and downregulating the LOX gene involved in jasmonic acid signaling. In summary, P. indica and its WDE inhibited C. capsici under in vitro conditions. Anthracnose incidence and severity were considerably reduced in the treated chilli seedlings and plants compared to control. In addition, P. indica enhanced growth as well as yield in colonized chilli plants, thereby enhancing the fruit quality. Further, the endophyte improved disease resistance in chilli plants by increasing the defense-related enzyme activities and expression of defense genes. Thus, the root fungal endophyte, P. indica can be considered as an efficient biocontrol agent in the management of chilli anthracnose.Item 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.Item Molecular characterisation of Ralstonia solanacearum (Smith) Yabuuchi et al causing bacterial wilt in solanaceous vegetables(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2001) Deepa James; KAU; Girija, DBacterial wilt incited by Ralstonia solanacearum is one of the most devastating diseases of solanaceous vegetable crops in Kerala. Crop losses due to the incidence of this disease may go upto 100 per cent. Existence of different strains, races and biovars has been responsible for breaking down of resistance of varieties evolved through breeding programmes. In view of wide variability, a study was undertaken to characterise the isolates of R. solanacearum collected from three different agro climatic zones ofKerala at molecular level. Nine isolates of R. solanacearum collected from three different locations from brinjal, chilli and tomato were used in the study. These were isolated, purified and maintained in sterile distilled water at room temperature. Inoculation techniques were standardised in brinjal, chilli and tomato plants for assessing the virulence and aggressiveness of the isolates. Virulence and aggressiveness of the isolates were studied on respective host plants and found them highly varying. Vellanikkara and Kumarakom isolates could cross inoculate, whereas Ambalavayal isolates did not. The isolates were characterised by various cultural, morphological and biochemical tests and the variability among them was studied. Biovars, III and IlIA and races, 1 and 3 were identified among the isolates. The isolates were resistant to ampicillin and sensitive to chloramphenicol. Plasmid DNA profile of the isolates were studied and no difference was found in the plasmid DNA profile of the nine isolates. Polymorphism among the isolates was studied using RAPD with ten decamer primers. RAPD profiles exhibited great diversity among biovars III and IlIA as well as among race 1 isolates. Race 3 isolates were less polymorphic with certain primers tested. OPF8 yielded a unique band specific to race 3 isolates. Dendrogram obtained from the pooled data of RAPD profiles also showed high genetic similarity between race 3 isolates. Dendrogram obtained from the pooled data of RAPD profiles also showed high genetic similarity between race 3 isolates. Restriction analysis could not characterise the isolates since no banding pattern was obtained with restricted DNA. No hybridization signal was detected after Southern hybridization in RFLP. Curing of plasmid DNA at high temperatures was found unsuccessful. Plasmid profiles of both mucoid and non-mucoid colonies were compared to assess the role of plasmid in EPS production and the plasmid could be observed in both types of colonies. In the latter, a reduction in size of the plasmid was noticed. Thus the study revealed that great diversity existed among strains of R. solanacearum at different locations of Kerala when molecular techniques, especially RAPD was used as a tool.