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Subject: search.filters.subject.Cassava mosaic disease

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    Role of microbes in the management of cassava mosaic disease
    (Department of molecular biology and biotechnology, college of agriculture , Vellayani, 2023-09-18) Athulya, V A
    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 11 2 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.
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    Role of microbes in the management of cassava mosaic disease
    (Department of molecular biology and biotechnology, college of agriculture, Vellayani, 2023-09-18) Athulya, V A.; Makeshkumar, T
    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 11 2 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.
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    Introgression of mosaic resistance in popular short duration cassava varieties of Kerala through marker assisted selection
    (Deparment of Plant Breeding and Genetics, College of Agriculture, Vellayani, 2018) Darshan, S; Arya, K
    The present study entitled “Introgression of mosaic disease resistance in to popular short duration cassava varieties of Kerala through marker assisted breeding”was conducted in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, Kerala Agricultural University and Division of Crop Improvement, ICAR- Central Tuber Crops research Institute, Sreekariyam, Thiruvananthapuram, Kerala during the period 2014 - 2017 with the core objective of introgression of cassava mosaic disease (CMD) resistance to short duration varieties of cassava through marker assisted selection (MAS) and to study the inheritance of early bulking nature. The research work was carried out as four experiments. In the first experiment, Five early bulking high yielding lines viz, Sree Jaya, Sree Vijaya, Vellayani Hraswa, CI 889 and 9S 75 and three testers viz, CR 54A3, IMS2-5 and CI 273 with resistance to cassava mosaic disease were selected and planted in a pollination block and crossed in Line x Tester (LxT) design to produce hybrid seeds of 15 F1 combinations. Experiment II was conducted in two parts. Screening of F1 seedlings for CMD resistance and early bulking nature was carried out in the first part of experiment II, where hybrids along with the parents were evaluated. Analysis of variance revealed significant differences among the genotypes for all the traits studied. All the agronomic traits were recorded and inheritance of early bulking and its correlation with other traits were studied. The CMD incidence expressed significant and negative correlation with tuber yield per plant where as significant and positive correlation for all other traits with tuber yield per plant was observed among the F1’s. As a part of experiment II (b), seedlings without the CMD visual symptoms were subjected to multiplex PCR and the results revealed that among the parents Sree Jaya, Sree Vijaya, Vellayani Hraswa expressed presence of Srilankan Cassava mosaic Virus (SLCMV) and Vellayani Hraswa expressed the presence of both SLCMV and Indian Cassava mosaic Virus (ICMV). Among the crosses, Sree Jaya x CR54 A3 (L1x T1), Sree Jaya x IMS2-5 (L1xT2), Vellayani Hraswa x IMS2-5 (L3xT2), CI 889 x CI 273 (L4xT3) expressed the presence of SLCMV. Real time PCR (qPCR) assay for seedlings identified CI 889 (L4), 9S 75(L5), CR 54A3 (T1), IMS2-5 (T2) and CI 273(T3) among the parents and Sree Jaya x CR54 A3 (L1x T1), Sree Jaya x IMS2-5 (L1xT2), Sree Jaya x CI 273(L1x T3) and 9S 75 x CR54 A3 (L5x T1), 9S 75 x IMS2-5 (L5xT2) and 9S 75 x CI 273 (L5x T3) among the crosses as highly resistant, based on viral load present in the DNA sample. Based on the previous report ten CMD resistance linked markers were screened through BSA and five of which SSRY 28, SSRY 44 SSRY 40, SSRY 106 and SSRY 235 were selected. Among the CMD linked SSR markers studied, the maximum polymorphism was elucidated by SSRY 28, SSRY 44 and followed by SSRY 235. SSRY 28 is a strongly linked marker to CMD2 which is a dominant gene conferring resistance among the clones of combinations (L1xT1, L2xT2, L3xT1 and L3xT3) three of five markers revealed alleles associated with CMD2 gene In the third experiment to evaluate the early bulking clones, field was laid out in randomized block design (RBD) with three replications consisting of CMD resistant clones along with parental clones using miniset technique. Analysis of variance revealed significant differences among the genotypes for all the traits. Measurement of heterosis was carried out considering parent Vellayani Hraswa (L3) as check and results revealed that standard heterosis was positive and significant in the combinations Sree Jaya x CR 54A3 (L1xT1) and Sree Jaya x CI 273 (L1xT3) for all the yield contributing traits. The crosses Sree Jaya x CR54 A3 (L1x T1) and Sree Jaya x CI 273 (L1xT3) exhibited negative standard heterosis for CMD. Combining ability analysis showed significant gca, sca variances and gca, sca effects for all the traits. Moreover gca/sca variance ratio indicated preponderance of dominance / non-additive gene action for the inheritance of all traits. Among the lines, Sree Jaya (L1) exhibited positive and significant gcaeffect for tuber yield and yield contributing traits. Among the testers, IMS2-5 (T2) exhibited negative and significant gca effect for CMD. Among the crosses Sree Jaya x CR54 A3 (L1x T1) exhibited positive and significant scaeffect for girth of tuber and stem girth, 9S 75 x CI 273 (L5xT3) exhibited positive and significant scaeffect for tuber yield per plant, CI 889 x CR 54A3 (L4xT1) exhibited negative and significant scaeffect for CMD. In the last experiment, through bulk segregants analysis using 5 SSR markers linked to early bulking in cassava were selected out of 9 SSR markers selected. Among 5 SSR markers of CMD and early bulking nature two SSR markers (SSRY 28 and SSRY 106) associated with resistance to CMD and One SSR marker, ESTs (SSRY) 292 associated to early bulking nature has been identified. Among the crosses, clones from Sree Jaya x CR54 A3 (L1xT1), Sree Jaya x CI 273 (L1x T3) and 9S 75 x CR 54A3 (L5xT1) are being confirmed with CMD resistance as well as early bulking nature.
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    Characterization of Bemisia tabaci (Gennadius) (hemiptera: aleyrodidae), for genetic variability, endosymbionts and vector-virus interactions in cassava
    (Department of Agricultural Entomology, College of Horticulture, Vellanikkara, 2018) Harish, E R; Mani Chellappan
    Cassava is one of the important tuber crops cultivated all over the World. Cassava Mosaic Disease (CMD) is the most important limiting factor in its production. Silverleaf whitefly, Bemisia tabaci (Gennadius) is the vector responsible for the transmission of Cassava mosaic virus in cassava, which causes CMD. Genetic variation among the members of B. tabaci, makes them very difficult to manage. Endosymbionts present in the whitefly system could be a factor responsible for making them a successful sucking pest. There are various kinds of interactions existing between whitefly and the CMV. Studying these interactions precisely will help to understand the behavioural and physiological variations in whiteflies. In this background the present study, “Characterization of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), for genetic variability, endosymbionts and vector-virus interactions in cassava” was proposed and carried out at the Department of Agricultural Entomology, College of Horticulture, Vellanikkara, during Mrch 2014 to April 2016, with the objectives to analyse the genetic variability in cassava whitefly, characterization of its endosymbionts and elucidation of cassava whitefly - cassava mosaic virus interactions. Various life stages of B. tabaci were collected from different cassava growing agro ecological zones of Kerala and reared in laboratory as well as in polyhouse at optimum conditions. Genetic variability study was conducted with 10 selected ISSR primers which had shown polymorphism in their banding pattern; with amplicon size ranged between 200bp to 2900bp. Phylogenetic analysis using NTsys software revealed the presence of two major clusters with Sultan Bathery population as out group. Similarity matrix had shown up to 49 per cent variation between the samples. Polymerase chain reaction using mitochondrial cytochrome oxidase1 primers, C1-J2195 and L2-N-3014 had given amplicon of 850bp. Nucleotide sequences had shown variation up to 16.5 per cent and dendrogram generated out of the sequences using MEGA-6 (Neighbor Joining Method) gave two clusters and one out group. Sequence similarity check using reference sequences from NCBI data base indicated the presence of two biotypes, AsiaI and AsiaII5 in cassava plants of Kerala. Morphometric studies were conducted to assess the variations in different pupal and adult characters of thirteen whitefly populations. Significant variations were found in pupal length and pupal width of the biotypes. Pupal length varied between 0.746 mm to 0.668 mm and pupal width varied between 0.539 mm to 0.468 mm in female pupa. Out of 14 characters of pupa studied, variations in length and width were found to be significant. Among seven characters of adults studied, variations in wing, antennal length, body length and width were significant. AsiaI biotype was found to have lesser body length, but more width compared to AsiaII5. AsiaII5 was found to be an important biotype of B. tabaci infesting cassava in 12 out of the 13 locations surveyed. Endosymbiont characterization from whitefly using Next Generation Sequencing (NGS) - Illumina platform revealed the variations in microbiota. At phylum level, Proteobacteria was found at 87.57 per cent in whitefly populations collected from plains. The populations from high ranges contained Firmicutes at 82.67 per cent. Arsenophonus, an ‘indirect helper’ for virus spread by protecting viral coat protein from degradation in insect system with their GroEL chaperones were found at 24. 69 per cent in B. tabaci populations collected from plains. Behavioural and life cycle variation study of B. tabaci using six cassava genotypes had shown that virus infection in B. tabaci altered the dispersal and settling. Speed of movement observed to be maximum at 16.25 cm/s in non- virulent female whiteflies on the genotype CMR-9. Life cycle of virulent and non-virulent whiteflies was found to vary between 19.57 days to 30.77 days. A thorough understanding of genetic variations, endosymbiont diversity and behavioural response to virus could help the researchers in planning proper management strategies for B. tabaci. In future, information generated of such kinds could also help the researchers and policy makers to foresee and manage any possible outbreak of the pest and avoid any havoc caused by them.
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    Molecular characterization of cassava mosaic disease (CMD) resistant varieties and wild relatives of cassava (Manihot esculenta Crantz) using SSR and SNP markers
    (Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Dhanya, O G; Mohan, C
    In the present study an attempt was made to estimate the extent of genetic diversity between 25 CMD resistant, 16 susceptible and seven wild relatives of cassava using 14 SSR and two SNP primers. Out of the 16 primers, nine primers was analysed using PAGE and remaining seven using capillary electrophoresis on genetic analyzer. The primers produced a total of 53 alleles across the 48 cassava accessions. NS198 was found to be highly polymorphic with 6 alleles followed by NS169, SSR36 and SSR39 (5allelles). The two SNP marker analysis on genetic analyzer namely SNPAPX3 and SNP ERF revealed that out of the two peaks generated, one of the peak at a range of 650-690 and 500-530bp respectively was common to all the 48 accessions and the other peak was variable between samples. The dendrogram constructed with 16 primers using UPGMA had four major clusters which clearly distinguished the resistant, susceptible and wild collections of cassava. The close observation made on one of the sub cluster within major resistant cluster revealed the resistant cultivars TME3 and TME4 were closely related with a similarity coefficient of 0.98. Clustering analysis was well supported by PCA, made representation of distinct location of CMD resistant, susceptible and wild relatives of cassava on 2D and 3D dimensions. Comparison of PIC value for all the 16 primers found out the PIC value for individual SSR markers is higher than the individual SNP PIC value at a range of 0.19 - 0.24. SSR PIC values ranged from 0.347 in SSR32 with observed heterozygosity (He) 0.447 to 0.72 in NS198 with He 0.76. Based on the PIC ranges NS198, NS169, SSRY39, RME-1, SSR106 and NS158 were selected as highly polymorphic markers.