Copper nanoparticles interfere with insecticide sensitivity, fecundity and endosymbiont abundance in olive fruit fly Bactrocera oleae (Diptera: Tephritidae).

BACKGROUND: The potential of copper nanoparticles (Cu-NPs) to be used as an alternative control strategy against olive fruit flies (Bactrocera oleae) with reduced sensitivity to the pyrethroid deltamethrin and the impact of both nanosized and bulk copper hydroxide (Cu(OH)2) on the insect's reproductive and endosymbiotic parameters were investigated. RESULTS: The application of nanosized and bulk copper applied by feeding resulted in significant levels of adult mortality, comparable to or surpassing those achieved with deltamethrin at recommended doses. Combinations of Cu-NPs or copper oxide nanoparticles (CuO-NPs) with deltamethrin significantly enhanced the insecticide's efficacy against B. oleae adults. When combined with deltamethrin, Cu-NPs significantly reduced the mean total number of offspring compared with the control, and the number of stings, pupae, female and total number of offspring compared with the insecticide alone. Both bulk and nanosized copper negatively affected the abundance of the endosymbiotic bacterium Candidatus Erwinia dacicola which is crucial for the survival of B. oleae larvae. CONCLUSION: The Cu-NPs can aid the control of B. oleae both by reducing larval survival and by enhancing deltamethrin performance in terms of toxicity and reduced fecundity, providing an effective anti-resistance tool and minimizing the environmental footprint of synthetic pesticides by reducing the required doses for the control of the pest. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

Fungicide resistance frequencies of Botrytis cinerea greenhouse isolates and molecular detection of a novel SDHI resistance mutation.

Sensitivity of B. cinerea to commonly used fungicides against Gray mold with emphasis to the newer quinone outside inhibitor (QoIs), and succinate dehydrogenase inhibitors (SDHIs) was assessed during a monitoring survey from vegetable greenhouses in four representative regions of Crete. 42% from a total of 168 isolates were simultaneously resistant to boscalid, fluopyram, pyraclostrobin and fenhexamid but not to fludioxonil making this phenylpyrrole fungicide an excellent anti-resistance antifungal agent. Isolates with double resistance to SDHIs and QoIs were found in very high frequencies indicating a selection towards double resistance due to the use of pyraclostrobin-boscalid mixtures. A number of sdhB resistance mutations (H272R, N230I and P225F/H) were found in isolates also carrying the G143A cytb resistance mutation in the above isolates. A novel sdhB point mutation (I274V) was identified for the first time in B. cinerea isolates collected from greenhouses with a fluopyram spray history with specific resistance to SDHIs. A PCR-RFLP diagnostic assay was developed for the detection of this mutation in the sdhB gene. Mutations P225F/H and I274V were found to be associated with fitness penalties in terms of mycelial growth, sporulation or pathogenicity. Results suggest that, in order to retain effective control of gray mold in Crete, appropriate anti-resistance strategies should be implemented taking into account the high double SDHI and QoI resistance frequencies. Additional studies for monitoring the already known and the new SDHI-resistance mutations, are necessary in order to hinder the further spread and establishment of single or double resistant isolates of B. cinerea detected in greenhouses in Crete.

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: Phytotoxicity on tomato and effect on symbiosis with the Fusarium solani FsK strain.

The effect of copper (Cu-NPs, CuO-NPs), silver (Ag-NPs) and zinc oxide (ZnO-NPs) nanoparticles (NPs) on plant growth, physiological properties of tomato plants and their symbiotic relationships with the endophytic Fusarium solani FsK strain was investigated. Fungitoxicity tests revealed that the FsK strain was significantly more sensitive to Cu-NPs and ZnO-NPs than CuO-NPs and Ag-NPs both in terms of mycelial growth and spore germination. All NPs were more toxic to FsK compared to their bulk counterparts except for AgNO3, which was 8 to 9-fold more toxic than Ag-NPs. Apart from AgNO3, NPs and bulk counterparts did not affect the number of germinated tomato seeds even in higher concentrations, while root length was significantly reduced in a dose dependent way in most cases. Dry weight of tomato plants was also significantly reduced upon treatment with NPs and counterparts with most pronounced effects in the cases of AgNO3, Cu-NPs, ZnO-NPs, and ZnSO4. Root and shoot length of grown tomato plants was also affected by treatments while differences between NPs and bulk counterparts varied. A marked oxidative stress response was recorded in all cases of NPs/bulk counterparts as indicated by increased MDA and H2O2 levels of treated plants. Treated plants had significantly reduced chlorophyl-a and carotenoid levels compared to the untreated control. NPs and counterparts did not affect FsK colonization of roots indicating a possible shielding effect of tomato plants once the endophyte was established inside the roots. Vice versa, a possible alleviation of CuO-NPs, ZnO-NPs, and ZnSO4 toxicity was observed in the presence of FsK inside tomato roots in terms of plant dry weight. The results suggest that phytotoxicity of NPs in tomato treated plants should be considered before application and while both FsK and tomato are sensitive to NPs, their reciprocal benefits may extent to resistance towards these toxic agents.

Copper nanoparticles against benzimidazole-resistant Monilinia fructicola field isolates.

Nano-fungicides are expected to play an important role in future plant disease management. Their unique properties include a broad antimicrobial action, increased effectiveness in lower doses, slower a.i. release and/or enhanced drug delivery and an ability to control drug-resistant pathogens, which makes them appealing candidates for use as eco-friendly antifungal alternatives to counter fungicides resistance. Copper nanoparticles (Cu-NPs) could suppress mycelial growth in both sensitive (BENS) and resistant (BEN-R) Monilinia fructicola isolates harboring the E198A benzimidazole resistance mutation, more effectively than copper oxide NPs (CuO-NPs) and Cu(OH)2. A significant synergy of Cu-NPs with thiophanate methyl (TM) was observed against BEN-S isolates both in vitro and when applied on plum fruit suggesting enhanced availability or nanoparticle induced transformation of TM to carbendazim. ATP-dependent metabolism is probably involved in the mode of fungitoxic action of Cu-NPs as indicated by the synergy observed between Cu-NPs and the oxidative phosphorylation-uncoupler fluazinam (FM). Copper ion release contributed in the toxic action of Cu-NPs against M. fructicola, as indicated by synergism experiments with ethylenediaminetetraacetic acid (EDTA), although the lack of correlation between nano and bulk/ionic copper forms indicate an additional nano-property mediated mechanism of fungitoxic action. Results suggested that Cu-NPs can be effectively used in future plant disease management as eco-friendly antifungal alternatives to counter fungicides resistance and reduce the environmental footprint of synthetic fungicides.

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.

Characterization of boscalid-resistance conferring mutations in the SdhB subunit of respiratory complex II and impact on fitness and mycotoxin production in Penicillium expansum laboratory strains.

Laboratory mutants of Penicillium expansum highly resistant (Rfs: 90 to >500, based on EC50s) to Succinate Dehydrogenase Inhibitors (SDHIs) were isolated after UV-mutagenesis and selection on media containing boscalid. A positive correlation was found between sensitivity of isolates to boscalid and other SDHIs such as isopyrazam and carboxin but not to fungicides affecting other cellular pathways or processes, such as the triazole flusilazole, the phenylpyrrole fludioxonil, the anilinopyrimidine cyprodinil and the benzimidazole benomyl. Most of the boscalid-resistant strains were more sensitive to the SDHI fluopyram and the QoI pyraclostrobin. In order to investigate the mechanism responsible for the observed resistance profiles, part of the SdhB subunit isolated the wild type and boscalid-resistant isolates, was genetically characterized. Comparison of the deduced amino-acid sequence between resistant and wild-type isolates revealed two point mutations at a position corresponding to codon 272 of the respective SdhB protein in Botrytis cinerea. The substitution of histidine by arginine was found in boscalid-resistant isolates which were equally sensitive to fluopyram compared with the wild-type whereas the replacement of histidine by tyrosine was found in strains with increased sensitivity to fluopyram. No adverse effects of resistance mutations were observed on fitness determining parameters such as osmotic sensitivity, sporulation and pathogenicity, while mycelial growth rate and spore germination was negatively affected in some of the mutants studied. P. expansum mutant strains displayed significantly perturbed patulin and citrinin levels as compared to the wild-type parent strain both in vitro and in vivo as revealed by thin layer (TLC) and high performance liquid chromatography (HPLC).

Effect of phenylpyrrole-resistance on fitness parameters and ochratoxin production in Aspergillus carbonarius.

The risk of resistance development to fludioxonil and the potential implications of resistance mutations to ochratoxin production in Aspergillus carbonarius were investigated. Mutants of A. carbonarius highly resistant to phenylpyrroles were isolated at a high mutation frequency after N-MNTG-mutagenesis and selection on media containing fludioxonil. A highly reduced sensitivity to fungicides belonging to the same cross-resistance group (AHDs and phenylpyrroles) such as the aromatic hydrocarbon tolclofos-methyl and the dicarboximide fungicides iprodione and vinclozolin was also observed. No cross-resistance relationships were found between fludioxonil and the triazole epoxiconazole, the anilinopyrimidine cyprodinil and the chloronitrile chlorothalonil. Interestingly, fludioxonil-resistant isolates were highly sensitive to the QoI fungicide pyraclostrobin compared to the wild-type parental strain. Fitness studies revealed that resistance mutation(s) had a negative effect on mycelial growth, resistance to osmotic stress and pathogenicity of the fludioxonil-resistant strains. Mycotoxin analysis showed that most fludioxonil-resistant strains produce less quantities of ochratoxin A (OTA) than the wild-type strain both when grown on artificial medium and on grapes. Increased osmotic sensitivity and reduced pathogenicity of the mutant strains were significantly correlated with reduced ochratoxin production in vivo but not in vitro. The above-mentioned data indicate that fludioxonil is an excellent fungicide for the control of A. carbonarius in grapes and a valuable asset for farmers in terms of resistance management and ochratoxin contamination of grapes, vine products and wines.

Molecular characterization, fitness and mycotoxin production of benzimidazole-resistant isolates of Penicillium expansum.

Penicillium expansum field-strains resistant to benzimidazole fungicides were isolated in high frequency from decayed apple fruit collected from packinghouses and processing industries located in the region of Imathia, N. Greece. In vitro fungitoxicity tests resulted in the identification of two different resistant phenotypes: highly (BEN-HR) and moderately (BEN-MR) carbendazim-resistant. Thirty seven percent of the isolated P. expansum strains belonged to the BEN-HR phenotype, carried no apparent fitness penalties and exhibited resistance levels higher than 60 based on EC50 values. Cross resistance studies with other benzimidazole fungicides showed that all BEN-HR and BEN-MR isolates were also less sensitive to benomyl and thiabendazole. Fungitoxicity tests on the response of BEN-HR isolates to fungicides belonging to other chemical classes revealed no cross-resistance relationships between benzimidazoles and the phenylpyrrole fludioxonil, the dicarboximide iprodione, the anilinopyrimidine cyprodinil, the QoI pyraclostrobin, the imidazole imazalil and the triazole tebuconazole, indicating that a target-site modification is probably responsible for the BEN-HR phenotype observed. Contrary to the above, some BEN-MR isolates exhibited an increased sensitivity to cyprodinil compared to benzimidazole-sensitive ones. BEN-MR isolates had fitness parameters similar to the benzimidazole-sensitive isolates except for conidia production which appeared significantly decreased. Analysis of mycotoxin production (patulin and citrinin) showed that all benzimidazole-resistant isolates produced mycotoxins at concentrations significantly higher than sensitive isolates both on culture medium and on artificially inoculated apple fruit. Comparison of the ß-tubulin gene DNA sequence between resistant and sensitive isolates revealed a point mutation resulting from the E198A substitution of the corresponding protein in most but not all HR isolates tested. Molecular analysis of the ß-tubulin gene in moderately resistant isolates did not reveal any amino acid substitution. This is the first report on the existence and distribution of highly mycotoxigenic field isolates of P. expansum resistant to the benzimidazoles indicating a high potential risk of increased mycotoxin contamination of pome fruit and by-products.

Effect of anilinopyrimidine resistance on aflatoxin production and fitness parameters in Aspergillus parasiticus Speare.

Mutants of Aspergillus parasiticus resistant to the anilinopyrimidine fungicides were isolated at a high mutation frequency after UV-mutagenesis and selection on media containing cyprodinil. In vitro fungitoxicity tests resulted in the identification of two predominant resistant phenotypes that were highly (R(1)-phenotype) and moderately (R(2)-phenotype) resistant to the anilinopyrimidines cyprodinil, pyrimethanil and mepanipyrim. Cross-resistance studies with fungicides from other chemical groups showed that the highly resistance mutation(s) did not affect the sensitivity of R(1)-mutant strains to fungicides affecting other cellular pathways. Contrary to that, a reduction in the sensitivity to the triazoles epoxiconazole and flusilazole, the benzimidazole carbendazim, the phenylpyrrole fludioxonil, the dicarboximide iprodione and to the strobilurin-type fungicide pyraclostrobin was observed in R(2)-mutant strains. Study of fitness parameters of anilinopyrimidine-resistant strains of both phenotypic classes showed that all R(1) mutant strains had mycelial growth rate, sporulation and conidial germination similar to or even higher than the wild-type parent strain, while these fitness parameters were negatively affected in R(2) mutant strains. Analysis of the aflatoxin production showed that most R(1) mutant strains produced aflatoxins at concentrations markedly higher than the wild-type parent strain. A considerable reduction in the aflatoxin production was observed on cultured medium and on wheat grains by all R(2) mutant strains, indicating a possible correlation between fitness penalties and aflatoxigenic ability of A. parasiticus. The potential risk of increased aflatoxin contamination of agricultural products and their byproducts by the appearance and predominance of highly aflatoxigenic mutant strains of A. parasiticus resistant to the anilinopyrimidines is discussed.