Research Projects

The aims of the research proposal are: (a) develop a simple and cost-efficient diagnostic immuno-strip based diagnostic kit in dip-stick format for detection of CTV strains in infected plant parts both in nursery and orchard; (b) Identification of CTV strains from various hotspot regions of NER and development of artificial Agroinfectious dimeric clones for Agroinoculation in various Citrus germplasm for testing CTV susceptibility; (c) development of a LAMP-based Point-of-care diagnostic kit for detection of Phytophthora Gummosis, Canker and citrus greening (HLB) through systematic genetic diversity studies and (d) formulation of liquid synthetic microbial consortium’ for addressing these diseases.

An extensive survey was undertaken during 2021-22 and 2022-23 across major citrus-growing regions of Northeast Indiax, Meghalaya, Tripura, Manipur, Arunachal Pradesh, Sikkim, Nagaland and Assam, along with Punjab, Haryana and Delhi to investigate the causal agent of citrus root rotand gummosis. A standardized isolation method for Phytophthora spp. was developed using young lemon-leaf baiting on corn meal agar supplemented with pimaricin(10μg/ml), ampicillin (250μg/ml), rifamycin (10μg/ml) and carbendazim(300μg/ml).A total of 101 isolates were successfully obtained, maintained on carrot agar and preserved in sterile water at 4 °C. Morphological characterization revealed fast-growing colonies with chrysanthemum and rosette patterns and whitish blooming mycelia. Pathogenicity assays on detached Kinnow leaves and fruits using a large number of isolates confirmed the disease-causing ability of several strains, and a standardized pathogenicity protocol for host plants was established. Molecular characterization using ITS-based sequencing placed there presentative isolates in a stable clade with Phytopythium vexans strain CBS 119.80. Conserved target sequences for the Citrus Tristeza Virus(CTV)have been identified. We conducted a literature survey and laborator yexperiments to evaluate previously validated LAMP primers. Currently, we are in the process of designing custom primers for strains from the North Eastern region. Optimization of the LAMP reaction is ongoing, and initial laboratory validations are planned. 46 number of HLB disease samples have been collected covering districts from four different North-Eastern states (Meghalaya, Assam, Arunachal Pradesh, Tripura, and Mizoram). CLas specific LAMP primers have been procured and optimization with gDNA of CLas collected from IARI New Delhi has been carried out. Molecular characterization of CTV isolates confirmed CTV infection in 6samples from Assam and Tripura by RT-PCR detection ofP25coatproteingeneandP23gene. These two genes of CTV (P25 and P23) were successfully cloned. Sequence analysis of these CTV genes revealed high similarity of Assam isolate with Bhutan isolate, and Tripura isolate with Sikkim isolate.