Silver nanoparticles green synthesized using leaf extract of Piper colubrinum for the management of foot rot in black pepper (Piper nigrum L.)

Abstract

The study entitled “Silver nanoparticles green synthesized using leaf extract of Piper colubrinum for the management of foot rot in black pepper (Piper nigrum L.)” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2022-2024 with the objectives to green synthesize silver nanoparticles with leaf extract of foot rot resistant Piper colubrinum, their characterization, test its efficacy in managing foot rot in black pepper (Piper nigrum L.) caused by Phytophthora capsici and estimation of defense related enzymes. Piper colubrinum and Piper nigrum leaf extract were used to synthesis silver nanoparticles (AgNPs), which were then physically characterized and tested for their antagonistic effect against P. capsici. Formation of silver nanoparticles was confirmed by colour change of the solution to dark brown after one hr incubation and by characterisation techniques. The green synthesized silver nanoparticles (GSAgNPs) using P. nigrum leaf extract (Pn-AgNPs) exhibited an absorbance maximum at 440 nm and GSAgNPs using P. colubrinum leaf extract (Pc-AgNPs) exhibited an absorbance maximum at 460 nm in UV Visible spectroscopy. The sharp peaks obtained in XRD analysis indicated the crystalline structure of the silver nanoparticles and the positions of the peaks suggest the presence of face-centered cubic (FCC) structures in both Pn-AgNPs and Pc-AgNPs. FTIR results revealed that more functional groups are present in Pn-AgNPs than Pc-AgNPs. FE-SEM showed the average size of Pn-AgNPs was 57 nm and that of Pc- AgNPs was 56 nm. The spherical morphology of Pn- AgNPs and Pc- AgNPs were revealed by HR-TEM images. Black pepper leaves with the foot rot symptoms of dark brown lesions with a fimbriate margin were selected for the isolation of the pathogen. The isolated colonies appeared as white to off white in stellate pattern on PDA medium. Mycelia were coenocytic with an average width of 3-8 μm. Lemon shaped papillate sporangia of the fungus with an average length 32-51 μm were observed microscopically. Molecular identification of the pathogen confirmed it as Phytophthora capsici and accession number of the culture is PP09178. The efficacy of Pn-AgNPs and Pc-AgNPs at different concentrations in inhibiting the growth of P. capsici was tested under in vitro conditions by poisoned food technique. Among treatments, commercial AgNPs, commercial chitosan nanoparticles (ChNPs), P. nigrum and P. colubrinum leaf extracts (5%) and copper oxychloride (0.2%), were also included. Highest in vitro mycelial growth inhibition (76.67 %) of the pathogen was observed with 750 ppm Pn-AgNPs followed by 750 ppm of Pc-AgNPs (73.33%). The better antifungal activity of Pn-AgNPs can be due to the type of phytochemicals present in P. nigrum leaf extract, which could provide better capping and stabilization of the silver nanoparticles. Copper oxychloride inhibited the mycelial growth completely. In detached leaf assay with black pepper variety Panniyur-1, lesion development was significantly reduced on leaves treated with the GSAgNPs (500 ppm and 750 ppm) compared to those treated with commercial AgNPs and ChNPs of the same concentrations on 3rd and 5th day after inoculation. No lesion was observed on leaves treated with 750 ppm of GSAgNPs. Leaf extracts (5%) were ineffective in disease suppression and pathogen growth. Mild symptoms were observed on leaves treated with the biocontrol agent (Pseudomonas fluorescens, 2%), while no symptoms were found on leaves treated with the fungicide. The best concentration of GSAgNPs (750 ppm) from in vitro assays was selected for in vivo pot culture study. In the in vivo assay, challenging with the pathogen after spraying the foliage of the foot rot susceptible black pepper variety Panniyur-1 with Pc-AgNPs at 750 ppm recorded the lowest percentage disease index (PDI) (15.55%) followed by that received 750 ppm Pn-AgNPs spray (17.77%), six days after inoculation. PDI for chemical control plants was 28.88% and that for plants treated with biocontrol agent was 37.77%. Highest PDI (77.77%) was observed for inoculated control plants. Peroxidase (PO), Phenylalanine ammonia lyase (PAL) and Polyphenol oxidase (PPO) from the treated plants were estimated on 2nd day, 4th day and 6th day after inoculation. Plants sprayed with Pc- AgNPs after pathogen inoculation showed the highest PO activity on the 4th day after inoculation (95.3 μg g-1min-1). Plants treated with 750 ppm of Pc-AgNPs after pathogen inoculation showed the highest PAL activity on 6 DAI (67.63 μg g-1min-1). The highest PPO activity was observed on 6 DAI and the corresponding values were in plants treated with 750 ppm of Pn AgNPs after pathogen inoculation (24.3 μg g-1min-1). The study revealed a significant increase in the defense enzyme activity in the plants sprayed with GSAgNPs (750 ppm), compared to the pathogen inoculated control plants without nanoparticle spray. This can be because of the action of GSAgNPs in triggering defense response. The present study suggests that AgNPs prepared with leaf extract of Piper spp. (Pn-AgNPs and Pc-AgNPs) effectively suppressed infection by P. capsici in black pepper at 750 ppm concentration, and these could be an effective and useful antifungal substitute for crop protection systems. However, multilocation field trials must be conducted to confirm the effectiveness of these nanoparticles. Studies to examine the residual toxicity of silver nanoparticles have also to be undertaken before field-level recommendations.