Management of postharvest anthracnose of banana using green nanoparticles

Abstract

A study entitled ‘Management of postharvest anthracnose of banana using green nanoparticles’ was conducted during 2021-23 at Department of Plant Pathology, College of Agriculture, Vellayani with the objective to characterize the major pathogen associated with anthracnose of banana fruits and its management using green nanoparticles. A survey was conducted in local markets of five agro-ecological units of Kerala viz., AEU 1, AEU 3, AEU 8, AEU 9 and AEU 12 covering Thiruvananthapuram, Kollam and Alappuzha districts. A total of 34 locations were surveyed during 2021-23 to collect the diseased specimens of banana Nendran variety (Musa AAB) and to study the symptomatology of the disease. Variations in symptoms were noticed from different locations such as black or brown sunken spots of various sizes, shriveling of fruits and spots having triangular shaped or angular edges. The pathogens were isolated from the collected specimens; a total of 62 isolates were obtained out of which 34 isolates were Colletotrichum sp. and Koch’s postulates were proved in matured harvested dehanded banana. All the 34 Colletotrichum sp. isolates were screened for its virulence and pathogenicity. The isolate K1B1 from Kollam corporation (AEU 1- Kollam district) recorded the highest Percent disease index (PDI) of 83.33 per cent with a highest lesion size of 5.10 x 4.95 cm on the 5th day of artificial inoculation; and hence concluded as the most virulent isolate. Cultural and morphological studies of isolate K1B1 were carried out. Initially white to grey floccose aerial mycelium was observed which turns orange colour with age. Microscopic studies revealed that mycelia were hyaline and septate, acervulus were brown without setae, conidia were hyaline aseptate with elliptical or cylindrical shape and appressorium were dark brown and irregular shaped. Based on the cultural and morphological studies, isolate K1B1 was identified as Colletotrichum musae. Further molecular characterization of the isolate K1B1 was done using ITS primers and the isolate was confirmed as Colletotrichum musae. In vitro evaluation of prepared essential oil nanoemulsions (NEs) viz., cinnamon oil NE, clove oil NE, basil oil NE, neem oil NE and mustard oil NE at 0.5 %, 1 % and 2 % against C. musae in PDA by poisoned food technique revealed that all the essential oil NEs significantly reduced the growth of C. musae over control. The highest inhibition (100 %) at the lowest concentration (0.5 %) was observed in cinnamon oil NE and clove oil NE followed by basil oil NE (96.66 %). In vitro evaluation of prepared green copper nanoparticles (CuNPs) synthesized using leaf extracts of neem, ocimum, clove, American mint, and cinnamon at 0.05 %, 0.1 % and 0.2 % against C. musae by poisoned food technique revealed that all the synthesized green CuNPs significantly reduced the growth of C. musae over control. The highest inhibition (100 %) at the lowest concentration (0.05 %) was observed in green CuNPs synthesized using leaf extracts of cinnamon and clove followed by green CuNPs synthesized using leaf extracts of neem (97.58 %). In vitro evaluation of chitosan NPs (60 nm) at 0.5 %, 1 % and 2 % in comparison with the best three treatments from essential oil NEs (cinnamon oil NE, clove oil NE and basil oil NE at 0.5 %) and green CuNPs (Synthesized using leaf extracts of cinnamon, clove and neem at 0.05 %) against C. musae by poisoned food technique revealed that the essential oil NEs and the green CuNPs significantly reduced the growth of C. musae over control. Chitosan NPs didn’t show any reduction in the growth of the pathogen.The highest inhibition (100 %) at the lowest concentration was observed in cinnamon oil NE (0.5%), clove oil NE (0.5%), green CuNPs synthesized using leaf extracts of cinnamon (0.05%) and clove (0.05%) followed by green CuNPs synthesized using leaf extracts of neem (97.58 % at 0.05%). Based on the results of in vitro evaluation, the best five treatments (cinnamon oil NE, clove oil NE, green CuNPs synthesized using leaf extracts of cinnamon, clove and neem) were taken for in vivo studies along with carbendazim (0.1 %), pathogen inoculated control and uninoculated control. All the tested green NPs significantly reduced the lesion formation in matured harvested dehanded banana. Lowest lesion size of 0.86 cm and 1.46 cm was recorded in green CuNPs synthesized using leaf extracts of cinnamon and clove respectively followed by cinnamon oil NEs (2.60 cm). Similarly, the highest percent disease reduction over control was observed in green CuNPs synthesized using leaf extracts of cinnamon (99.01 %) and clove (98.28 %) followed by cinnamon oil NE (97.56 %). The best three green NPs (Green CuNPs synthesized using leaf extracts of cinnamon and clove and cinnamon oil NE) from in vivo studies were characterized. The formation of green CuNPs synthesized were confirmed by a characteristic peak obtained at 800 nm by UV-Vis spectroscopy.The results from FT-IR (Fourier transform infrared spectroscopy) analysis conclude that the surface of synthesized CuNPs were capped and stabilized by flavonoids and other phenolic compounds in the leaf extracts. The morphological characterization of CuNPs using FESEM (Field emission scanning electron microscopy) and HRTEM (High resolution transmission electron microscopy) revealed the presence of spherical particles with some agglomeration and the size of the particles was found to be in the range of 20 – 60 nm. DLS (Dynamic light scattering) analysis was used to find out the surface charge of NPs and the negative zeta potential was found at -22.2 mV, -21.7 mV and -25.8 mV for green CuNPs synthesized using leaf extracts of cinnamon, clove and cinnamon oil NE respectively. The X-ray diffraction (XRD) analysis of green CuNPs revealed the crystalline structure of CuNPs. The shelf life and organoleptic properties (appearance with and without skin, colour with and without skin, texture, taste, flavor and overall acceptability) of the banana fruits treated with green nanoparticles was evaluated along with the uninoculated control and pathogen inoculated control and the green CuNPs synthesized using leaf extracts of cinnamon was noticed with highest shelf life (9 days) and excellent organoleptic properties followed by green CuNPs synthesized using leaf extracts of clove (8 days) and cinnamon oil NE (7 days). Based on the results of in vivo evaluation, the best three treatments (Green CuNPS synthesized using leaf extracts of cinnamon, clove and cinnamon oil NE) were taken for the study of biochemical changes (Reducing sugar, ascorbic acid, titratable acidity, protein, moisture and pH) in comparison with the uninoculated control and pathogen inoculated control. The green CuNPs synthesized using leaf extracts of cinnamon was noticed with less biochemical changes with a decrease in reducing sugar (8.30 %), decrease in ascorbic acid (62.56 mg/100g), decrease in titratable acidity (0.59 %), decrease in protein (5.08 mg/g fresh weight), increase in moisture (56.24 %) and acidic in pH (3.40) when compared with the uninoculated control with reducing sugar (8.87 %), ascorbic acid (62.77 mg/100g), titratable acidity (0.60 %), protein (5.25 mg/g fresh weight), moisture (55.88 %) and pH (3.41) followed by green CuNPs synthesized using leaf extracts of clove and cinnamon oil NE. The present study revealed that the major pathogen associated with anthracnose of banana fruits is Colletotrichum musae. The postharvest spraying of green CuNPs synthesized using leaf extracts of cinnamon at 0.05 % is proved to be an effective novel strategy for the management of banana anthracnose with higher shelf life (9 days) and excellent organoleptic properties. The results may be subjected to multi-location and multi seasonal field trials and the residual toxicity of CuNPs on the fruits have to be undertaken.