PhD Thesis
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Item Management of banana bract mosaic virus using beneficial fungal root endophyte, Piriformospora indica(Department of Plant Pathology, College of Agriculture ,Vellayani, 2024-03-20) Sinijadas, K.; Joy, MThe research work entitled “Management of Banana bract mosaic virus using beneficial fungal root endophyte, Piriformospora indica” was carried out at the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during the academic year 2018-2022. The objective was to evaluate the beneficial fungal root endophyte, P. indica for the management of Banana bract mosaic virus (BBrMV); and to elucidate the role of antioxidants and plastids in this tripartite interaction. The characteristic symptoms of BBrMV viz., reddish spindle shaped streaks on pseudostem, black necrotic streaks on peduncle, chlorotic spindle lesions on leaves and underdeveloped fruits were observed during the survey conducted in five agro-climatic zones (ACZ) of Kerala. Percent disease incidence (PDI) and vulnerability index (VI) of the disease recorded from different zones showed the highest in banana var. Nendran (PDI - 61.66 & VI – 44.03) from southern zone and lowest in var. Poovan (PDI – 7.29 & VI – 6.29) in central zone. Serological and molecular detection confirmed the presence of BBrMV in banana var. Nendran. The sequence similarity analysis of the coat protein (CP) gene of BBrMV southern zone isolate (Vellayani) showed 99.12 percent genetic closeness to its Tamil Nadu isolates compared to the Kerala isolates (98.38 percent). Screening of the most virulent strain of BBrMV from five different ACZ of Kerala was done based on early symptom development in TC banana plants var. Nendran. The BBrMV strain from southern zone could produce the symptoms on var. Nendran within 30 days (lowest) followed by central zone (55 days) on artificial inoculation with viruliferous aphids, Pentalonia nigronervosa. The cross-infection study of virulent strain of BBrMV (southern zone) of Nendran on Nendran developed chlorotic spindle lesions on leaves at 30 days after inoculation followed by Nendran on Robusta (75 days). P. indica-colonization in banana var. Nendran was carried out using standardized medium. The chlamydospores of the fungus were observed in roots at 20 days after colonization (DAC). In both in vitro and in vivo experiments, P. indicacolonized plants showed reduced disease severity irrespective of the virusinoculation stage with a vulnerability index of 6.7 percent in BBrMV (+Pi / +V); and 20.0 percent in the virus-infected plants post-colonized with P. indica (+V / +Pi) compared to 53.3 percent in the virus alone infected plants. Further, PCR analysis with BBrMV coat protein specific primer yielded amplicon of low intensity in P. indica-colonized plants inoculated with the virus compared to the control plants indicating the ability of the fungus to inhibit the virus. Further, P. indica precolonized plants inoculated with BBrMV had improved growth and yield parameters compared to non-colonized plants. Field trial was laid out with two treatments (P. indica-colonized and noncolonized banana plants var. Nendran) at Instructional Farm, College of Agriculture, Vellayani. P. indica-colonized plants recorded a drastic reduction in the severity of BBrMV by 33 to 58 percent compared to non-colonised control plants. Enhanced plant height (30 percent), collar girth (45 percent), number of leaves (25 percent), leaf length (30 percent), leaf width (27 percent), fresh weight of shoot (34 percent), number of secondary roots (62 percent), number of tertiary roots (76 percent) and root weight (86 percent) were observed in P. indica-colonized plants at 90 days after treatment. P. indica-colonization also improved the bunch weight (32.9 percent) and fruit quality. Biochemical detection of superoxides using nitroblue tetrazolium (NBT) and H2O2 with diamino benzidine (DAB) stains at 5, 10, 15, 30 and 45 days revealed a reduction in reactive oxygen species (ROS) accumulation in both P. indica-colonized plants challenged with BBrMV (+Pi / +V) and virus infected plants post-colonized with P. indica (+V / +Pi) compared to BBrMV alone. The decrease in ROS production and disease severity in the endophyte-colonized plants inoculated with the virus were attributed to the increased activities of antioxidant enzymes viz., peroxidase, superoxide dismutase, catalase, glutamate synthase and ascorbic acid oxidase. The molecular analysis of genes involved in the symptom development indicated the beneficial effect of P. indica on BBrMV infection in banana. P. indica reduced the symptoms by up-regulating chlorophyll biosynthesis gene (chlorophyll synthase-CHLG) and down-regulating chlorophyll degradation genes (chlorphyllase CLH1 & CLH2; and pheophytin pheophorbide hydrolase - PPH), carotenoidbiosynthesis genes (phytoene synthase-PSY1 and PSY2), carotenoid degradation gene (Phytoene desaturase - PDS) and the virus specific genes responsible for symptom development (Hc-Pro and P3). Thus, the present study reveals that P. indica enhances tolerance against BBrMV in addition to improved growth promotion, yield and fruit quality in banana plant.Item Characterisation and management of sugarcane bacilliform virus (SCBV) causing leaf fleck disease in sugarcane(Department of Plant Pathology, College of Agriculture Vellanikkara, 2021) Sanju Balan; Anita CherianSugarcane (Saccharum officinarum) is a monocotyledonous perennial cash crop cultivated worldwide both under tropical and sub tropical conditions. It is being cultivated in more than 120 countries in the world. Like any other crops, it is also susceptible to biotic stress. Of which, diseases caused by viruses not only pose serious threat to sugarcane cultivation but also result in deterioration and exclusion of elite varieties of the germplasm. One of the major viral disease which affects global exchange of sugarcane germplasm is leaf fleck disease caused by Sugarcane bacilliform virus (SCBV). The research project entitled ‘Characterization and management of Sugarcane bacilliform virus causing leaf fleck in sugarcane’ was initiated with purposive sampling surveys in selected sugarcane fields in districts of Kerala and Tamil Nadu in order to document the symptoms under natural conditions, to assess the disease incidence, severity and to collect infected samples for further studies. The per cent disease incidence of the leaf fleck disease in Kerala ranged from 12 to 51 per cent whereas severity ranged from 10 to 36.5%. In Tamil Nadu the per cent disease incidence ranged from 28 to 56 per cent while severity ranged from 28 to 50.41%. Major symptoms observed on leaves were mottling, chlorotic flecks, chlorotic patches streaks and stripes with general yellowing of the canopy. In the case of severely affected clones, there was reduction in tillering, internodal length, number of internodes and appearance of deep longitudinal cracks. In highly susceptible clones, stunted growth with bunchy top appearance was noticed. On the basis of phenotypic variability of symptom expression, genotypes were classified into five groups. The development of the symptoms was also studied under artificial condition through insect transmission of the virus using pink mealy bug, Saccharicoccussacchari. Morphological characterisation of the virus done using electron microscopy revealed the presence of bacilliform virus particles of size 30 X 130–150 nm which indicated that the virus belongs to genus BADNA and family Caulimoviridae and the etiology of the disease was confirmed as Sugarcane bacilliform virus. The molecular detection of SCBV was also standardized through polymerase chain reaction (PCR). PCR amplification of RNaseH/RT gene was done using BADNA specific and SCBV129 specific primers. The amplicons were sequenced and in silco analysis of sequences showed sequence homology of 99 to 100 percent identity to SCBV. Widespread occurrence of the disease was observed even in the early generation of varietal development and in newly developed varieties. The transmission of the virus was suspected through true seed (fluff) developed by biparental crossing during sugarcane varietal development programme. Hence, the study was conducted to establish possible transmission of the virus from sugarcane parents to their progenies and the role of maternal and paternal parents in disease transmission through true seeds to the progenies. Samples from eight months old seedlings, three months old seedlings and parental clones were tested positive to the virus in PCR assays. Real time PCR was also standardized to assay these clones. Immunodiagnostic technique was validated using DAC ELISA. The technique of immunocapture PCR was also standardized. Minimal dilution of antisera with which SCBV could be detected was 2:1000 (V/V). Plant extract (antigen) at a dilution of 1:5 was found to be optimal for the detection of SCBV. Molecular detection of SCBV from mealy bug vector was also standardized. Both phenotypic and molecular methods were utilized to identify potential sources of natural resistance against SCBV. Based on the severity of symptom expression and PCR assays these were further classified as highly susceptible (HS), moderately susceptible (MS) moderately resistant (MR) and resistant (R). For generation of RNAi hair pin construct, initially forward (SF) and reverse primer (SR) were used to amplify 700 bp fragment of RT/RNase H gene to be cloned in sense orientation of the vector, pHANNIBAL. The linearized vector and the insert were ligated, and the ligation mixture was used to transform competent cells of Escherichia coli and the transformants were selected. Later antisense forward (AF) and reverse (AR) primer pairswere used to amplify 700 bp fragment of RT/RNase H gene to be cloned in antisense orientation. PCR product ligated into antisense direction of the vector and transformed into competent cells of E. coli. The recombinant pHANNIBAL vector was digested with restriction enzymes. The recombinant pHANNIBAL vector harbouringRNase H /RT gene was released from the vector through Not I site and subcloned into plant expression binary vector. Thus, cassette for RNA silencing was prepared.130 Meristem tip culture was also standardized with antiviral chemical tenofovir. Recovery percentage of meristem varied from 70 to 75 per cent and the viral load was quantified using real time PCR. The outcome of the study would facilitate early detection and elimination of the source of infection and prevent the spread of the disease in the field. Information generated in the study could be utilized while planning biparental crossing and reduce the spread of the virus in varietal development programmes. The hair pin construct developed in this study could be further utilized to generate transgenic disease resistant plants.