Phytomediated zinc oxide and sulfur nanoparticles for management of soft-rot causing pathogenic fungi in ginger Pramod U. Ingle a, Mahendra Rai a,b, Patrycja Golińska c, Aniket K. Gade a,c,d,* a Nanobiotechnology Laboratory, Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati, Maharashtra, India, 444602 b Department of Chemistry, Federal University of Piaui (UFPI), Teresina, Brazil c Department of Microbiology, Nicolaus Copernicus University, 87-100 Torun, Poland d Department of Biological Sciences and Biotechnology, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai, Maharashtra, India, 400019 A R T I C L E I N F O Handling Editor: Dr. Ching Hou Keywords: Nanofungicides Pythium Fusarium Aspergillus Zone of inhibition Minimal inhibitory concentration A B S T R A C T Diseases management has been the greatest challenge to agriculture leading to huge crop failure, represented in low-profit production. Phytopathogenic fungi are among pathogens causing a variety of agricultural diseases. Various nanoparticles (NPs) have previously been employed to combat phytopathogenic fungi. Post-harvest deterioration due to fungal invasion is the most imperative cause of loss of ginger (Zingiber officinale) production. In the present study, four fungal pathogens isolated from soft-rot infected ginger were identified as Pythium spp. (isolate I and II), one Fusarium sp. (isolate III) and one Aspergillus sp. (isolate IV). Moringa oleifera mediated zinc oxide (ZnONPs) and sulfur nanoparticles (SNPs) were characterized by UV–Visible spectropho tometry and showed absorption maxima at 356 nm and 295 nm. Average size of NPs under Nanoparticle Tracking Analyzer was 78 nm and 26 nm respectively, confirmed by FESEM. Stable NPs with zeta potential of − 14.1 mV (Standard deviation = 4.51 mV) and − 21.1 mV (Standard deviation = 9.56 mV) were synthesized. FTIR detected the presence of various functional groups in NPs. In vitro antifungal activity was assessed by Kirby-Bauer disc diffusion assay and MIC values compared to antibiotic ketoconazole and fungicide mancozeb. Time kill assay represented the minimum time required by selected NPs to inhibit the growth of test pathogenic fungi. ZnONPs and SNPs can be used for further antifungal studies in the field as novel nanofungicides. Extrapolated and uncontrolled use can lead to the inhibition of germination, reduction in photosynthesis rate, and disruption of the plant roots. Thorough small-scale experimentation is necessary before commercialization. 1. Introduction Ginger (Zingiber officinale Rosc.) (Family: Zingiberaceae) is a perennial herb. The ginger rhizomes are consumed as a spice. India being a foremost producer of ginger, during 2012–13 the production in country was 7.45 lakh tonnes of ginger from 157,839 ha area. Ginger is cultivated in almost all the states in India. However, states mainly Karnataka, Meghalaya, Orissa, Arunachal Pradesh, Assam, and Gujarat contribute 65% to the country’s production (ICAR- IISR, 2013; IISR-Annual ICAR-IISR-Annual Report, 2022). * Corresponding author. Nanobiotechnology laboratory, Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati, Maha rashtra, India – 444602. E-mail addresses: aniketgade@sgbau.ac.in, aniket.gade@umk.pl, ak.gade@ictmumbai.edu.in (A.K. Gade). Contents lists available at ScienceDirect Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab https://doi.org/10.1016/j.bcab.2024.103229 Received 1 February 2024; Received in revised form 1 May 2024; Accepted 13 May 2024 Biocatalysis and Agricultural Biotechnology 58 (2024) 103229 Available online 17 May 2024 1878-8181/© 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). mailto:aniketgade@sgbau.ac.in mailto:aniket.gade@umk.pl mailto:ak.gade@ictmumbai.edu.in www.sciencedirect.com/science/journal/18788181 https://www.elsevier.com/locate/bab https://doi.org/10.1016/j.bcab.2024.103229 https://doi.org/10.1016/j.bcab.2024.103229 http://crossmark.crossref.org/dialog/?doi=10.1016/j.bcab.2024.103229&domain=pdf http://creativecommons.org/licenses/by/4.0/ Ginger is an essential spice crop in India and world as well. Besides it use in preparation of curry recipes, ginger is widely used in processed and unprocessed foods, medicines and cosmetic products. Ginger as a nutraceutical product is used in the pharmaceutical and food industries for a long time. Growing health concerns among all classes of people and awareness on the use of ginger in pharmaceutical products, the demand for organic as well as value-added ginger is increasing with time. Ginger is mostly cultivated by the marginal and small farmers in India and it is cultivated in small patches of land. Potential increase in ginger production in the country is needed for changing the socio-economic situation of a section of people through ginger farming (NAIP, 2014). Globally, many countries are also facing problems during the cultivation and post-harvest storage and maintenance of ginger. In the case of country like Ethiopia, due to higher chances of fungal attacks, production of ginger is lowered. There are few varieties available around the world, limiting their extensive species based yield development studies. Majority of the cultivation methods are conventional accompanied by the recent intrusion of bacterial wilt in the agricultural field (Zakir et al., 2018; Kifile et al., 2023). Sri Lanka being a minor producer of ginger, land availability has always been a constraint. In addition, marketing issues, unavailability of quality seeds, higher inputs values, scarcity of knowledge of cultivation and proper procedures of post-harvest handling and storage unfortunately limit the ginger production (Adegboye, 2011; Dewanarayana and Wimalaratana, 2018). Post-harvest damage and decay are the most significant reasons for the loss of Zingiber officinale production which is mainly due to fungal attack. Various Pythium spp. And the Fusarium spp. Are the major phytopathogenic fungi causing soft-rot in ginger (Sharma et al., 2022; Tilahun et al., 2022; Nejad et al., 2023). Despite the lack of recent data, during the year 2012–2013 the country produced 0.745 million tons of ginger from 157,839 ha of land (Jayashree et al., 2016; Prasath et al., 2023). Plant pests and pathogens cause projected worldwide losses of 20%–40% per year (Flood, 2010). Thus, there is an increased motivation to develop high-performance and cost-efficient, control agents that are less toxic to the environment. Fungi are the main cause of post-harvest deterioration in ginger. Post-harvest spoilage of ginger can be reduced by proper growing methods, harvesting, and post-harvest handling techniques. The aforementioned problems with ginger production can be addressed with the development of nanotechnology. Nanotechnology in agriculture is presently being explored in the delivery of plant hormone, water management, seed germination, targeted gene transfer, nanobarcoding, nanosensors, and controlled release of agrichemicals. Researchers have engineered nanoparticles (NPs) with antici pated properties, such as pore size, shape, and surface functionalities, to use them as protectants or for targeted and precise dissemination via encapsulation, adsorption, and/or conjugation of a dynamic ingredient, such as a pesticide (Khandelwal et al., 2016; Hayles et al., 2017; Mahmad et al., 2023; Nevado-Velasquez et al., 2023). A new generation of fungicides is being developed in agricultural nanotechnology that has potential to manage plant diseases. The most usually explored nanoparticle carriers are silica, polymer mixes, and chitosan. Various fungi are continuously tested for antifungal efficacy of the nanofungicides against them (Worrall et al., 2018; Yadav et al., 2023a). Amid the different microbial pathogens, phytopathogenic fungi are main cause of potent diseases in agriculture. Fungi easily adopt with any medium and are capable of growing different substrates or media in precarious environmental surroundings. They can disturb different growth stages of the crop, right from sowing to vegetative and from production to postharvest. Today, phytopathogenic fungi are controlled with chemical fungicides, which are costlier and can be obtained from the market. They have resulted the development of fungi with more resistance, becoming sturdier against chemical products. However, their undiscerning use have created several problems such as pollution of environmental factors, diseases in animals and humans, and ecological imbalances. Such as, aquatic habitats are contaminated via wastewater and agriculture use discharges. While the non-point sources like drift, drainage and surface run-off can cause water pollution (Bereswill et al., 2012; Zubrod et al., 2019). In case of wetlands, removal efficacy of fungicides also plays a vital role in damaging environment (Gikas et al., 2022). Traditional methods of controlling mycological infections in ginger crop includes chemical methods, physical methods, and bio logical methods. The physical method involves suppressive soil with higher clay content and lower pH for cultivation, mulching of soil via soil solarization, covering moist fields with plastic sheets, followed by phyto-sanitization (Bennett et al., 1991; Chérif et al., 1994; Dake, 1995; Rai et al., 2018). Chemical method applies various synthetic fungicidal chemical agents like metalaxyl (fosetyl-alumi num/Ridomil), Apron 35 WS, Dithane M 45, or their combination as Chloropyrifos 20 EC + Mancozeb 80 W P (Diathane M-45) and Carbendazim 50 DF (as Bavistin) for synergistic activity (Luong et al., 2010; Gautam and Mainali, 2016; Rai et al., 2018). Biological approaches includes the application of plant extracts like M. oleifera, Azadiracta indica, Aegel marmelos, etc. For controlling ginger soft-rot (Parveen and Sharma, 2014). Various bacterial species like Bacillus, Rhizobium, Pseudomonas, etc. And fungal species like Fusarium and Trichoderma are used as biocontrol agents in ginger soft-rot management (Yadav et al., 2023a). M. oleifera leaf and seed extracts has been reported for its fungistatic potential against many crop pathogenic fungi like Botrytis cinerea (He et al., 2011; Ahmadu et al., 2021). Also, the NPs synthesized using M. oleifera extracts reduce the radial growth of fungi (Jenish et al., 2022). These studies imposed a choice of moringa for the production of NPs in the present study. Nanotechnology offers an alternate eco-friendly method for plant disease management and overcomes drawbacks associated with ecotoxicity. Plant-based synthesis methods are called as green technologies as they are less polluting, cost-effective, helps in ensuring human health and environmental safety (Ying et al., 2022). Nanomaterials can increase crop development and yields, improve identification and management of pests and diseases, helps in genetic modification of plants, and ease in postharvest managements (Ghidan and Al Antary, 2020). Active agrochemicals are delivered in agriculture fields in a smart way using nanotechnology (Gogos et al., 2012). Thus, sustainably strengthen the new paradigm in agriculture and alleviate prevailing complexities (Fraceto et al., 2016). Various NPs are applied in agriculture, and their use depends upon their mode of action. The most widely used NPs are silver, titanium, gold, copper, zinc, aluminum, chitosan, silica, and sulfur (Ghazy et al., 2021; Banerjee and Rajeswari, 2024; Sabir et al., 2014; Mustafa et al., 2023; Sivakumar et al., 2024). There is a need of a sustainable product with antifungal activity with minimal losses of the product and the least damage to the environment. Nanotechnology provides a sustainable and advanced approach to replace con ventional fungicides with biogenic nanoparticles. The present study encompasses the in vitro evaluation of zinc oxide and sulfur NPs P.U. Ingle et al. Biocatalysis and Agricultural Biotechnology 58 (2024) 103229 2 against fungal pathogens in ginger and their management (Tandon et al., 2023). The present study will play an important role in management of plant pathogenic fungi and related infections based upon the plant mediated nanoparticles in a sustainable way, and with future studies aimed at developing nano-based antifungal formulation for agriculture application. 2. Materials and methods 2.1. Survey and sample collection A market survey was made alongside the field survey in pursuit of soft-rot infected ginger for isolation of fungal pathogens. The infected ginger samples were collected from the street vendors and agro-food markets of Amravati district. Some ginger samples were collected from the nearby agricultural fields of farmers (Location coordinates: 20◦ 56′ 14.7264″ N and 77◦ 46′ 46.3764″ E). In total about 35 samples of ginger were collected. The ginger samples were further subjected to surface cleaning and fungal pathogen isolation and identification as mentioned in section 2.2. 2.2. Isolation and morphological identification of fungi Ginger rhizome samples (100 g) were washed with sterile distilled water followed by surface sterilized by using 0.1 % sodium hypochlorite for 3 min and followed by three times successive sterile distilled water washes. Ginger samples were washed with sodium hypochlorite to remove any bacterial contaminants on the ginger surface that could interfere with the isolation of fungal pathogens from inside the ginger. The samples were chopped into small, thin slices of 1–2 mm thickness with the help of a sterile knife or a scalpel. The ginger slices were aseptically inoculated onto a Potato Dextrose Agar (PDA) (pH, 5.6) using sterile forceps. The plates were incubated for 4 days at 25 ◦C. When the mycelia arise, the organisms were sub-cultured on fresh sterile agar media to get a pure culture of each isolate. Ginger decomposing fungi were isolated and identified by microscopy (Fig. 1). Seven to fifteen-day-old fungal cultures grown on PDA were aseptically taken using sterile inoculating needle, mounted on to clean microscopic slide and stained using lactophenol cotton blue (HiMedia, Mumbai, India). Identification of fungi was carried out to the genus level based on morphological characteristics Fig. 1. Fungi isolated from soft-rot infected ginger, (A) Pythium isolate I, (B) Pythium isolate II, (C) Fusarium oxysporum isolate III, (D) Aspergillus isolate IV. P.U. Ingle et al. Biocatalysis and Agricultural Biotechnology 58 (2024) 103229 3 using compound microscope and colony characteristics on PDA and compared with structures in Samson et al. (2009) and Dugan (2006) and colored plates in Samson et al. (2009). 2.3. Preparation of aqueous Moringa oleifera leaf extract Zinc oxide and sulfur NPs were synthesized from the aqueous Moringa oleifera leaf extract which was prepared from freshly har vested leaves. Fresh moringa leaves (10 g) were cleaned by multiple washing under running tap water trailed by surface sterilization with 5 % sodium hypochlorite for 2 min. The surface sterilized leaves were finally washed with sterile distilled water and kept for boiling in distilled water (100 ml) for 15 min with continuous stirring. The extract was cooled to room temperature, filtered through Whatman filter paper followed by passing through 0.2 μm nitrocellulose filter to get rid of any impurities. The final volume was made up with sterile distilled water to 100 ml and the resulting extract was stored at 4 ◦C for further use. 2.4. Synthesis of biogenic NPs zinc oxide NPs (ZnONPs) and sulfur NPs (SNPs) The ZnONPs were synthesized by equal volumes of 10 mM ZnSO4 with diluted (1:20) M. oleifera extract in distilled water. The reaction mixture was continuously stirred for 30 min at room temperature with the dropwise addition of 0.1 N NaOH. The reaction mixture was observed for the formation of off-white precipitate of ZnONPs. For SNPs synthesis 50 mM of sodium thiosulphate (Awwad et al., 2015) was mixed with 100 ml of M. oleifera extract. Few drops of concentrated hydrochloric acid were added with continuous stirring for 30 min at room temperature or until pale yellow precipitate of SNPs was observed. The precipitates for both ZnONPs and SNPs were purified by centrifugation at 15,000 rpm for 15 min at 4 ◦C. The pellet was washed multiple times to assure no impurities in the final product (El-Gebaly et al., 2024), followed by overnight drying of the pellet in oven at 40 ◦C. 2.5. Characterization of NPs The M. oleifera mediated ZnONPs and SNPs were further detected in the colloidal form by visualization of any color change before and after synthesis. Characterization was done by UV–Visible spectrophotometry (NanoDrop, 2000c, Thermo Scientific, India; 1 mg/ ml for both NPs, spectrum range 200–800, at room temperature) to determine absorbance maxima for respective NPs. The average size of NPs was determined using Nanoparticle Tracking Analyzer (NTA LM 20, Malvern, UK), which was further confirmed with FESEM (Field Emission Scanning Electron Microscopy, FESEM, JEOL JSM-7100 F, Singapore; 18 kV and 30,000× magnification) with dwell time of 6 μs for ZnONPs and 10 μs for SNPs. The stability based on the surface potential for NPs was determined by zeta potential measurements (Zetasizer Nano-ZS 90, Malvern, UK). The biochemical composition of the capping layer of ZnONPs and SNPs was elucidated by detecting the various functional groups exposed using Fourier Transform Infrared Spectrometry (FTIR Spectrometer, Bruker Optics ATR, GmbH, Germany). The purity of NPs and their crystalline or amorphous nature was predicted by X-ray diffraction studies (Rigaku X-ray diffractometer, USA). The percent elemental constitution of NPs was determined by EDX (Energy Dispersive X- ray Microanalysis) which confirms specimen composition and distribution of the NPs through spectrum and elemental mapping. The characterized ZnONPs and SNPs were further evaluated for their antifungal activity by various. 2.6. In vitro antifungal activity assessment In vitro antifungal activity of ZnONPs and SNPs was evaluated against isolates of Pythium and Fusarium obtained from infected ginger samples by disc diffusion and Minimum Inhibitory Concentration (MIC) determination methods using microtiter plate assay as per guidelines of the Clinical Laboratory Standards Institute (CLSI, 2021) (https://www.treata.academy/wp-content/uploads/2021/ 03/CLSI-31-2021.pdf). 2.6.1. Kirby-Bauer Disc Diffusion assay Kirby-Bauer Disc Diffusion susceptibility test was performed to determine whether the given test fungal culture is susceptible or resistant to the given set of NPs tested. Inoculum of all four isolates were prepared growing of fungal biomass on potato dextrose broth for 8–10 days which was suspended in Rosewell Park Memorial Institute medium (RPMI 1640 medium). The optical density (OD) was upheld between 0.15 and 0.17 at 530 nm. The fungal suspensions were spread with the help of a sterile cotton swab on sterile PDA plates. Sterile discs obtained from HiMedia (HiMedia Pvt. Ltd. Mumbai, Maharashtra, India) were positioned onto the surface of inoculated PDA plate. Each sterile disc was loaded with 20 μl of respective NPs (zinc oxide and sulfur) suspension (2 mg/ml for both the NPs). The precursor salts solutions ZnSO4 (10 mM) and Na2S2O3 (50 mM) were taken as a control set individually (same as used for the synthesis of NPs). A separate controls were maintained as water (a negative control), antifungal antibiotic ketoconazole and fungicide mancozeb (1 mg/ml) as a positive controls. Along with this a separate disc for diluted moringa leaf extract (1:20) and finally a blank disc was maintained with sterile distilled water. The activity of these NPs was assessed using diverse concentrations including 2 mg/ml. The plates were incubated for 24–48 h at 28 ± 2 ◦C till visible growth appeared. The zones of inhibition (ZOI) were measured in mm with scale. The experiment was accomplished in triplicate. 2.6.2. Determination of MIC by microdilution method The MIC of NPs (zinc oxide and sulfur) was determined by micro-broth dilution method against all isolates. For evaluation of MIC, fungi were inoculated in potato dextrose broth (PDB) and incubated at 28 ± 2 ◦C for 7 days. The OD of the fungal suspension was P.U. Ingle et al. Biocatalysis and Agricultural Biotechnology 58 (2024) 103229 4 https://www.treata.academy/wp-content/uploads/2021/03/CLSI-31-2021.pdf https://www.treata.academy/wp-content/uploads/2021/03/CLSI-31-2021.pdf attuned between 0.15 and 0.17 b y using RPMI medium. The absorbance was measured by using colorimeter at 530 nm and the fungal spore load was kept between 0.4 × 104 to 5 × 104 CFU/ml. The concentrations of NPs tested were in the range of 50–450 μg/ml for both the NPs. 50 μl of fungal inoculum was added to each well. The negative control was maintained with sterile PDB broth to check sterility. Similarly, the positive control was maintained with PDB inoculated with 50 μl of fungal spore suspension with the standard fungicide i.e. mancozeb, 1 mg/ml. The micro dilution plates were incubated at 28 ± 2 ◦C for 3 days. The plates were observed at every 24 h interval. MIC recorded was the lowermost concentration of NPs that resulted in visual inhibition of fungal growth. Each assay was performed in triplicate. 2.6.3. Time kill assay The fungal cultures were grown in PDB medium for 7 days to get visible growth. The nanoparticle concentration represented at MIC was taken to determine the minimum time required to inhibit 100 % of the fungal growth in liquid medium at 28 ± 2 ◦C. The fungal cultures treated with respective NPs for the time period of 60 h. An aliquot (10 μl) was taken after every 6 h interval and spread on the PDA plate followed by counting the number of colonies after 3 days of incubation at 28 ± 2 ◦C. The plate showing the complete in hibition of fungal growth was selected for the respective treatment time as a minimum time of inhibition for the given set of fungus and nanoparticle treatment. 2.7. Statistical analysis Statistical significance of the data obtained were analyzed and verified using the standard one way analysis of variance method (one-way ANOVA) for the test results for both the nanoparticles. 3. Results 3.1. Isolation and identification of fungi The fungal isolates stained with lactophenol cotton blue were identified on the basis of their respective shapes of conidia and the respective genus for the given culture was determined. Isolate I and II grew as off-white colored colonies that showed the dark blue stained prominent nuclei and non-septate, cenocytic cell growth, which is a peculiar character of genus Pythium. Pythium isolates were identified on the basis of classification given by Patterson (1989) and Dick (2001). Isolates I and II were identified as Pythium spp. On the basis of their white colored colonies with irregular margins with radial growth pattern (Navi et al., 2019) and empty bursiform sporangium with short exit tube (Fig. 1 A) and oogonium surrounded by several antheridia (Fig. 1 B) respectively (Ho, 2018). Isolate III was identified as Fusarium oxysporum (Fig. 1 C) (Leslie and Summerell, 2006) on the basis of fluffy growth and creamy white colored upper surface of colonies with slightly pink colored pigmentation at the bottom (Rafique et al., 2015). Conidiogenous cell was long and monophilides. Macroconidia were slender, slightly curved, showed 5-7 septations. Isolate IV was identified as Aspergillus sp. (Fig. 1 D) (Thom and Raper, 1945). Colonies showed green/yellow colored pigmentation and the bottom was shiny yellow to golden in color (Moslem et al., 2010; Awad et al., 2023). Microscopic structures showed flask-shaped phialides, with series of vesicles on the top of Fig. 2. Biological synthesis of nanoparticles from M. oleifera extract (A), white precipitate of Zinc oxide nanoparticles (ZnONPs) (B) and pale yellow precipitate of Sulfur nanoparticles (SNPs) (C). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) P.U. Ingle et al. Biocatalysis and Agricultural Biotechnology 58 (2024) 103229 5 conidiophore (Raper and Fennell, 1965). The identified fungal cultures were maintained in a pure form on PDA medium and stored at 4 ◦C for further in vitro assessment. 3.2. Synthesis of biogenic NPs The ZnONPs which were synthesized from M. oleifera leaf extract (Fig. 2 A) in the form of a white colloidal suspension (Fig. 2 B) which was collected by ultracentrifugation at 15,000 rpm for 10 min, followed by washing pellet with distilled water (3 times) and overnight drying in the oven at 40 ◦C. Off-white colloidal SNPs (Fig. 2 C) were also collected by centrifugation (15,000 rpm for 15 min) followed by washing with distilled water (3 times) and overnight oven drying to obtain the dry pellet. The dried pellets were ground in to a fine powder and collected in an air-tight vials for further characterization and in vitro assessment. 3.3. Visual detection of nanoparticles The aqueous M. oleifera extract which was pale yellow in color (Fig. 2 A) when mixed with the 10 mM zinc sulphate solution changed to the color to white precipitate of zinc oxide NPs (Fig. 2 B). This indicated the synthesis of ZnO NPs. The synthesis of sulfur NPs was confirmed by the formation of off-white precipitate (Fig. 2 C) of SNPs after the addition of sodium thiosulphate and a few drops of HCl to the moringa extract (Paralikar and Rai, 2018). 3.4. Characterization of NPs 3.4.1. Characterization of zinc oxide nanoparticles UV–visible spectrophotometric measurement of ZnONPs indicated the absorbance maxima of 356 nm (Fig. 3 A). NTA analysis indicated the average size of 78 nm and 35 nm standard deviation with particle size distribution over a broad range (Fig. 3 B and 3 D). Particle concentration was found to be 1.5 × 107 particles/per ml of test sample. The zeta potential measurement indicated the average potential of − 14.1 mV with the standard deviation of 4.51 mV (Fig. 3 C). The FTIR spectrometry indicated the peaks present for various functional groups exposed in the capping layer of ZnONPs (Fig. 4 A). The transmittance bands were observed at 3265 cm− 1 which is assigned to O–H group in hydrogen bonded alcohols and phenols, 1722 cm− 1 assigned to C––O group in aldehydes, ketones, carboxylic acids and esters. The peaks at 1565 cm− 1, 1320 cm− 1 represented NO2 group in nitro compounds. Transmittance peak at 1398 cm− 1 was assigned to C–H stretch of alkanes. The minor peaks at 1153 cm− 1, 1025 cm− 1 and 819 cm− 1 represented C–N stretch of secondary Fig. 3. Characterization of M. oleifera mediated ZnONPs, (A) UV–visible spectrum, (B) size determination using NTA (Nanoparticle Tracking Analysis), (C) Zeta potential determination, and (D) particle size distribution in 2D under NTA. P.U. Ingle et al. Biocatalysis and Agricultural Biotechnology 58 (2024) 103229 6 amines, C–O stretch of ethers and esters and C–Cl stretch of aliphatic chloro compounds respectively. Energy dispersive x-ray analysis (Fig. 4 B) indicated the presence of 62 % of Zn by weight and 28.83 % by atomic weight, and rest was constituted by oxygen only. There were no other elemental impurities detected in EDX. X-ray diffraction pattern of ZnONPs (Fig. 4 C) showed Miller indices as 102, 110, 103 and 112 which corresponds to the 2 theta angle of diffraction of 47.51, 56.53, 62.83 and 67.89◦. The Bragg’s values and Miller indices indicated that the zinc oxide NPs were crystalline in nature with face centered cubic structure of ZnONPs crystals. FESEM of ZnONPs (Fig. 4 D) indicated the formation of polymorphic ZnONPs i.e. roughly circular and bar shaped ZnONPs crystals with size range in resemblance as per shown by NTA analysis. The roughly circular disc shaped crystal of ZnONPs have smooth but irregular margins with variable diameter ranging approximately between 10 and 60 nm. While bar shaped crystals are similar to quartz crystals with slender (with length 5–7 times the width) structure, fine edges and soft corners. 3.4.2. Characterization of sulfur nanoparticles The sulfur nanoparticles (SNPs) indicated the absorption maxima at 295 nm under UV–visible light (Fig. 5 A). Average size of 52 nm was observed by NTA analysis with standard deviation of 32 nm (Fig. 5 B). The particle concentration was found to be 1.3 × 109 particles/per ml. The stable SNPs were confirmed by average zeta potential value of − 21.2 mV and standard deviation of 9.56 mV (Fig. 5 C). The SNPs were observed to disperse over a broad range from 10 nm and up to 100 nm and very less particles with size above 100 nm (Fig. 5 D). The transmittance peaks were observed at 3412 cm− 1 represented the presence of N–H stretch of amines and amides, 1644 cm− 1 C––O groups of aldehydes and ketones, 1428 cm− 1 C–H stretch of alkanes, 1251 cm− 1 represented presence of C–N stretch of amines and amides. The other minor peaks at 1113 cm− 1, 986 cm− 1, 789 cm− 1 and 652 cm− 1 were assigned to C–O alcoholic stretch, C–H of alkenes, aliphatic chloro- and aliphatic iodo-compounds respectively were present in capping layer (Fig. 6 A). EDX represented the presence of sulfur (79.29 % by wt. And 65.63 % atomic weight) and oxygen only and no other elemental contamination (Fig. 6 B). XRD spectrum (Fig. 6 C) indicated the typical Miller indices for SNPs at 113 (15.38), 131 (22.68), 222 (23.07), 026 (25.83), 311 (27.71), 044 (31.39), 404 (37.35), 062 (42.78), 066 (47.88) and 517 (51.32). The miller indices were assigned to a specific Bragg’s diffraction angle as given herewith. The Miller indices indicated the formation of face centered cubic crystalline SNPs (Joint Com mittee on Powder Diffraction, Standard file no. 04–0836). As shown in the figure (Fig. 6 D) FESEM disclosed the wrinkled with almost spherical sulfur NPs with polydispersed morphology with variable range of diameters were synthesized by M. oleifera. The size range of SNPs detected in FESEM corroborated with the NTA results. Some are slight ellipsoidal with rough or wrinkled surface. Accumulation of SNPs might have resulted in larger sized spheres as observed under FESEM. Fig. 4. Characterization of M. oleifera mediated ZnONPs, (A) Fourier Transform Infrared spectrum, (B) EDX pattern, (C) X-ray diffraction pattern, and (D) FESEM images. P.U. Ingle et al. Biocatalysis and Agricultural Biotechnology 58 (2024) 103229 7 3.5. In vitro antifungal activity assessment In vitro antifungal activity of both the synthesized NPs namely zinc oxide and sulfur NPs were evaluated at concentrations 2 mg/ml, against two Pythium spp., F. oxysporum and Aspergillus sp. Isolated from infected ginger samples by Kirby–Bauer disc diffusion method (Bauer et al., 1966; Sales et al., 2016; Sharma et al., 2022). 3.5.1. Kirby-Bauer Disc Diffusion assay The ZnONPs and SNPs post-characterization were evaluated for their antifungal activity against identified fungi by in vitro method i.e. Kirby-Bauer disc diffusion method. The diameter of zone of inhibition (ZOI) was measured, which determined the antifungal potential of test compound. The negative control sets viz. Plant extract (moringa leaf extract), sodium thiosulphate and zinc sulphate did not show any zone of inhibition against all four fungal cultures, indicating no antifungal activity exhibited by the precursor molecules used for the synthesis of NPs. The zones of inhibition for Pythium isolate I, were observed (Fig. 7) in the decreasing order as Mzb + ZnONPs > Mzb > Mzb + SNPs > Kz (ketoconazole) + SNPs > ZnONPs ≈ Kz + ZnONPs ≈ Kz > SNPs. For the Pythium isolate II, the order was observed as: Mzb + ZnONPs > Mzb ≈ Mzb + SNPs ≈ Kz + SNPs ≈ KznONPs > SNPs. Isoloate III i.e. Fusarium sp. Showed highest ZOI by SNPs in combination with fungicide, mancozeb. The order of ZOI for Fusarium isolate was found in the order as: Mzb + SNPs > Mzb ≈ Mzb + ZnONPs > Kz + SNPs ≈ Kz + ZnONPs > SNPs ≈ Kz > ZnONPs. Finally, the Aspergillus isolate IV largest ZOI with SNPs in combination of antibiotic, ketoconazole which was equivalent to the ZOI shown by Mancozeb. The order of ZOI were found to be in the order as follows: Kz + SNPs ≈ Mzb > Kz + ZnONPs > Kz > Mzb + SNPs > Mzb + ZnONPs > ZnONPs > SNPs. The smallest ZOI were observed for ZnONPs against, which might be the result of lower antifungal potential and less diffusion in medium from the sterile disc. 3.5.2. Determination of MIC by micro titer plate assay The minimal inhibitory concentration for all test fungi were observed. ZnONPs showed the highest value of MIC against Pythium isolate I.e. 450 μg/ml and lowest for the Aspergillus sp. Pythium II and Fusarium sp. Showed the same MIC values. SNPs showed the equivalent MIC against both Pythium isolate I and Aspergillus sp. The order of MIC followed by both ZnONPs and SNPs was observed as Pythium I > Pythium II ≈ Fusarium sp. > Aspergillus sp. And Pythium I > Aspergillus sp. > Pythium II ≈ Fusarium sp. Respectively (Supplenemtary file, Fig. S1 – S4). Fig. 5. Characterization of M. oleifera mediated SNPs, (A) UV–visible spectrum, (B) size determination using NTA (Nanoparticle Tracking Analysis), (C) Zeta potential determination, and (D) particle size distribution in 2D under NTA. P.U. Ingle et al. Biocatalysis and Agricultural Biotechnology 58 (2024) 103229 8 3.5.3. Time kill assay The time of inhibition (TOI) of fungal growth at respective MIC was calculated for all selected fungi. The minimum treatment time of selected NPs was considered as TOI where no visible growth was observed after the given treatment time. In case of the treatment with biogenic ZnONPs treated set of fungi, a slow decrease in the viable spore count was observed up to ≈18 h of incubation followed by rapid decline till ≈36 h incubation (Supplementary file, Figs. S5 and S6). The complete growth was inhibited nearby ≈42 h of incubation and no visible growth was observed at ≈ 48 h PDA plates (Fig. 8). The complete inhibition of two isolates of Pythium (I and II), was observed at ≈ 42 h, as no growth was observed on PDA plates. The Aspergillus and Fusarium species showed complete inhibition Fig. 6. Characterization of M. oleifera mediated SNPs, (A) Fourier Transform Infrared spectrum, (B) EDX pattern, (C) X-ray diffraction pattern, and (D) FESEM images. Fig. 7. Zones of inhibition against all the test fungi Level of significance ***P ≤ 0.05, NS= Non-significant. P.U. Ingle et al. Biocatalysis and Agricultural Biotechnology 58 (2024) 103229 9 ≈36 h. In the SNPs treated set of fungi, Pythium I and Fusarium were shown to be completely inhibited at ≈ 42 h. The other fungal subjects, Pythium II and Aspergillus was fully inhibited by ≈ 30 h (Fig. 9). Statistical analysis of time kill assay predicted the P-value of 0.000495 and 0.000462 for ZnONPs and SNPs respectively (both are ≤0.05). The change in variance for time kill assay was found to be a function of the time of contact as indicated by the increased value of variance as the time increases and downfall observed at the highest (≈100%) removal or killing of fungal spores in the suspension. The statistical analysis thus validates the results of experimental results. 4. Discussion Ginger is globally consumed almost daily as a culinary spice as a fresh rhizome or in the form of a dried powder. It has medicinal properties as well as being an important spice cash crop its losses due to fungal infections need to be minimized. Authors chose to work under the growth inhibition of fungi that grow in ginger, because the fungi cause 60–90% losses in ginger post-harvest. The present study thus focused on developing a sustainable approach for management of deteriorating fungal pathogens in ginger and help farmers to enhance the crop conditions and crop yield in order to improve their economic status (Rai et al., 2018; Bazioli et al., 2019; Wang, 2020). The phytopathogenic fungi isolated from soft-rot infected ginger were obtained in pure culture on PDA medium and were subjected to morphological microscopic examination. The morphological structures like the sizes, shapes, and locations of the sporangium on the mycelium, antheridium, oogonium and oospores were observed to identify the genus of given isolated fungi. The first two isolates were showing filamentous (thread-like), coenocytic (non-septate threads lacking cross walls) cell growth, which is a peculiar characteristic of Pythium spp. The asexual or vegetative stage of Pythium produces thick walled resting stage i.e. chlamydospores, as shown in Fig. 1 A. the standard key given by Dick (1990) was referred to identify the genus of the given isolates. On the basis of monograph (Van der Plaats-Niterink, 1981) of Pythium the isolate I and II (Fig. 1 A and B) were confirmed to belong to genus Pythium. The third isolate (III) was identified as a species of Fusarium genus (Fig. 1 C), an important phytopathogen associated with the soft-rot in ginger (Li et al., 2014; Rai et al., 2018). It was reported that the Fusarium can cause rhizome rot in ginger for the first time in China. Rhizome rot infected cultivar Laiwu Big Ginger, was reported in Anqiu, Shandong Province, with yield losses of up to 70% due to Fusarium oxy sporum associated (Li et al., 2014; Prasath et al., 2023; Kaningini et al., 2023). Finally, the forth isolate i.e. isolate IV, was found to belong to another major genus of phytopathogenic fungi i.e. Aspergillus (Fig. 1 D). The genus was determined based on Aspergillus manual by Thom and Raper (1945). In the present study, M. oleifera aqueous extract was used for the synthesis of NPs because they are rich in various bioactive secondary metabolites, such as zeatin, caffeoylquinic acid, quercetin, kaempferol, and b-sitostero, etc. Many of the reported metab olites are growth regulators, insecticides and are fungicidal in nature (Nizioł-Łukaszewska et al., 2020; Jenish et al., 2022). The huge array of compounds with in vitro antifungal, antiviral, antibacterial and antioxidant properties present in moringa extract, improves shelf life of crops and their high antioxidant concentration allows its preferential use over other plants. The ease of cultivation of moringa plants in temperate regions of Indian subcontinent is another advantage (Mncube et al., 2021; Oniha et al., 2021; Baldis serotto et al., 2023; Yadav et al., 2023b). It is observed that there is a significant decrease in the phytochemical constitution of moringa leaves during drying depending on the mode of drying i.e. freeze, air, sun or oven drying. On the contrary, fresh leaf extracts show higher contents of components like flavonoids, saponins, phenolics, vitamins, etc. That are most probably lost during drying. These phytochemicals play an important role during the synthesis and stabilization of nanoparticles. Hence, it is recommended to prefer entially use the fresh leaf extract for nanoparticle synthesis due to its higher phytochemical composition. Moringa leaf extracts (fresh) was utilized in the present study as it is rich in several antifungal compounds such as tannins and flavonoids that have already demonstrated inhibitory activity against fungi (Yadav et al., 2023b). This makes moringa as a suitable candidate to be used in the synthesis of antifungal nanoparticles (Ademiluyi et al., 2018; Rajput, 2017; Vergara-Jimenez et al., 2017). This suggests the use of Fig. 8. Time kill assay of ZnONPs against all test isolates Level of significance **P ≤ 0.05, NS= Non-significant. P.U. Ingle et al. Biocatalysis and Agricultural Biotechnology 58 (2024) 103229 10 moringa extract as a stabilizing agent and reducing material for the synthesis of antifungal nanoparticles. Various modes of antifungal materials are responsible for imparting fungicidal activity t the nanoparticles synthesized. Further, the zinc oxide nanoparticle syn thesis by M. oleifera was confirmed with the formation of white colored colloid (Fig. 2 B) after addition of a few drops of alkali (0.1 N NaOH). The synthesis from aqueous extract of moringa leaves as a capping material stabilized the ZnONPs in the colloidal state. The spectrometric measurement showed absorption maxima at 356 nm (Fig. 3 A), which is an excellent match with the earlier report and within the range of typical Surface Plasmon Resonance Band for metal oxide NPs without any kind of aggregation (Venkatesham et al., 2012; Duque et al., 2019; Githala and Trivedi, 2023). The absorption maxima is an attribute of their huge excitation binding energy. The blue-shifted absorption spectrum and larger particle size is a function of quantum imprisonment effect exhibited by colloidal zinc nanomaterials (Abel et al., 2021; Degefa et al., 2021; Hazaa et al., 2021). The nanoparticle tracking analysis revealed the size and distribution of ZnONPs determined on the basis of their Brownian motion. Fig. 3 B and 3 D, revealed the average size of ZnONPs as 78 nm, with standard deviation of indicating polydispersed nanoparticle formation by moringa extract and distribution over a broad size range with very less amount of NPs with larger size (Jadhao et al., 2020; Irfan et al., 2021). The zeta potential of − 14.1 mV and a single narrow peak revealed the stable nature of ZnONPs (Fig. 3 C), due to the stabilization of NPs by numerous metabolites present in the moringa extract. The high negative value indicated the stable ZnONPs which bypassed aggregation at room temperature in a colloidal state. The zeta potential values shown thus corroborated with the earlier studies based upon theories of stability of hydrophobic colloids (Derjaguin and Landau, 1941; Verwey and Overbeek, 1948). The stability of the synthesized NPs can be explained on the basis of presence of various biomolecules as predicted by Fourier Transform Infrared spectrometry (Fig. 4 A), in terms of percent trans mittance and respective wavenumber (cm− 1) assigned to specific functional groups in the capping layer of NPs. The presence of transmittance bands assigned to –OH group, aldehydic C––O stretch, NO2 group, and the C–Cl stretch of chloro compounds probably contributed the negative vale of zeta potential and the aliphatic C–H stretch have formed the core of ZnONPs. The strong peaks be tween 400 and 600 cm− 1 indicated the presence of metal oxides in the sample, confirming the presence of Zn–O stretching vibrations and formation of zinc oxide NPs (Matinise et al., 2017; Balan et al., 2019; Espenti et al., 2020; Mondal et al., 2024). In case of calcined ZnONPs the results are similar with the formation of prominent peaks in the range of 400–600 cm− 1 (Adam et al., 2021). EDX analysis of ZnONPs (Fig. 4 B) represented the presence of major peaks for percent weight of Zn and oxygen, along with the smaller peaks belonging to the traces of impurities belonging to other elements. The lower atomic percent yield can be stipulated due to presence of Zn ions primarily in the core of NPs and outer surface is covered with the metabolites from the moringa extract. The results were in accordance with the previous reports by Ansari and colleagues (2020) and Espenti et al. (2020). The peculiar diffraction peaks in XRD spectrum (Fig. 4 C) of ZnONPs at Miller indices of 101, 102, 110, and 103 indicated the presence of face centered cubic crystal for mation (JCPDS- File No. 89–0510). The sample showed the other minor peaks might be due to the formation of polymorphic ZnONPs crystals as represented in SEM images at 30000x resolution (Fig. 4 D). The SEM images signifies polymorphic nature of ZnONPs showing firstly the discoid crystals and secondly the multifaceted cuboidal diamond shaped crystals. This clarifies the variation in the size range of ZnONPs. Quartz like and disc shaped polymorphic crystals of ZnONPs were confirmed by FESEM images (Irfan et al., 2021). The disc like depositions of ZnONPs were reported previously which also showed the similar results as quoted in the present study (Abel et al., 2021). The size of ZnONPs was below 100 nm i.e. 78 nm (Iranbakhsh et al., 2021). As per the literature available, there are various modes of antifungal activity of nanoparticles has been reported. This includes the adsorption of NPs on the cell surface leading to membrane depolarization. Endocytosis of NPs ultimately results in cytosolic release of NMs and triggers the release of various reactive oxygen species (Lipovsky et al., 2011). On the other hand diffusion of NPs through the cell wall may also result in the intracellular damage including the damage to genetic material of the cell. This ultimately decides the fate of the cell to undergo the process of apoptosis, and death of the cell. Fig. 10 below represents the probable mechanism of antifungal activity of nanoparticles Fig. 9. Time kill assay of SNPs against all test isolates Level of significance **P ≤ 0.05, NS= Non-significant. P.U. Ingle et al. Biocatalysis and Agricultural Biotechnology 58 (2024) 103229 11 (Gurunathan et al., 2022). Sulfur being an important element in soil is widely used in industry, pharmaceutical and agriculture as well. It is used as an active component of nitrogenous fertilizer. Sulfur is a key prosthetic group in many of the enzymes, co-enzymes and proteins. It also helps plants in mitigating biotic stress and disease resistance (Dubuis et al., 2005; Najafi et al., 2020; Kaur et al., 2023; Bhaskar et al., 2023). A green synthesis of Sulfur NPs had previously been reported by various methods using many kinds of plant extracts and without application of any hazardous additives (Banerjee and Rajeswari, 2024). In present study extract of fresh leaves of moringa plants were used to synthesize SNPs. Other researchers have confirmed the synthesis from various parts of plants like, fresh and dried leaves, fruits, bark and peels. Other plants explored for the biogenic synthesis of SNPs are Alianthus altissima, Albizia julibrissin, Azadirachta indica, Cinnamomum zeylanicum, Punica granatum, Sophora japonica, etc. (Ghotekar et al., 2020). Sodium thiosulphate is a common precursor material used for the plant mediated synthesis of SNPs. Synthesis of SNPs using sodium thiosulphate and bark extract of Cinnamomum zeylanicum resulted in formation of yellow colored precipitate (Najafi et al., 2020). The absorption maxima of SNPs i.e.295 nm (Fig. 5 A), was reported to be in the same range of wavelength as reported by Paralikar and Rai (2018). A study by Chaudhuri and Paria (2011), elaborated the growth kinetics in aqueous medium for SNPs that has similar absorption range. Further analysis of SNPs by NTA reported the size in the range below 100 nm. In NTA particles are sized one-by-one under laser light according to their Brownian motion in the area focused. The mean displacement is calculated for the particles moving in the same planes (Paralikar and Rai, 2018). NTA also predicted the crowding of SNPs more frequently in the range between 10 and 60 nm as per the 2D distribution graph (Fig. 5 D). Negative zeta potential values indicated null negative charge on the surface of the SNPs and the average zeta potential value away from zero viz. − 21.2 mV (Fig. 5 C) indicated the stability of SNPs in colloidal systems. The FTIR spectrum of SNPs showed overlapping peaks in approximately the same wavenumber range as control i.e. M. oleifera extracts. Negative zeta potential supported the presence of negatively charged biomolecules from extract in the capping layer of SNPs (Coates, 2006; Choudhury et al., 2013). This confirms the presence of functional groups that are common to both, SNPs and control samples. The crystal lattice of SNPs was compared with the standard dataset. This indicated the SNPs with lattice corresponding to the JCPDS file No. 08247. The assigned peaks were presenting the structure of SNPs and the unassigned peaks in XRD were considered due to the impurities and amorphous matters in the test sample (Suleiman et al., 2013; Awwad et al., 2015; Suleiman et al., 2015). Majority of the SNPs reported are spherical, which supports the present findings. The results obtained from FESEM were analogous with the previous reported studies (Kouzegaran and Farhadi, 2017; Khairan et al., 2019; Ghotekar et al., 2020). The previous reports also supports the synthesis of spherical SNPs (Tripathi et al., 2018; Baloch et al., 2023). EDX analysis showed a higher percentage of sulfur in the matrix followed by oxygen (Ismalita et al., 2022). The FESEM imaging as shown in Fig. 4 D and 6 D shows, that both the nanoparticles have distinct morphologies. The variation in the morphology of ZnONPs and SNPs is probably due to the difference in the reduction rate because of the presence of different biomolecules in moringa extract which are responsible for the different nucleation rates of the nanoparticle growth during the syn thesis process. The reduction and stabilization of NPs by different biomolecules from moringa extract will decide the nanoparticle morphology and stability. Also, the nanoparticles’ shape and size are defined by the surface energy required which is attended for electro- and thermodynamic stability during their stabilization (Pinchuk and Schatz, 2008; Yao et al., 2015; Vollath et al., 2018; Anand et al., 2020). Several studies have shown the promising antifungal and antibacterial property of biogenic SNPs. For example, Catharanthus roseus extract mediated green synthesized SNPs were potential antifungal agents against Microsporum canis (Chantongsri et al., 2021). In the present study, the antifungal activity of SNPs was confirmed by visible inhibition of visible fungal growth on PDA plates. Larger zones of inhibition indicated the higher fungicidal potential of SNPs. MIC i.e. minimum inhibitory concentration was considered as the lowest concentration where visible fungal growth inhibition is observed on PDA plates with nitrocellulose plate loaded with the SNPs. In the present study both the nanoparticles i.e. ZnONPs and SNPs individually as well as synergistic activity in combination with fungicide mancozeb and antibiotic ketoconazole showed significant fungicidal activity against test fungi. This indicated the enhanced fungicidal effect of standard antifungal agents. In time kill assay for ZnONPs and SNPs, time dependent antifungal activity was observed i.e. as the time of nanoparticle exposure to fungus increases the viable spore count as well as the colony count decreases. The earlier reports also suggests that enhancement of antifungal activity of SNPs was possibly due to the C. roseus extract which was used for their synthesis (Chantongsri et al., 2021; Abdel-Maksoud et al., 2023). Similarly, in the present study M. oleifera leaf extract was used which contains secondary metabolites which were the primary reason for stabilization of SNPs increasing surface to volume ratio. Here, both the nanoparticles ZnONPs and SNPs were thoroughly washed to remove any residual components from the moringa extract. This indicated the antifungal activity of the NPs was not the direct function of extract, instead the secondary metabolites involved in their stabilization. The fungal load decreased as the time of exposure with NPs increases. Thus, the fungal load was minimized at 42 and 36 h after exposure with ZnONPs and SNPs (Figs. 8 and 9). The results were in cohesion with the reports of Chantongsri et al. (2021) and Mohammed and Hawar (2022). The antifungal activity of NPs was an attribute of their small particle size, higher surface area to volume ratio which facilitates the adhesion as well as entry of NPs into the fungal mycelium. Thus rupturing the cell membrane and decreasing the integrity of the cell wall may be due to the decreased lipid content (Roy Chaudhary et al., 2013; Mohammed et al., 2022; Mahmad et al., 2023). The synthesized ZnONPs and SNPs showed significant antifungal activity and will aid in formulation of nano-based fungicides. Various methods are available for the preparation of nanofrmulations, and can be applied by various modes such as sprays, dusting, soil applicants, etc. Mode of application will differ based upon the desired activity of the nanoformulations. Nanoformulations developed by biological approach and a green methods will be more beneficial and economic with respect to their scaling up and public use. But, before their agriculture application, there is a need for thorough field trials, and pilot scale studies followed by the large-scale field experimentation in natural environment (Krishnamoorthy and Mahalingam, 2015). The present study was aimed to P.U. Ingle et al. Biocatalysis and Agricultural Biotechnology 58 (2024) 103229 12 evaluate in vitro antifungal potential of two nanoparticles ZnONPs and SNPs against fungal pathogens from ginger. This approach of applying two or more antifungal nanoparticles with variable biochemical composition (as elucidated by FTIR analysis) will minimize the risk of development of resistance and effective killing and management of fungal pathogens in the agricultural fields (Alghuthaymi et al., 2021). In addition to the fungicidal potential of NPs, composition as well as chemical and biological properties of soil are greatly affected by the application and accumulation of the NPs. This may impact the microbial ecosystem of soil which plays an important role in the soil fertility maintenance. Uncontrolled dispersal and accumulation of various NPs will change the microbial and functional diversity of the soil. Thus controlled and more supervised application of NMs in agriculture is recommended (Ghidan and Al Antary, 2020; Chauhan, 2023). NMs can increase soil fertility significantly. But on the other hand, excess amounts can contaminate the soil, and finally migrate to ground waters. To prevent the damage to aquatic systems by rainwater runoff and soil erosion along with the transportation of the manufactured NMs from production sites is recommended (Ray et al., 2009). In order to prevent these com plexities, it is advised to use NMs as per the recommendations of government agencies and in approved limits. Thorough field trials, in vitro and in vivo studies based upon the dose-dependent activities for their beneficial as well toxicity aspects will be required in the future to examine and anticipate the highest potential of NMs in agriculture (Tarafdar, 2015; DBT, 2020; Kalia et al., 2020; Cruz-Luna et al., 2021). 5. Conclusion India being an agriculture-based country, it is important to fulfill the ever increasing requirements of food and feed. At the same time, it is required to follow safe practices for food production, storage and consumption. This can only be achieved through increased production and reduced pest infestation using a sustainable approach. Nanobiotechnology has proven its record as a new generation of a sustainable approach to mitigate problems in various fields including pharmaceutical, medicine, agricultural, etc. ZnONPs and SNPs reported in the present study were synthesized by a plant-based method. The phyto-stabilization of ZnONPs and SNPs was possible using M. oleifera crude leaf extract. The ZnONPs and SNPs were biologically synthesized, which makes them easily available for interaction with fungal counterparts and timely killing the target fungus. It can be concluded that the in vitro antifungal assessment of ZnONPs and SNPs against fatal phytopathogenic fungi of ginger such as, Aspergillus, Fusarium and Pythium signifies their application in development of alternative fungicidal materials for agricultural application can be produced and applied on a commercial scale for the pre-treatment of seed ginger before sowing. It can be concluded from the statistical analysis that, the significant inhibition of the fungal growth was observed with the increase in time of contact between fungal species with ZnNPs as well as SNPs. This will aid in the preparation of nanoformulation based upon these tested NPs against tested pathogenic fungal isolates from ginger, which can be publicly distributed after critical laboratory and field-based studies. The majority of the NPs are reported for their novel benefits in various fields like medicine and agriculture. However, their overuse may impose serious threats to the plants reducing the germination rate, bioaccumulation and environmental toxicity. It is thus necessary to evaluate their toxicity prior to public use based on the guidelines and standard application methods which will benefit farmers. CRediT authorship contribution statement Pramod U. Ingle: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Mahendra Rai: Visualization, Validation, Supervision, Project administration, Formal analysis, Data curation, Conceptualization. Fig. 10. Mechanism of antifungal activity of nanoparticles, Nanoparticles inhibit growth of fungal cell by entering into cell by endocytosis, release ROS and ultimately kills the cell by apoptosis. P.U. Ingle et al. Biocatalysis and Agricultural Biotechnology 58 (2024) 103229 13 Patrycja Golińska: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis. Aniket K. Gade: Visualization, Validation, Supervision, Project administration, Formal analysis, Data curation, Conceptualization. Declaration of competing interest Authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgement PG and AKG would like to acknowledge this research as part of the project No. UMO-2022/45/P/NZ9/01571 co-funded by the National Science Centre and the European Union Framework Programme for Research and Innovation Horizon 2020 under the Marie Skłodowska-Curie grant agreement No. 945339. For the purpose of Open Access, the author has applied a CC-BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bcab.2024.103229. References Abdel-Maksoud, G., Abdel-Nasser, M., Hassan, S.E.D., Eid, A.M., Abdel-Nasser, A., Fouda, A., 2023. Biosynthesis of titanium dioxide nanoparticles using probiotic bacterial strain, Lactobacillus rhamnosus, and evaluate of their biocompatibility and antifungal activity. Biomass Conv. Bioref. https://doi.org/10.1007/s13399- 023-04587-x. 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