MSc

Cassava (Manihot esculenta Crantz), the staple or subsidiary food for about one fifth of the world’s population, is an important source of dietary calories. Among the diseases and pests of cassava, cassava mosaic disease (CMD) is a serious factor limiting the productivity of cassava and the variety, Vellayani Hraswa is found to be highly susceptible to the disease. CMD is caused by viruses belonging to the genus Begomovirus of the family Geminiviridae. Since the crop is vegetatively propagated, the CMD is carried from one crop cycle to the next, through the use of infected cuttings used as planting material.
Studies were conducted for the diagnosis of cassava mosaic geminivirus and production of disease free planting material of cassava variety, Vellayani Hraswa causing the mosaic disease of cassava in Kerala. The characteristic symptoms observed include chlorotic areas on the leaf and bright mosaic pattern of yellow or pale green patches. In severely affected plants, leaf distortion and twisting, reduction in size of leaf lamina and stunting of plant growth were observed. Host range studies were conducted and the virus was found to infect only Datura stramonium belonging to the Solanaceae family. The sap transmission of the virus using 0.1 M sodium phosphate buffer (pH 7.0) was found not successful from cassava to cassava. Chlorotic lesions were produced on the local lesion host, Datura which later turned necrotic. Insect transmission studies revealed that only whiteflies (Bemisia tabaci) were able to transmit the virus whereas the spiralling whiteflies (Aleurodicus dispersus) and mealy bugs (Ferrisia virgata) associated with cassava could not transmit the virus. Graft transmission of the virus was also found successful.
The pathophysiological studies revealed that the total carbohydrate and phenol contents in graft inoculated plants were significantly higher compared to the uninoculated cassava plants. The content of chlorophyll a, b and total chlorophyll were lower in inoculated cassava plants. There was a decrease in total protein content in inoculated plants compared to the healthy plants but was found to be increasing at different stages of inoculation. The activity of the defense related enzymes were higher in case of inoculated plants than that of the control plants. Protein profile study of virus infected cassava plants using SDS- PAGE showed an extra novel protein with approximate molecular weight of 40 KDa corresponding to the coat protein of the virus. Isozyme analysis of polyphenol oxidase produced three isoforms in both healthy and inoculated plants with relative mobility (Rm) values of 0.68, 0.75 and 1. Two isoforms with Rm values 0.63 and 0.74 were observed in case of peroxidase isozyme analysis in inoculated plants whereas only one in healthy plants. The activities of the isoforms were more in the inoculated plants.
Serological and molecular characterization of the virus was also done. The virus was partially purified from the infected leaves and antiserum with a titre of 1: 512 was produced. Serological characterization of the virus using ELISA showed the close relationship of ACMV and ICMV with the disease. Presence of the virus in the whitefly vector B. tabaci was confirmed through DAC- ELISA. Detection of the virus was also done using Dot Immunobinding Assay (DIBA). A field level diagnostic kit using DIBA was developed. Multiplex PCR using ICMV and SLCMV specific primers produced an amplicon of size 600 bp corresponding to the DNA- A fragment of SLCMV. Sequencing of the PCR product obtained 584 bp long nucleotide sequences. The cluster dendrogram constructed by multiple alignment of sequences showed three major clusters and the isolated viral sequence shared the same cluster with SLCMV isolates collected from Kerala.
The meristem from the infected cassava plants were regenerated into complete plantlets within 30 days in a suitable MS media supplemented with benzyladenine (0.5 ml l-1), naphthalene acetic acid (0.1 ml l-1) and gibberellic acid (0.1 ml l-1) of millimolar concentration. Root induction was better in the basal MS media and was tested for the presence of the virus by subjecting it to DAC- ELISA. The absorbance of the plantlet regenerated from healthy and infected meristem were found to be 0.22 and 0.31 respectively which was on par with the healthy field sample (0.28) but much lower than that of the infected field sample (1.23) which was used as the positive control. The virus free plantlets were transferred to pots containing sand, soil and compost in 1:1:1 ratio and were grown in a green house.
Meristem culture and virus indexing by use of protein and nucleic acid based detection thus, can be used as a viable strategy for the early detection and elimination of the virus and hence for the production of virus free planting materials.