1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Viricidal activity of nanoparticles against Cowpea aphid borne mosaic virus in cowpea(Department of Plant Pathology, College of Agriculture,Vellanikkara, 2025-03-12) Anirudh, S.; Ayisha, RThe study entitled “Viricidal activity of nanoparticles against Cowpea aphid borne mosaic virus in cowpea” was carried out at the College of Agriculture, Vellanikkara during 2022-2024 to evaluate the antiviral activity of nanoparticles against Cowpea aphid-borne mosaic virus (CABMV) in cowpea. Cowpea leaf samples exhibiting symptoms such as vein-banding and mosaic patterns were collected from different locations in Vellanikkara (AEU10) and Palakkad (AEU23). The virus was maintained through mechanical inoculation for further studies. The virus is sap- transmissible and was mechanically inoculated using 0.01 M phosphate buffer. Successful transmission of virus by mechanical inoculation (98%) to the local lesion host, Chenopodium amaranticolor, and the propagative host, cowpea, was possible. The virus maintained in Chenopodium and cowpea was further isolated and characterized molecularly using RT-PCR. RT-PCR yielded an expected amplicon size of 812 bp with specific primers targeting the coat protein gene of Potyvirus. Sequence analysis through BLAST revealed 95.81% similarity with Cowpea aphid-borne mosaic virus (CABMV) and the sequence was deposited in NCBI with accession number PQ511232. Screening of nanoparticle (NP) formulations using silver nanopowder (SRL Catalog No: 44416) and silicon dioxide nanopowder (SRL Catalog No: 69294), was done to evaluate their antiviral activity against CABMV. Simultaneous inoculation of virus and nanoparticles (NPs), as well as pre-and post-inoculation of virus followed by NP foliar spray, was done to evaluate the efficacy of NPs at different concentrations. Direct inoculation/simultaneous inoculation of virus and NPs was done to evaluate the virucidal activity of NPs on the virus. In these studies, viral sap was mixed with NPs and incubated for 10 minutes before application to Chenopodium. A reduced number of diffused local lesions were observed on leaves inoculated with viral sap and NPs. A comparatively higher number of intact local lesions (34) were observed on plants treated with viral sap without NPs. Transmission electron microscopic (TEM) analysis of viruses from diffused and intact local lesions revealed aggregated viral particles in diffused local lesions. To further characterize viral particle aggregation, dynamic light scattering (DLS) analysis was performed on viral sap treated with NPs and untreated viral sap. The hydrodynamic radius of the particles in the NP- treated viral sap was found to be larger, with a mean size of 956.46 nm, compared to viral sap (512.56 nm) and nanosuspension alone (421 nm). The increase in size is likely due to the aggregation of viral particles on the NPs, facilitated by electrostatic attraction between the virus and the NPs. Pre- and post-inoculation studies using various concentrations of silver and silicon NPs on Chenopodium revealed that treatments T8 and T16, pre- and post- inoculation of the virus combined with a foliar spray of silicon NPs at 1000 ppm were themost effective, showing no local lesions compared to 34 local lesions observed in the viral control. This was followed by T14 (silicon NPs at 200 ppm), which also demonstrated significant efficacy. Pre- and post-inoculation studies on cowpea confirmed that foliar application of silicon NPs at 1000 ppm after challenge inoculation with the virus significantly reduced disease severity, with a zero-vulnerability index (VI), compared to 94.44 in the viral control. The reduction in disease severity was further validated by measuring the viral titre using DAS-ELISA with CABMV-specific antiserum (DSMZ PV RT–0417, DAS-ELISA set). Treatments T14 (Si 200 ppm) and T16 (Si 1000 ppm) showed 80% and 86% reduction in viral titre with less absorbance of 0.023 and 0.031 respectively. Based on the VI and viral titre, silicon 1000ppm was used for further studies. Various application methods for silicon NPs were standardized, and their efficacy was assessed in combination with the promising endophyte Bacillus pumilus VLY17. The applications include seed treatment, foliar application, and their combinations for silicon NPs at 1000 ppm. For Bacillus pumilus, seed treatment and soil drenching were done. A total of 19 treatments involving NP applications, both individually and in combination with Bacillus pumilus, were developed and tested. Based on the VI and reduced viral titre, treatment T9 (Bacillus seed treatment followed by Si NP foliar 1000ppm) was identified as the most effective. Evaluation of yield and plant growth parameters also highlighted T9 as the best performing treatment. T9 resulted in a 2.5-fold increase in pod yield per plant (138.7 g/plant) compared to the viral control (52.3 g/plant). Additionally, root length (56.5cm), fresh weight, and dry weight of both root (14.9g and 6.63g) and shoot (91.8g and 43.7g) were significantly higher than those in the viral control. Furthermore, T9 exhibited a substantially reduced viral titre (OD at 405nm -0.126) compared to the viral control (OD at 405nm-1.248). Analysis of defense related enzymes was conducted across all treatments to investigate the underlying mechanisms of reduced disease severity in treated plants. Treatment T9 showed a 21% increase in peroxidase activity (0.418 μmol/min/ml), a 46% increase in polyphenol oxidase activity (0.57 change in absorbance per minute/g), and a 2.36% increase in PAL activity (1.30 mg of cinnamic acid g-1 fresh weight) compared to viral control. Additionally, the activity of antioxidant enzymes, superoxide dismutase (SOD) and catalase, was analyzed. T9 exhibited enhanced SOD activity, with 358 units/ml at 20 days after treatment (DAT), and a 25% increase in catalase activity (121.42 enzyme units/g) compared to the viral control at 20 DAT. The research highlights the potential of silicon NPs in reducing disease severity caused by CABMV, promoting growth and yield parameters, and enhancing the activity of antioxidant and defense related enzymes. Seed treatment with Bacillus pumilus, combined with foliar application of silicon NPs at 1000 ppm, emerged as an effective management strategy for controlling CABMV in cowpea.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 Engineering banana bract mosaic virus resistance in banana by targeted editing of elF4E gene using crispr/Cas9 system(Department of Plant Biotechnology, College of Agriculture, Vellanikkara, 2024-05-23) Pandiaraj, S; Smitha NairItem 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.