Browsing by Author "Virendra Kumar Baranwal"
Now showing 1 - 1 of 1
Results Per Page
Sort Options
ThesisItem Open Access Use of conventional and deep sequencing methodologies to understand the etiology of leaf crinkle disease in urdbean (Vigna mungo L.)(Division of Plant Pathology Indian Agricultural Research Institute New Delhi-, 2019) ABHISHEK KUMAR DUBEY; Virendra Kumar BaranwalVigna mungo (L.) Hepper, (urdbean) is an important leguminous crop of Indian subcontinent severely affected by Urdbean leaf crinkle disease (ULCD). The disease is characterized by the appearance of extreme crinkling, curling, puckering and rugosity of leaves, stunting and malformation of floral organs. Most recently, viruses such as CPMMV, SoYMMV, GBNV and MYMIV were found associated with urd and mung field samples showing leaf crinkle symptoms using next-generation sequencing approaches, but constant association of viruses has not been established yet. During our study, we studied epidemiology and etiology of the disease by multipronged approaches from conventional to recent advanced techniques.The disease has significant effect on seed characteristics such as 23.7 % to 26.1% reduction in seed index was observed in diseased plants compared to healthy while only 58% germination was observed for diseased seeds. Temperature influence on symptom expression was determined by mechanical inoculation in seeds and exposure to a set of temperature under glasshouse conditions and it was found that temperature range 30–35°C was most favorable for symptom development. Non-linear beta model study revealed potential ULCD distribution in central India and northern India are prone to severe and low leaf crinkle disease during kharif and summer season respectively. Prediction of potential geographical region for small disease incidence is a key component in tactical management of the ULCD under changing climates. The disease is reported to be transmitted through sap, seed, grafting and wide range of insect vectors. In present study, the rate of seed transmission was found upto 100 %. To find out the association of viruses viz.,CPMMV, SoYMMV and GBNV, a large number of symptomatic field samples from Delhi were tested using electron microscopy, ELISA and RT-PCR, but could not find a consistent association of any of these viruses even on severely symptomatic samples. A culture of ULCD was established by seed transmission in the greenhouse under controlled insect-proof conditions. To test the samples from glasshouse for the presence of already reported and unreported viruses again TEM, ELISA, PCR and RT-PCR was done. But no confirmation of viruses associated with ULCD was found. The next ϭϯϭ generation sequencing using Illumina platform of small RNA and transcriptome was also done. The small RNA sequences from samples collected from the greenhouse culturewas aligned with all known viral sequences present in the GenBank both manually as well as using programs specially designed to identify viruses. None of the reads were matching with any of the known plant viruses including the ones which were already reported (CPMMV, SoYMMV and GBNV). The possibility of novel viruses was also checked using conserved domain searches, but could not find any virus related sequence. However, most of the reads were matching viroids in all symptomatic samples, collected both from field and greenhouse. The consensus sequences identified from reads matching viroids had extensive secondary structures, high G-C content and the conserved domain, which is signature evidence of association of viroids. Few of the reads were also aligning with viroids from healthy samples, but were insignificant compared to that of symptomatic samples. Possible association of viroids identified from small RNA sequencing was supported by findings from two separate experiments. The experiments include thermal inactivation point determination which lies between 70°C and 75°C and upto 100% seed transmission. The high thermal inactivation point due to extensive secondary structures and seed transmission are characteristic of viroids. Thus our findings strongly point to viroid etiology of the disease. In response to pathogenic infections, physiological changes are often observed in plants. In the present study, the fundamental understanding of urdbean response to ULCD infection was extended through comparing transcriptome changes between healthy and ULCD infected urdbean. Transcripts were assigned with GO terms and functionally categorized into three ontology group viz., biological process, molecular function and cellular component.Transcriptome analysis revealed that a total of 727 genes were found to be differentially expressed out of which 460 were up-regulated and 267 were downregulated. Out of total up-regulated genes 74 genes were of unknown functions while 42 genes with unknown function among down-regulated genes. It is evident from our study that no virus association with ULCD, every possibility of association of viroid which needs further validation and characterization. ϭϯϮ Keywords: ULCD, sequencing, etiology, crinkle, transcriptome, small RNA, seed transmission, thermal inactivation point, potential geographical distribution, viroid