BSc-MSc

The study entitled “Role of microbes in the management of Cassava Mosaic Disease” was carried out at the Division of Crop Protection, ICAR- Central Tuber Crops Research Institute and the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani in the year 2022-2023 to analyse the role of microbial biocontrol agents in reducing the incidence of Cassava Mosaic Disease.
Infected cuttings of two susceptible cassava varieties H226 and Sree Prakash were collected from the fields at ICAR-CTCRI. They were identified to be infected with Cassava Mosaic Virus by visual observation of disease symptoms. DNA was isolated from the leaves of these plants by CTAB method and PCR was performed to confirm infection using the coat protein primer pair.
Ten isolates of Trichoderma asperellum and Bacillus sp. were used. They were cultured in Potato Dextrose Broth and Nutrient Broth respectively. The infected cuttings were immersed in the microbial suspensions and treated using a sett treating device for 15 min at 15 lbs pressure. They were then planted in trays in a mixture of coir pith and soil, along with untreated control. Six replicates were used for all.
Biometric observations like number of leaves, branch length and symptom scores were recorded for three months from the date of planting. DNA was isolated at one and three months after planting and PCR was performed to confirm infection. qPCR was performed on this DNA to check the viral load in the plants.
Three isolates of Trichoderma asperellum (T4, T7 and T8) showed reduction in viral load as compared to control in H226 plants. After the first month, plants treated with T4 had a viral load of 1.36x108 copies/10ng and T7 had 1.26x105 copies/10ng while control had 105 copies/10ng. After three months, plants treated with T4 had 53.2 copies/10ng, T7 had 58.5 copies/10ng and T8 had 68.5 copies/10ng while control had 3.9x109 copies/10ng.
Two isolates (B3 and B8) of Bacillus sp. showed reduction in viral load in Sree Prakash variety. One month after planting, plants treated with B3 (Bacillus subtilis) had a viral load of 1.27x109 copies/10ng while untreated control had 7.4x105 copies/10ng. After three months, plants treated with B3 had a viral load of 1.74x104 copies/10ng and those treated with B8 had 8.1x103 copies/10ng while control had 8.7x105 copies/10ng.
The biometric observation did not always show correlating trends. In Trichoderma treated H226 plants, the percentage changes in disease severity were 9.1% reduction for T7 and 10.9% reduction for T8 over control after one month. After three months, there was a 25% increase for T4, 8.3% increase for T7 and 4.2% increase for T8 in disease severity over control. In Sree Prakash plants treated with Bacillus sp., the percentage change in disease severity was 24% increase for both treatments B3 and B8 after one month, over control. After three months, the percentage change in disease severity was 26.75% increase with B3 over control.
Primers were designed for two plant defense genes ETR1 and PAL1 using Primer3 online tool for future study of the expression of these genes. This may help understand the mechanism of action of these biocontrol agents in reducing viral load.
Thus, certain isolates of Trichoderma asperellum and Bacillus sp. were found to have effective biocontrol activity against Cassava Mosaic Disease in this preliminary study. Further studies with a larger number of replicates and longer growth period is necessary for proper validation of the results.