The potential of copper nanoparticles (Cu-NPs) to counter fungicide resistance was investigated using five spontaneous Altrernaria alternata mutants highly resistant to fludioxonil. Resistant isolates carried mutations causing premature termination of AaHK1, the target gene of phenylpyrrole fungicides. Resistance mutations had no adverse effects on mycelial growth and virulence while fludioxonil-resistant isolates were more sensitive to osmotic stress and produced less conidia compared to the wild-type parental strains. Cu-NPs exerted a significant fungitoxic effect against both wild-type and resistant isolates, which was superior to that exerted by a reference fungicide containing Cu(OH)2. A profound synergistic effect against wild-type and resistant A. alternata isolates resulted from the combination of Cu-NPs with fludioxonil or iprodione, probably due to enhanced fungicide bioavailability. The fungitoxic mode of action of Cu-NPs extended beyond copper ion release as indicated by both the synergistic rather-than-antagonistic effect of the strong chelating agent EDTA when mixed with Cu-NPs and the lack of any correlation between Cu-NPs and Cu(OH)2. The enhanced antifungal activity of Cu-NPs + EDTA may stem from a reduction of NP size due to a capping effect of the chelating agent. Furthermore, ATP-dependent ion efflux may contribute to the fungitoxic effect of Cu-NPs against A. alternata, as indicated by the additive effect between NPs and the oxidative phosphorylation uncoupler fluazinam. Concluding, Cu-NPs can serve as an effective fungicide alternative against A. alternata, a promising anti-resistance strategy when combined with fludioxonil, and a means for minimizing the environmental impact of synthetic fungicides.

Anthracnose, caused by Colletotrichum fungal species, is among the most prevalent and severe both pre- and postharvest diseases affecting various fruits, including olive, citrus, and avocado. In an era where chemical control is being challenged by fungicide resistance development and strict EU legislation, causing the withdrawal of many established active ingredients used for disease management, metallic nanoparticles (MNPs) represent promising, eco-friendly fungicide-alternatives for controlling pathogens. In this study, the effectiveness and potential fungitoxicity mechanisms of copper nanoparticles (Cu-NPs) against Colletotrichum gloeosporioides strains isolated from orange and avocado fruit were investigated both in vitro and in vivo. Cu-NPs outperformed their ionic counterpart Cu(OH)2—used as a reference fungicide- in reducing mycelial growth and disease symptoms. Pearson correlation analysis revealed a positive cross-resistance between Cu-NPs and the demethylase inhibitor (DMI) fungicide difenoconazole. A significant antagonism between Cu-NPs and detoxification inhibitors diethyl malate, piperonyl butoxide as well as DMI fungicides difenoconazole, prochloraz, and prothioconazole used against the pathogen was observed. Furthermore, an induced C-14 alpha-demethylase gene (CgCyp51) overexpression was found, at least partly, to account for the observed superior fungitoxic action of Cu-NPs against C. gloeosporioides. Overall, Cu-NPs demonstrate exceptional potential as alternative fungicides against C. gloeosporioides, outperforming conventional treatments through multiple mechanisms. A key mode of action involving disrupting ergosterol biosynthesis, as evidenced by the significant upregulation of the CgCyp51 gene in response to Cu-NP exposure was identified for the first time.

The insecticide lambda-cyhalothrin a type II synthetic parathyroid. It is a hydrophobic and highly effective broadspectrum insecticide commonly used in pest management. However, its presence in subsurface formations poses environmental toxicity risks and potentially may have adverse effects on humans. Chitosan, a polymer with unique physicochemical and absorption properties, is utilized in numerous various industrial applications. In this study, the interactions of lambda-cyhalothrin with chitosan in the presence and absence of quartz sand were investigated under static, dynamic (batch), and column transport conditions at 25 degrees C. The experimental adsorption data were effectively modeled using a pseudo-second-order adsorption kinetic model, while the transport experiments were simulated with an advection dispersion model incorporating two-site linear or nonlinear particle attachment. The results indicate that the observed lambda-cyhalothrin adsorption onto either Chitosan or quartz sand is governed by chemisorption. Cotransport experiments revealed bidirectional interactions, where chitosan forms aggregates with lambda-cyhalothrin, enhancing chitosan attachment and significantly reducing lambda-cyhalothrin retention. This, highlights chitosan's potential as a remediation agent for mitigating pesticide contamination in porous media.

Sensitivity assessment of 300 Cercospora beticola isolates collected from North Greece revealed that 38 % of the population was highly resistant to at least one of the demethylase inhibitors (DMIs) difenoconazole, epoxiconazole and flutriafol. Resistance factors greater than 50, 100 and 100 were calculated for the most resistant C. beticola isolates to flutriafol, epoxiconazole and difenoconazole, respectively. DMI-resistant isolates carried fitness penalties in terms of mycelial growth, sporulation and pathogenicity, probably accounting for their low frequencies in the field. Pearson correlation analysis revealed a positive cross-resistance relationship between all three DMIs tested suggesting the existence of a target-gene (CbCyp51) related resistance mechanism in the observed phenotypes. Sequence analysis of the CbCyp51 gene from sensitive and DMI-resistant isolates revealed a novel mutation (E149K) present only in C. beticola isolates resistant to all three fungicides tested. To the best of the author's knowledge, this is the first report of this target-site mutation associated with high resistance levels to all flutriafol, epoxiconazole and difenoconazole fungicides. No target-site mutations were found in the rest of DMI-resistance phenotypes. A compound-specific induced CbCyp51 overexpression was observed in C. beticola isolates with specific resistance to flutriafol, epoxiconazole, and difenoconazole, while no indication of constitutive overexpression was found. Overall, the results of the present study provide insights on the mechanisms responsible for the occurrence of high DMI-resistance in the field and a basis for establishing effective strategies for the control of C. beticola in sugar beet fields in Greece.

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. (c) 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Human activities in modern life are contributing significantly to global environmental pollution. With the need for clean drinking water ever increasing, so does the need to find new water-cleaning technologies. The ability of nanoparticles (NPs) to remove persistent pollutants from aqueous solutions makes them very important for use in water treatment technology. Titanium dioxide (TiO2) is recognized as an NP with unique optical, thermal, electrical, and magnetic properties and is widely used as an adsorbent material. Due to the extensive use of pesticides, their removal from the aquatic environment has gained widespread attention from the scientific community. In the present work, the transport of pesticide thiophanate methyl (TM), as well as the cotransport of TM and TiO2 nanoparticles, in a water-saturated column packed with quartz sand under various water conditions were investigated. Several ionic strengths (1, 10, 50, and 100 mM) and pH values (3, 5, 7, and 10) were examined. The results from the transport experiments were fitted and analyzed with the use of the ColloidFit software, while the results from the cotransport experiments were fitted with a modified version of a recently developed mathematical cotransport model. The results of this study suggested that the lowest mass recovery rate was for the cotransport experiments with the addition of NaCl. Furthermore, it was shown that TM has a weak affinity for sand but a relatively strong affinity for TiO2 at high ionic strength and acidic pH, probably accounting for the reduced mass recovery of TM in cotransport experiments.

<p>This study investigates the transport of pesticide thiophanate methyl (TM) as well as the co-transport of TM and titanium dioxide (TiO<sub>2</sub>) nanoparticles in a water saturated column packed with quartz sand under various water conditions. Several ionic strengths (Is) (1, 10, 50 and 100 mM) and pH (3, 5, 7, 10) values were examined. The results from the transport experiments  were fitted and analyzed with the use of the ColloidFit software, while the results from cotransport experiments were fitted with a modified mathematical model of Katzourakis and Chrysikopoulos (2015). The results suggested that the lowest mass recovery rate was for the co-transport experiments with the addition of NaCl. It was shown that TM has a weak affinity for sand but a relatively strong affinity for TiO<sub>2</sub> at high Is and acidic pH. Furthermore, salinity was shown to have significant effects on TM removal.</p>

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.

Metal nanoparticles constitute promising, eco-compatible alternatives to be used as nano-fertilizers or nano-fungicides although their potential impact on the agroecosystem is poorly studied. In the present study, the impact of copper (Cu-NPs, CuO-NPs), silver (Ag-NPs) and zinc oxide (ZnO-NPs) nanoparticles (NPs) on tomato plant development, physiological properties and the symbiotic relationship with the endophytic Fusarium solani FsK strain was assessed in comparison with their respective bulk/ionic counterparts. Both NPs and their counterparts did not affect the number of germinated tomato seeds even at higher concentrations except for AgNO3, which significantly decreased seed germination rates. On the contrary, a dose dependent decrease of root length was observed in most NP/bulk treatment cases. This was also the case for dry weight of tomato plants which was also significantly reduced upon treatment with NPs and counterparts especially in the cases of AgNO3, Cu-NPs, ZnO-NPs, and ZnSO4. Although differences between NPs and bulk counterparts varied, root and shoot length of grown tomato plants was also negatively affected by treatments. NPs/bulk counterpart treatments resulted in a marked oxidative stress response as indicated by increased MDA and H2O2 levels of treated plants. Photosynthetic pigments were also significantly affected by NP/bulk treatments, a fact evident from the reduced chlorophyl-a and carotenoid levels recorded. The FsK tomato-symbiotic strain was significantly more sensitive to Cu-NPs and ZnO-NPs than CuO-NPs and Ag-NPs as revealed in both mycelial growth and spore germination fungitoxicity tests. With the exception of AgNO3,which was 8 to 9-fold more toxic than Ag-NPs, all NPs were more fungitoxic to FsK than their bulk/ionic counterparts. FsK colonization of roots was not significantly affected by treatments with NPs and counterparts indicating that, once established inside the roots, the endophyte is shielded against the toxic effect of metals. At the same time, an alleviation of CuO-NPs, ZnO-NPs,and ZnSO4 phytoxicity was observed when FsK was present inside tomato roots in terms of plant dry weight. Concluding, results suggest that phytotoxicity of NPs in tomato treated plants should be considered before nano-fertilizer/fungicide treatments while the benefits of FsK inoculation of tomato plants may extent to resistance towards these toxic agents for both organisms.

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.

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.

Sensitivity and inherent resistance risk of Alternaria solani to fludioxonil, cross-resistance profiles and the potential implications of resistance mutations on fitness parameters were investigated. Fludioxonil was highly effective against a wild type A. solani field strain both in vitro (EC50 = 0.05 μg/mL) and in preventive applications on artificially inoculated tomato fruit. Mutants with low [Resistance factor (Rf): 15 based on EC50], medium (Rf: 150–300) and high (Rf: > 1000) levels of phenylpyrrole resistance were isolated from the wild type strain at high frequencies following mutagenesis with UV irradiation and selection on fludioxonil containing medium. Resistant isolates retained their resistance levels even after 9 subcultures on fungicide-free growth medium while they could express their resistant phenotypes in planta. Investigation of cross-resistance relationships showed that fludioxonil resistance mutations also reduce the sensitivity of mutant strains to the aromatic hydrocarbon fungicide quintozene as well as the dicarboximides iprodione and vinclozolin. No cross-resistance was observed between fludioxonil and fungicides with different modes of action such as the sterol biosynthesis inhibitors (DMIs) imazalil and flusilazole and the carboxamide boscalid. All fludioxonil resistant isolates were more sensitive to the anilinopyrimidine pyrimethanil, while only two isolates were less sensitive to the QoI pyraclostrobin compared to the wild-type strain. Study of fitness determining parameters showed that resistance mutation(s) had no adverse effects on mycelial growth, conidial germination and sensitivity to osmotic stress while they had a pleiotropic effect on virulence and conidia production in resistant mutants. Results of the present study indicate that fludioxonil is a highly effective fungicide against A. solani, while the risk of resistance development to this fungicide is considered to be medium making fludioxonil an ideal alternative to high risk fungicides such as boscalid and pyraclostrobin whose performance against early blight has already been compromised by resistance development.

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.

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 (BEN?S) and resistant (BENR) 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.

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 β-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.

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 β-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.

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.

Field isolates of Alternaria alternata collected from tomato processors were characterized for sensitivity to respiration inhibitors using in vitro mycelial growth assays. Pyraclostrobin (QoI), boscalid, fluopyram and isopyrazam (SDHIs) mean EC50 values were 0.32, 1.43, 2.21, and 3.53 μg/ml respectively. Of the 42 isolates, 36 were sensitive to all respiration inhibiting fungicides tested whereas three isolates were less sensitive to boscalid, one to pyraclostrobin and two were simultaneously resistant to both inhibitors and isopyrazam. Correlation analysis between fungicide sensitivities revealed a positive cross-resistance between pyraclostrobin and tebuconazole, and between cyprodinil and mancozeb. There was no cross-resistance between QoIs, SHDIs or any other mode of action. Sequencing of the QoI and SDHI targets revealed the G143A cytochrome b resistance mutation in all pyraclostrobin-resistant isolates while analysis of the succinate dehydrogenase coding gene revealed point mutations in two of three of the gene subunits analyzed in boscalid-resistant isolates. Specifically, two isolates carried the H277Y and three the H133Q resistance mutations located in the sdhB and sdhD subunits of the respiration complex II, respectively. Isolates bearing the H277Y mutation also carried the G143A cytochrome b resistance mutation. Boscalid and pyraclostrobin-resistant isolates exhibited greater pathogenicity and sporulation compared to sensitive isolates, respectively. Isolates with cross-resistance exhibited greater pathogenicity and sporulation but slower mycelial growth compared to sensitive isolates. This is the first report of field isolates of A. alternata with single or double resistance to QoIs and SDHIs in Greece and should be considered in planning and implementing effective anti-resistance strategies.

 Introduction: The agrochemical industry represents the backbone of the agricultural sector world-wide. The improvement of food production, increasing concerns over food and environmental safety, and the emergence of resistant to pesticides pests and pathogens, are among the major challenges that the sector is facing. The latter results in heavy yield losses due to the decreased efficacy of the applied crop protection agents. Within this context, the understanding of changes at the metabolome level and their correlation to the observed resistance is important for combating this issue, and represents a newly emerged and promising field in pesticide R&D. Here, we have developed a robust H NMR metabolomics-based protocol for the discovery and study of Fusarium graminearum resistance mechanism(s) to benzimidazole fungicides.