Viricidal activity of nanoparticles against Cowpea aphid borne mosaic virus in cowpea

Abstract

The 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.