New perception about the use of nanofungicides in sustainable agriculture practices.

Protecting plants from pathogens using synthetic nanofungicides is not very effective, because it is harmful to the environment. However, it is synthetic fungicides that farmers are familiar with and commonly use. In this modern era, nanotechnology offers a smart solution to environmental issues at the nanoscale level. It is an emergent field and nanoparticles can be synthesized through various methods. Nanofungicides are efficient due to their solubility and permeability, low dose-dependent toxicity, low dose, enhanced bioavailability, targeted delivery, enhanced bioavailability, and controlled release. There are many metallic compounds, such as Cu, Zn, Ag, and TiO2 available which are used as nanofungicides. There is a contrary relationship between the size of the nanoparticles and their efficacy and antifungal potential. This review article offers a wide knowledge about formulation of nanomaterials as nanofungicides and their role in disease management in plants.

Engineered nanomaterials in plant diseases: can we combat phytopathogens?

Engineered nanomaterials (ENM) have a high potential for use in several areas of agriculture including plant pathology. Nanoparticles (NPs) alone can be applied for disease management due to their antimicrobial properties. Moreover, nanobiosensors allow a rapid and sensitive diagnosis of pathogens because NPs can be conjugated with nucleic acids, proteins and other biomolecules. The use of ENM in diagnosis, delivery of fungicides and therapy is an eco-friendly and economically viable alternative. This review focuses on different promising studies concerning ENM used for plant disease management including viruses, fungi, oomycetes and bacteria; diagnosis and delivery of antimicrobials and factors affecting the efficacy of nanomaterials, entry, translocation and toxicity. Although much research is required on metallic NPs due to the possible risks to the final consumer, ENMs are undoubtedly very useful tools to achieve food security in the world. KEY POINTS: • Increasing global population and fungicides have necessitated alternative technologies. • Nanomaterials can be used for detection, delivery and therapy of plant diseases. • The toxicity issues and safety should be considered before the use of nanomaterials.

Phytofabricated zinc oxide nanoparticles as a nanofungicide for management of Alternaria blight of Brassica.

Plant pathogens resistant to the commercially available fungicides and bactericides even at higher concentrations are the biggest challenge for the farmers to control the losses due to plant diseases. The antibacterial and antifungal potential of nanomaterials makes them a suitable candidate for the control of plant diseases. Thus, the present study reports the phytofabricated zinc oxide nanoparticles (ZnO Np's) using aqueous plant leaf extract of Terminalia bellerica (Baheda). Characterization of ZnO nanoparticles was done by ultraviolet-visible (UV-Vis) studies, X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infra-red (FT-IR) analysis, and transmission electron microscopy (TEM). The presence of pure hexagonal wurtzite crystalline structure of ZnO nanoparticles was confirmed by XRD analysis. The TEM images revealed the spherical to hexagonal shaped ZnO nanoparticles with sizes ranging from 20 to 30 nm. The stabilization of synthesized ZnO nanoparticles through the interactions of terpenoids, steroids, phenylpropanoids, flavonoids, phenolic acids, and enzymes present in the leaf extract was suggested by FTIR analysis. The mechanism of the formation of ZnO nanoparticles using Terminalia bellerica (Baheda) (Tb-ZnO Np's) as a bioactive compound is proposed. These phytofabricated ZnO nanoparticles (Tb-ZnO Np's) have shown significant antifungal potential against Alternaria brassicae the causal agent of Alternaria blight disease/leaf spot disease in Brassica species. The microscopic results confirm the changes in mycelium morphology and reduction in the number of spore germination at 0.2 mg/mL concentration Tb-ZnO Np's.

Green synthesis of metal nanoparticles using microorganisms and their application in the agrifood sector.

The agricultural sector is currently facing many global challenges, such as climate change, and environmental problems such as the release of pesticides and fertilizers, which will be exacerbated in the face of population growth and food shortages. Therefore, the need to change traditional farming methods and replace them with new technologies is essential, and the application of nanotechnology, especially green technology offers considerable promise in alleviating these problems. Nanotechnology has led to changes and advances in many technologies and has the potential to transform various fields of the agricultural sector, including biosensors, pesticides, fertilizers, food packaging and other areas of the agricultural industry. Due to their unique properties, nanomaterials are considered as suitable carriers for stabilizing fertilizers and pesticides, as well as facilitating controlled nutrient transfer and increasing crop protection. The production of nanoparticles by physical and chemical methods requires the use of hazardous materials, advanced equipment, and has a negative impact on the environment. Thus, over the last decade, research activities in the context of nanotechnology have shifted towards environmentally friendly and economically viable 'green' synthesis to support the increasing use of nanoparticles in various industries. Green synthesis, as part of bio-inspired protocols, provides reliable and sustainable methods for the biosynthesis of nanoparticles by a wide range of microorganisms rather than current synthetic processes. Therefore, this field is developing rapidly and new methods in this field are constantly being invented to improve the properties of nanoparticles. In this review, we consider the latest advances and innovations in the production of metal nanoparticles using green synthesis by different groups of microorganisms and the application of these nanoparticles in various agricultural sectors to achieve food security, improve crop production and reduce the use of pesticides. In addition, the mechanism of synthesis of metal nanoparticles by different microorganisms and their advantages and disadvantages compared to other common methods are presented.

Optimization of green silver nanoparticles as nanofungicides for management of rice bakanae disease.

Rice bakanae, a devastating seed-borne disease caused by Fusarium species requires a more attractive and eco-friendly management strategy. The optimization of plant-mediated silver nanoparticles (AgNPs) as nanofungicides by targeting Fusarium species may be a rational approach. In this study, Azadirachta indica leaf aqueous extract-based AgNPs (AiLAE-AgNPs) were synthesized through the optimization of three reaction parameters: A. indica leaf amount, plant extract-to-AgNO3 ratio (reactant ratio), and incubation time. The optimized green AgNPs were characterized using ultraviolet-visible light (UV-Vis) spectroscopy, field emission scanning electron microscopy (FESEM) with energy dispersive X-ray (EDX) spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), and powder X-ray diffraction (XRD) techniques. The optimal conditions for producing spherical, unique, and diminutive-sized AgNPs ranging from 4 to 27 nm, with an average size of 15 nm, were 2 g AiLAE at a 1:19 ratio (extract-to-AgNO3) and incubated for 4 h. Fusarium isolates collected from infected soils and identified as F. fujikuroi (40) and F. proliferatum (58 and 65) by PCR were used for seed infestation. The AgNPs exhibited concentration-dependent mycelial growth inhibition with EC50 values ranging from 2.95 to 5.50 µg/mL. The AgNPs displayed exposure time-dependent seed disinfectant potential (complete CFU reduction in F. fujikuroi (40) and F. proliferatum (58) was observed at a concentration of 17.24 µg/mL). The optimized green AgNPs were non-toxic to germinating seeds, and completely cured bakanae under net-house conditions, suggesting their great nano-fungicidal potency for food security and sustainable agriculture.

Nanocapsules of ZnO Nanorods and Geraniol as a Novel Mean for the Effective Control of Botrytis cinerea in Tomato and Cucumber Plants.

Inorganic-based nanoparticle formulations of bioactive compounds are a promising nanoscale application that allow agrochemicals to be entrapped and/or encapsulated, enabling gradual and targeted delivery of their active ingredients. In this context, hydrophobic ZnO@OAm nanorods (NRs) were firstly synthesized and characterized via physicochemical techniques and then encapsulated within the biodegradable and biocompatible sodium dodecyl sulfate (SDS), either separately (ZnO NCs) or in combination with geraniol in the effective ratios of 1:1 (ZnOGer1 NCs), 1:2 (ZnOGer2 NCs), and 1:3 (ZnOGer2 NCs), respectively. The mean hydrodynamic size, polydispersity index (PDI), and ζ-potential of the nanocapsules were determined at different pH values. The efficiency of encapsulation (EE, %) and loading capacity (LC, %) of NCs were also determined. Pharmacokinetics of ZnOGer1 NCs and ZnOGer2 NCs showed a sustainable release profile of geraniol over 96 h and a higher stability at 25 ± 0.5 °C rather than at 35 ± 0.5 °C. ZnOGer1 NCs, ZnOGer2 NCs and ZnO NCs were evaluated in vitro against B. cinerea, and EC50 values were calculated at 176 µg/mL, 150 µg/mL, and > 500 µg/mL, respectively. Subsequently, ZnOGer1 NCs and ZnOGer2 NCs were tested by foliar application on B. cinerea-inoculated tomato and cucumber plants, showing a significant reduction of disease severity. The foliar application of both NCs resulted in more effective inhibition of the pathogen in the infected cucumber plants as compared to the treatment with the chemical fungicide Luna Sensation SC. In contrast, tomato plants treated with ZnOGer2 NCs demonstrated a better inhibition of the disease as compared to the treatment with ZnOGer1 NCs and Luna. None of the treatments caused phytotoxic effects. These results support the potential for the use of the specific NCs as plant protection agents against B. cinerea in agriculture as an effective alternative to synthetic fungicides.

Zinc nanoparticles: Mode of action and efficacy against boscalid-resistant Alternaria alternata isolates.

The antifungal potential of ZnO-NPs against Alternaria alternata isolates with reduced sensitivity to the succinate dehydrogenase inhibitor (SDHI) boscalid, resulting from target site modifications, was evaluated in vitro and in vivo. ZnO-NPs could effectively inhibit mycelial growth in a dose-dependent way in both boscalid (BOSC) sensitive (BOSC-S) and resistant (BOSC-R) isolates. The fungitoxic effect of ZnO-NPs against the pathogen was significantly enhanced when combined with boscalid compared to the individual treatments in all phenotype cases (BOSC-S/R) both in vitro and in vivo. Fungitoxic effect of ZnO-NPs could be, at least partly, attributed to zinc ion release as indicated by the positive correlation between sensitivities to the nanoparticles and their ionic counterpart ZnSO4 and the alleviation of the ZnO-NPs fungitoxic action in the presence of the strong chelating agent EDTA. The superior effectiveness of ZnO-NPs against A. alternata, compared to ZnSO4, could be due to nanoparticle properties interfering with cellular ion homeostasis mechanisms. The observed additive action of the oxidative phosphorylation-uncoupler fluazinam (FM) against all phenotypes indicates a possible role of ATP-dependent ion efflux mechanism in the mode of action of ZnO-NPs. A potential role of ROS production in the fungitoxic action of ZnO-NPs was evident by the additive/synergistic action of salicylhydroxamate (SHAM), which blocks the alternative oxidase antioxidant action. Mixture of ZnO-NPs and boscalid, resulting in a "capping" effect for the nanoparticles and significantly reducing their mean size, probably accounted for the synergistic effect of the mixture against both sensitive and resistant A. alternata isolates. Summarizing, results indicated that ZnO-NPs can be effectively used against A. alternata both alone or in combination with boscalid, providing an effective tool for combating SDHI-resistance and reducing the environmental fingerprint of synthetic fungicides.

Metal nanoparticles against fungicide resistance: alternatives or partners?

Chemical control suffers from the loss of available conventional active ingredients due to strict environmental safety regulations which, combined with the loss of fungicide efficacy due to resistance development, constitute major problems of contemporary crop protection. Metal-containing nanoparticles (MNPs) appear to have all the credentials to be next-generation, eco-compatible fungicide alternatives and a valuable anti-resistance management tool. Could the introduction of MNPs as nano-fungicides be the answer to both reducing the environmental footprint of xenobiotics and dealing with fungicide resistance? The potential of MNPs to be utilized as nano-fungicides, both as alternatives to conventional fungicides or/and as partners in combating fungicide resistance, is discussed in terms of effectiveness, potential antimicrobial mechanisms as well as synergy profiles with conventional fungicides. However, their "golden" potential to be used both as alternatives and partners of conventional fungicides to combat resistance and reduce environmental pollution is challenged by undesirable effects towards non-target organisms such as phytotoxicity, toxicity to humans and environmental ecotoxicity, constituting risks that should be considered before their commercial introduction as nano-pesticides at a large scale. © 2022 Society of Chemical Industry.

Differential Antimycotic and Antioxidant Potentials of Chemically Synthesized Zinc-Based Nanoparticles Derived from Different Reducing/Complexing Agents against Pathogenic Fungi of Maize Crop.

The present study aimed for the synthesis, characterization, and comparative evaluation of anti-oxidant and anti-fungal potentials of zinc-based nanoparticles (ZnNPs) by using different reducing or organic complexing-capping agents. The synthesized ZnNPs exhibited quasi-spherical to hexagonal shapes with average particle sizes ranging from 8 to 210 nm. The UV-Vis spectroscopy of the prepared ZnNPs showed variation in the appearance of characteristic absorption peak(s) for the various reducing/complexing agents i.e., 210 (NaOH and NaBH4), 220 (albumin, and thiourea), 260 and 330 (starch), and 351 nm (cellulose) for wavelengths spanning over 190-800 nm. The FT-IR spectroscopy of the synthesized ZnNPs depicted the functional chemical group diversity. On comparing the antioxidant potential of these ZnNPs, NaOH as reducing agent, (NaOH (RA)) derived ZnNPs presented significantly higher DPPH radical scavenging potential compared to other ZnNPs. The anti-mycotic potential of the ZnNPs as performed through an agar well diffusion assay exhibited variability in the extent of inhibition of the fungal mycelia with maximum inhibition at the highest concentration (40 mg L-1). The NaOH (RA)-derived ZnNPs showcased maximum mycelial inhibition compared to other ZnNPs. Further, incubation of the total genomic DNA with the most effective NaOH (RA)-derived ZnNPs led to intercalation or disintegration of the DNA of all the three fungal pathogens of maize with maximum DNA degrading effect on Macrophomina phaseolina genomic DNA. This study thus identified that differences in size and surface functionalization with the protein (albumin)/polysaccharides (starch, cellulose) diminishes the anti-oxidant and anti-mycotic potential of the generated ZnNPs. However, the NaOH emerged as the best reducing agent for the generation of uniform nano-scale ZnNPs which possessed comparably greater anti-oxidant and antimycotic activities against the three test maize pathogenic fungal cultures.

Synergistic Antifungal Activity of Graphene Oxide and Fungicides against Fusarium Head Blight In Vitro and In Vivo.

Plant pathogens constantly develop resistance to antimicrobial agents, and this poses great challenges to plant protection. Therefore, there is a pressing need to search for new antimicrobials. The combined use of antimicrobial agents with different antifungal mechanisms has been recognized as a promising approach to manage plant diseases. Graphene oxide (GO) is a newly emerging and highly promising antimicrobial agent against various plant pathogens in agricultural science. In this study, the inhibitory activity of GO combined with fungicides (Mancozeb, Cyproconazol and Difenoconazole) against Fusarium graminearum was investigated in vivo and in vitro. The results revealed that the combination of GO and fungicides has significant synergistic inhibitory effects on the mycelial growth, mycelial biomass and spore germination of F. graminearum relative to single fungicides. The magnitude of synergy was found to depend on the ratio of GO and fungicide in the composite. In field tests, GO-fungicides could significantly reduce the disease incidence and disease severity, exhibiting a significantly improved control efficacy on F. graminearum. The strong synergistic activity of GO with existing fungicides demonstrates the great application potential of GO in pest management.

Synthesis and Characterization of Novel Copper Nanoparticles for the Control of Leaf Spot and Anthracnose Diseases of Olive.

Olive crop is frequently treated with copper fungicides to combat foliar and fruit diseases such as olive leaf spot caused by Fusicladium oleagineum and anthracnose caused by Colletotrichum spp. The replacement of copper-based products with more eco-friendly alternatives is a priority. Metal nanoparticles synthesized in several ways have recently revolutionized crop protection with applications against important crop pathogens. In this study, we present the development of four copper-based nanoparticles (CuNP Type 1 to 4) synthesized with a wet chemistry approach. The CuNPs were characterized using Transmission Electron Microscopy, Dynamic Light Scattering, Laser Doppler Electrophoresis, and Attenuated Total Reflection measurements. In addition, the activity of the four CuNP types was tested in vitro and in planta against F. oleagineum and Colletotrichum spp. In vitro sensitivity measurements showed that for both pathogens, mycelial growth was the most susceptible developmental stage to the tested compounds. Against both pathogens, CuNP Type 1 and Type 2 were found to be more active in reducing mycelial growth compared to the reference commercial compounds of copper oxide and copper hydroxide. In planta experiments showed that CuNP Type 3 and CuNP Type 4 exhibited a strong protectant activity against both F. oleagineum and Colletotrichum acutatum with control efficacy values significantly higher than those achieved by the applications of either reference product.

Use of silver nanoparticles to counter fungicide-resistance in Monilinia fructicola.

The potential of Ag-NPs to suppress Monilia fructicola isolates and to broaden the effectiveness of fungicides to overcome resistance was tested in vitro and in vivo. Twenty-three M. fructicola isolates were subjected to fungitoxicity screening with a number of fungicides in vitro, which resulted in the detection of 18 isolates resistant to benzimidazoles (BEN-R) thiophanare methyl (TM) and carbendazim (CARB). DNA sequencing revealed the E198A resistance mutation in the ß-tubulin gene, target site of the benzimidazole fungicides in all resistant isolates. Ag-NPs effectively suppressed mycelial growth in both sensitive (BENS) and resistant isolates. The combination of Ag-NPs with TM led to a significantly enhanced fungitoxic effect compared to the individual treatments regardless resistant phenotype (BEN-R/S) both in vitro and when applied on apple fruit. The above observed additive/synergistic action is probably associated with an enhanced Ag-NPs activity/availability as indicated by the positive correlation between Ag-NPs and TM + Ag-NPs treatments. No correlation was found between AgNO3 and Ag-NPs suggesting that difference(s) exist in the fungitoxic mechanism of action between nanoparticles and their ionic counterparts. Synergy observed between Ag-NPs and the oxidative phosphorylation-uncoupler fluazinam (FM) against both resistance phenotypes indicates a possible role of energy (ATP) metabolism in the mode of action of Ag-NPs. Additionally, the role of released silver ions on the fungitoxic action of Ag-NPs against M. fructicola was found to be limited because the combination with NaCl revealed a synergistic rather than the antagonistic effect that would be expected from silver ion binding with chlorine ions. The results of this study suggested that Ag-NPs can be effectively used against M. fructicola and when used in combination with conventional fungicides they could provide the means for countering benzimidazole resistance and at the same time reduce the environmental impact of synthetic fungicides by reducing doses needed for the control of the pathogen.

Synergy between Cu-NPs and fungicides against Botrytis cinerea.

Combating drug-resistance is a daunting task, especially due to the shortage of available drug alternatives with multisite modes of action. In this study, the potential of copper nanoparticles (Cu-NPs) to suppress 15 Botrytis cinerea isolates, which are sensitive or resistant to fungicides, alone or in combination with conventional fungicides, was tested in vitro and in vivo. Sensitivity screening in vitro revealed two fungicide resistance phenotypes, resulting from target site mutations. DNA sequencing revealed three B. cinerea isolates highly resistant to benzimidazoles (BEN-R), thiophanare methyl (TM), and carbendazim, bearing the E198A resistance mutation in the ß-tubulin gene, and four isolates highly resistant to the QoI pyraclostrobin (PYR-R) with a G143A mutation in the cytb gene. Cu-NPs were equally effective against sensitive and resistant isolates. An additive/synergistic effect was observed between Cu-NPs and TM in the case of BEN-S isolates both in vitro and when applied in apple fruit. A positive correlation was observed between TM and TM + Cu-NPs treatments, suggesting that an increased TM availability in the target site could be related with the observed additive/synergistic action. No correlation between Cu(OH)2 and Cu-NPs sensitivity was found, indicating that different mechanisms govern the fungitoxic activity between nano and bulk counterparts. A synergistic profile was observed between Cu-NPs and fluazinam (FM) - an oxidative phosphorylation inhibitor - in all isolates regardless of resistance phenotype, suggesting that ATP metabolism could be involved in the mode of action of Cu-NPs. Furthermore, the observed cross sensitivity and antagonistic action between Cu-NPs and NaCl also provided evidence for copper ions contribution to the fungitoxic action of Cu-NPs. The results suggested that Cu-NPs in combination with conventional fungicides can provide the means for an environmentally safe, sustainable resistance management strategy by reducing fungicide use and combating resistance against B. cinerea.

Use of copper, silver and zinc nanoparticles against foliar and soil-borne plant pathogens.

Nano-fungicides are expected to play an important role in future plant disease management as eco-friendly alternatives of conventional synthetic fungicides. In the present study, the sensitivity of seven fungal species, known to cause foliar and soil-borne diseases, to nanoparticles (NPs) containing copper (Cu-NPs, CuO-NPs), silver (Ag-NPs) and zinc (ZnO-NPs) was assessed in vitro. Mycelial growth assays revealed that Cu-NPs with mean inhibition rates, EC50, ranging between 162 and 310 µg/mL were most effective among the NPs tested in inhibiting fungal growth, followed by ZnO-NPs with EC50 ranging between 235 and 848 µg/mL. All fungal species were practically insensitive to CuO-NPs and Ag-NPs except for B. cinerea, which was equally sensitive to Ag-NPs and Cu-NPs (EC50 = 307 µg/mL). Cu-NPs were more fungitoxic in terms of mycelial growth, to almost all species tested, than a protective fungicide containing Cu(OH)2, which was used as a reference. Fungitoxicity experiments with the NPs tested and bulk size reagents containing the respective metals revealed that ZnO-NPs were more toxic to all fungal species tested than ZnSO4, whereas Cu-NPs were more fungitoxic than CuSO4 in all cases, except for B. cinerea, A. alternata and M. fructicola. The existence of a positive correlation between Cu-NPs and CuO-NPs toxicity and, at the same time, the absence of any correlation between NPs tested and their respective bulk metal counterparts indicated potential differences in the mode of action between bulk and nanosized antifungal ingredients. Although there was considerable variation between fungal species, all NPs were generally 10 to 100 fold more fungitoxic to spores than hyphae and in the majority of cases more effective than Cu(OH)2, as revealed by colony formation bioassays. NPs significantly suppressed grey mold symptoms on plum fruit, especially Ag-NPs, which completely inhibited disease development. Consequently, tested NPs have the potential to be used as protective antifungal agents.

ZnO-based nanofungicides: Synthesis, characterization and their effect on the coffee fungi Mycena citricolor and Colletotrichum sp.

In this work we compare the antifungal capacity of zinc oxide nanoparticles (ZnO-NPs) synthesized by a chemical route and a ZnO-based nanobiohybrid obtained by green synthesis in an extract of garlic (Allium sativum). To find out the characteristics of the materials synthesized, X-ray diffraction (XRD), IR spectroscopy and absorption in UV-Vis were used, as well as both scanning (SEM) and transmission (TEM) electron microscopy. The results showed that the samples obtained were of nanometric size (<100 nm), with a predominance of the wurtzite crystal phase of ZnO and little crystallization of the nanobiohybrids. Their antifungal capacity on two pathogenic fungi of coffee, Mycena citricolor (Berk and Curt) and Colletotrichum sp. was also evaluated. Both nanomaterials showed an efficient antifungal capacity, particularly the nanobiohybrids, with ~97% inhibition in growth of M. citricolor, and ~93% for Colletotrichum sp. The microstructural study with high resolution optical (HROM) and ultra-structural microscopy (using TEM) carried out on the fungi treated with the synthesized nanomaterials showed a strong nanofungicidal effect on the vegetative and reproductive structures and fungal cell wall, respectively. The inhibition of the growth of the fungi and micro and ultra-structural affectations were explained considering that the size of the nanomaterials allows them to pass easily through the cell membranes. This indicates that they can be absorbed easily by the fungi tested here, causing cellular dysfunction. Nanofungicide effects are also attributable to the unique properties of nanomaterials, such as the high surface-to-bulk ratio of atoms and their surface physicochemical characteristics that could directly or indirectly produce reactive oxygen species (ROS), which affect the proteins of the cell wall.