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20235
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Subject: Plant Pathology × End date: 2023 × Start date: 2023 × Type: Thesis × Thesis Type: Ph.D ×
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    Understanding sunflower - Macrophomina phaseolina interaction under water stress conditions
    (Punjab Agricultural University, 2023) Navkiran Kaur; Sharma,Pankaj
    A total of 35 isolates of Macrophomina phaseolina causing charcoal rot were collected from eleven kharif and rabi crops from different districts of Punjab and adjoining states. Macrophomina phaseolina isolated from sunflower field, Ludhiana was characterized on cultural and morphological and molecular basis. The isolates infecting sunflower showed highest sequence homology of ITS genomic region with M. phaseolina isolate from Indore, Madhya Pradesh NCBI accession no. MT127404. This isolate was further used to standardize the inoculation techniques under field and polyhouse conditions. Tooth pick inoculation technique was found most effective for large scale screening while sorghum grain inoculation method was proficient under polyhouse conditions. A set of 71 sunflower genotypes were evaluated using tooth pick inoculation technique against charcoal rot under irrigated and restricted irrigation conditions. Four genotypes viz; 75B, EC6078261, OPH137, and OPH 172 were found moderately resistant under irrigated conditions whereas only two genotypes 75B and EC6078261 showed moderately resistant reaction in restricted irrigated conditions. The present study was first attempt to investigate the water stress-Macrophomina phaseolina interaction on different components of physiological, plant growth and yield parameters along with disease incidence. A significant reduction in total chlorophyll content (54.78 %), relative water content (47.06 %), membrane stability (51.75 %), sugar (69.00 %), protein (13.90 %) and proline (56.66 %) content was observed with inoculum density of 4 g/kg soil at soil moisture levels of 40 % and 60 %. The inoculum density of 4 g/kg of dry soil resulted in maximum mortality during seedling stage and growth period. Thus, varied inoculum density and water stress conditions made the sunflower plants more prone to dismissive physiological alterations. It led to biomass reduction viz; root length (41.27 %), root width (76.74 %), root weight (85.37 %), shoot length (56.71 %) and shoot weight (79.06 %). Thus it resulted in per plant seed yield reduction (95.45 %) at 52.62 to 100 % disease severity at higher inoculum density (4g, 6g, and 8g/kg dry soil). The expression levels of the defense related genes LOX, ACCO1, PAL, SOD, and APX were also modified under varying degree of water stress and inoculum density. The strong correlation between low soil moisture and disease severity implies that the proper irrigation of the field can be suggested as cultural management practices for the farmers.
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    Detection, variability and translocation of Potato virus Y (PVY) infecting potato in Punjab and identification of host plant resistance
    (Punjab Agricultural University, 2023) Belludi, Rakesh; Sharma, Abhishek
    This study aimed to investigate the variability of Potato virus Y (PVY) infecting potato crop of Rabi season in Punjab, encompassing symptomatology, serology and molecular aspects. The study revealed prevalent symptoms, such as mild mosaic, chlorotic patches, interveinal chlorosis, puckering, veinal necrosis and stem necrosis, on potato plants caused by various potato viruses including PVY. ELISA tests confirmed the presence of six out of seven viruses in potato samples. Among them, PVX exhibited the highest incidence (78.08%), followed by PVYN (51.37%), PVM (20.55%), PVYO/C and PVS (13.01%). PLRV exhibited a lower incidence (2.74%) and no samples tested positive for PVA. Notable regional variations noticed, with Ludhiana showing high incidences of PVYN (78.43%), PVYO/C (31.37%), PVM (33.3%) and PVS (35.29%). Conversely, Kapurthala displayed a 100% PVX incidence, while Jalandhar had the highest PLRV (19.05%) incidence. In nucleic acid-based detection through RT-PCR, the primer NIb2F/3R effectively amplified the desired ~350bp amplicon in all PVYN and PVYO/C seropositive samples. Whereas, the qPVY NIb F2/CP R2 primer generated the ~1.5kb fragment in only 24 out of 67 samples, further confirmed via BLASTn sequence analysis as PVYO, PVYNTN and PVYN-Wi strains. Further virus translocation studies revealed a wide range of carryover rates, spanning from 40% to 92.46%, from infected mother plants to daughter tubers, exhibiting significant variation in viral titre across genotypes and among tubers. Spatial translocation within the tuber's root zone revealed that the bottommost tuber displayed a relatively lower PVY titre. Interestingly, freshly harvested tubers exhibited a higher viral titre at the heel end (stem end) compared to the rose end eyes in all examined tubers. After storage, a contrasting trend emerged in sprouted tubers, where the highest PVY titre was found in rose end eyes and the lowest in heel end eyes. In germplasm screening against resistance to PVY, one germplasm (KP -16-19-14) displayed high resistance (HR), while 16 exhibited resistant (R), 20 moderately resistant (MR), 22 moderately susceptible (MS), 10 susceptible (S) and two were highly susceptible (HS) to PVY. Following PVY inoculation, significant reduction was observed in chlorophyll fluorescence parameters, viz., Fv/Fm, Y(II), qP, and qL values in susceptible cultivars, accompanied by notable increases in NPQ, Y(NO) and qN values. Susceptible genotypes displayed yield losses ranging from 13.64 to 58.84% with viral titre ranging from 0.014 mc to 419.673 mc. Finally, a farmer-friendly and cost-effective vertical flow assay (VFA) was developed for rapid PVY detection in potato leaf samples. The sensitivity of the developed assay was equivalent to commercially utilized ELISA. Specificity test confirmed assays precision for prevalent PVY strains and its non-specificity to other potato viruses. Developed assay exhibited 95% accuracy compared to RT-PCR.
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    ThesisItemRestricted
    Characterization of citrus greening pathogen(s) and its integrated management
    (Punjab Agricultural University, 2023) Katoch, Sonali; Arora, Anita
    Surveys conducted during 2019-21 to assess the distribution and intensity of greening disease in citrus orchards of Punjab revealed that its prevalence was high in the district Ludhiana (33.33%) followed by Hoshiarpur (32.60%), Fazilka (27.59%) and Faridkot (19.31%). The maximum mean disease incidence (16.60%) was observed in Hoshiarpur and the maximum mean disease index (11.98%) was observed in Ludhiana. Leaf mottling, yellowing, green islands and bi-coloured fruit symptoms of disease were observed on different cultivars of citrus. Multilocus sequence typing using 16S rDNA, CLIBASIA gene locus and tufB-secE-nusG-rplKAJL-rpoBC gene cluster was followed to identify and characterize the pathogen. Three primers (OA1/OI2c, CGB F/R and Las606/LSS) corresponding to bacterial 16S rDNA sequence were used on a total of 153 samples, and positive reactions were observed with all the three primer pairs. However, CGB F/R and Las606/LSS had higher detection rates (69.9%) than the OA1/OI2c primer (37.8%), indicating more robustness of the former. Similar results were observed with nursery samples of Punjab. The root samples from greening positive plants collected in the month of February were also found positive with five primer pairs (OA1/OI2c, CGB F/R, rplJ/K F/R, Las606/LSS and LapGP F/R) whereas, the root samples collected in May and September months showed no amplification. In addition, the CLIBASIA gene locus based primer pairs corresponding to sequences coding for hypothetical, heamolysin and type-I secretary system protein and one primer based on tandem repeats were used on 15 isolates of greening bacterium. All the samples showed positive reaction with five primer pairs. The disease was characterized by sequencing of 16S rDNA and CLIBASIA gene locus from five isolates with CGB F/R, three with Las606/LSS, two clones with OA1/OI2c and four with CLIBASIA gene locus which showed 99-100% similarity with reported isolates of Candidatus Liberibacter asiaticus (Ca. Las) from Asia. The phylogenetic analysis of these isolates showed 99-100% similarity with Ca. Las. The isolation of Ca. Las from leaves of PCR positive Kinnow plants was attempted on Liber A media, but it could not be cultured. The cultivars viz., Kinnow, Daisy, Musambi and Kagzi Lime were observed to be the best indicator plants for indexing. For integrated management of citrus greening, the treatment consisting of zinc sulphate + manganese sulphate + boric acid, tetracycline hydrochloride and 2,4-dichlorophenoxyacetic acid (2,4-D sodium salt) was proved significantly superior to other treatments in reducing the percent disease index (13.99) and providing 59.29 percent disease control with significant increase in yield (54.87 kg/tree) as compared to control (38.32 kg/tree).
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    ThesisItemOpen Access
    Mapping quantitative trait loci and biochemical analysis of sheath blight resistance in rice (Oryza sativa L.)
    (Punjab Agricultural University, 2023) Thesiya Mayur Rajeshbhai; Lore, Jagjeet Singh
    Sheath blight caused by Rhizoctonia solani Kühn, is one of the most prevalent diseases of rice in South East Asia. Use of resistant sources is an eco- and farmer friendly and more economically approach to mitigate yield losses due to this disease. The aim of the present study was to identify potential quantitative trait loci (QTLs) linked with resistance to sheath blight and associated with temporal spike in defense related enzymes as well as histopathological patterns during infection process of R. solani. Next generation sequencing (NGS) assisted bulk segregant analysis (BSA) was integrated with R package i.e. QTLseqr to map QTLs associated with sheath blight resistance in F3 mapping population derived from the cross between susceptible rice cultivar PR121 and resistant parent ShB1. The five QTLs namely qShB1, qShB3, qShB5.1, qShB5.2 and qShB6 were identified on chromosome 1, 3, 5 and 6, respectively. Activity of defense related enzymes such as phenylalanine ammonia lyase, polyphenol oxidase and peroxidase were estimated in parental lines along with three sets of progeny lines which showed significantly high activity in resistant parent as compared to the susceptible parent after R. solani inoculation. The moderately resistant (MR) lines expressed maximum enzyme activity than the highly susceptible (HS) lines. The pathogen R. solani exhibited more profuse and dense hyphal growth with close contact to surface grooves, lobate appressoria-like structures, higher number of microsclerotial development and longer lesion length on HS line as compared to the MR line. The present results provided better understanding of host-pathogen interaction and novel QTLs identified may be used in rice breeding program for the development of durable resistance to sheath blight in rice cultivars.
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    Pathogenic and genetic diversity in Fusarium species causing rice Bakanae in Punjab and its management
    (Punjab Agricultural University, 2023) Bag, Dipanjali; Hunjan, Mandeep Singh
    Bakanae disease, primarily caused by Fusarium fujikuroi has been reported from nearly all ricegrowing countries across the world, especially in Asian countries like India and is known to cause high yield losses. The disease incidence is especially high on Basmati rice cultivars Pusa Basmati 1121 and Pusa Basmati 1509. Studies on cultural and morphological characters of 25 isolates showed that, the isolates produced milky white, to cream colored and cottony to fluffy colonies. The size of the spores also varied from 3-21µm X 1-5µm for non-septate micro-conidia and 13-18µm X 2-7µm for macroconidia with 3-5 septa. Molecular identification and characterization of the isolates were carried using TEF-1α gene, ITS gene and RPB2 genes. All the isolates showed amplification, and these genes from four representative isolates from different districts of Punjab were sequenced. The BLAST and phylogenetic analysis of three isolates i.e., FR4, FR14, and FR24 showed 99-100% similarity with Fusarium fujikuroi, and one isolate (FR11) with Fusarium sacchari. Pathogenic diversity analysis of these isolates revealed that they produced typical Bakanae symptoms in the different basmati cultivars. The most virulent isolate of Fusarium was FR8 from Ropar, and the least virulent was FR7 from Badshahnagar (Patiala), with the highest disease incidence recorded in the most susceptible varieties Pusa Basmati 1121 and Pusa Basmati 1509. In molecular diversity analysis revealed, all the isolates showed positive amplification for FUM1 gene for Fumonisins production loci and 24 isolates for des gene (Gibberellic acid production loci). Sequence analysis of representative isolates showed 99-100% similarity with Fusarium fujikuroi. Genetic diversity of the isolates resolved using twenty SSR primers revealed high degree of polymorphism (PIC value ranging from 0 to 0.89) inferring intra- and interspecific genetic variation, clustering the pathogen population into two major clusters. It was observed that the pathogen does not survive in soil; infected seeds being the source of primary inoculum. The level of seed infection and age of nursery plays a very important role in development of Bakanae in both nursery and field. Maximum disease was observed when the level of seed infection was 30 per cent and when 35 days old seedlings were transplanted. The mitigation studies demonstrated that presowing seed treatment with either Sprint 75WS or Trichoderma harzianum greatly reduced the disease during Kharif 2021 and 2022. Fungicide sprays in field could also curb the seed infection in succeeding crop, however, these mitigation strategies were adaptable only upto a certain level of seed infection.