Molecular analysis of phylogeography of cassava mosaic disease

Abstract

A study on “Molecular analysis of phylogeography of cassava mosaic disease” conducted at the ICAR- Central Tuber Crop Research Institute, Sreekariyam, Thiruvananthapuram during 2015-2016. During the study, a survey was conducted in all districts of Kerala to identify the different cassava mosaic viruses present in major cassava growing areas. The survey conducted revealed that cassava mosaic disease (CMD) is widespread in Kerala having high symptom severity and increased aggressiveness as compared to earlier years. Maximum intensity of CMD was observed in Wayanad followed by Malppuram, Alappuzha and Ernakulam districts. From the 53 fields visited across all districts, 115 samples were collected and their symptomatology was recorded. Serological tests like ELISA and DIBA were done to detect the presence of cassava mosaic viruses in them and all the samples were further diagnosed using multiplex PCR to differentially detect ICMV and SLCMV. From the 115 samples, 9 samples were ICMV positive, 68 samples SLCMV positive, 18 samples had both ICMV and SLCMV (mixed infection) and 20 samples had neither ICMV nor SLCMV infection. These 20 samples were selected and PCR were employed using specific primers of ACMV, EACMV, UgV to detect any such virus presence and the results showed that 3 samples from Pathanamthitta had suspected ACMV infection which were also positive for SLCMV. From PCR analysis, it was revealed that SLCMV is widespread in all districts of Kerala while ICMV infection was restricted to 9 districts only viz., Thiruvananthapuram, Kollam, Alappuzha, Kottayam, Idukki, Ernakulam, Palakkad, Malappuram and Kasargod. To identify the variation of cassava mosaic viruses within different districts, each ICMV and SLCMV representative samples from all districts were used for PCR-RFLP analysis using 6 different restriction enzymes viz., EcoRV, HindIII, XhoI, ClaI, BamHI and KpnI. The results revealed that SLCMV samples from districts viz., Kollam, Pathanamthitta, Kottayam, Idukki, Kannur and Kasargod shown variation within the EcoRV, HindIII, XhoI, ClaI sites wheras ICMV samples from Kollam and Malappuram showed variation within HindIII and XhoI sites. To identify the molecular level variations within the conserved region of both ICMV and SLCMV, multiplex PCR amplified products of 5 representative samples (3 ICMV and 2 SLCMV) were sequenced and the result indicates that the SLCMV samples had more variability than the ICMV samples and also more number of SNPs were found within the conserved region of all these samples. Whole genome amplification of selected samples was done using RCA to identify the variation within the DNA-A genome and the sequence result disclosed that high level of variability is present in the form of SNPs in the conserved Rep region within DNA-A of selected samples. To identify the phylogenetic relationship of the sequenced samples with that of available accessions, dendrograms were made using MEGA 9.0 software and the tree showed that sequences has variability eventhough lies within the group. To identify the presence of beta satellite molecules in the samples collected during the survey, multiplex positive samples and samples showing unusual symptoms and increased aggressiveness were selected and PCR was employed using beta specific primer. Only one sample (Thiruvananthapuram mixed infection) shown positive results for suspected beta molecule and this is the first report of identification of beta satellite molecules associated with cassava mosaic disease in India making the situation more crucial.