Comparative fitness of Monilinia fructicola isolates with multiple fungicide-resistant phenotypes.

This study characterized 52 isolates of Monilinia fructicola from peach and nectarine orchards for their multi-resistance patterns to thiophanate-methyl (TF), tebuconazole (TEB), and azoxystrobin (AZO) using in vitro sensitivity assays and molecular analysis. The radial growth of M. fructicola isolates was measured on media amended with a single discriminatory dose of 1 µg/ml for TF and AZO and 0.3 µg/ml for TEB. Cyt b, CYP51, and ß-tubulin were tested for point mutations that confer resistance to quinone outside inhibitors (QoIs), demethylation inhibitors (DMIs), and methyl benzimidazole carbamates (MBCs), respectively. Eight phenotypes were identified including isolates with single, double, and triple in vitro resistance to QoI, MBC, and DMI fungicides. All resistant phenotypes to TF and TEB presented the H6Y mutation in ß-tubulin and the G641S mutation in CYP51. None of the point mutations typically linked to QoI resistance were present in the Monilinia isolates examined. Moreover, fitness of the M. fructicola phenotypes was examined in vitro and detached fruit assays. Phenotypes with single-resistance displayed equal fitness in in vitro and fruit assays compared to the wild-type. In contrast, the dual and triple-resistance phenotypes suffered fitness penalties based on osmotic sensitivity and aggressiveness on peach fruit. In this study, multiple resistance to MBC, DMI, and QoI fungicide groups was confirmed in M. fructicola. Results suggest that Monilinia populations with multiple resistance phenotypes are likely to be less competitive in the field than those with single resistance, thereby impeding their establishment over time and facilitating disease management.

MOF-based Ag NPs/Co3O4 nanozyme for colorimetric detection of thiophanate-methyl based on analyte-enhanced sensing mechanism.

Silver nanoparticles supported on metal-organic framework (ZIF-67)-derived Co3O4 nanostructures (Ag NPs/Co3O4) were synthesized via a facile in situ reduction strategy. The resulting materials exhibited pH-switchable peroxidase/catalase-like catalytic activity. Ag NP doping greatly enhanced the catalytic activity of Ag NPs/Co3O4 towards 3,3',5,5'-tetramethylbenzidine (TMB) oxidation and H2O2 decomposition which were 59 times (A652 of oxTMB) and 3 times (A240 of H2O2) higher than that of ZIF-67, respectively. Excitingly, thiophanate-methyl (TM) further enhanced the peroxidase-like activity of Ag NPs/Co3O4 nanozyme due to the formation of Ag(I) species in TM-Ag NPs/Co3O4 and generation of more radicals resulting from strong interaction between Ag NPs and TM. The TM-Ag NPs/Co3O4 nanozyme exhibited lower Km and higher Vmax values towards H2O2 when compared with Ag NPs/Co3O4 nanozyme. A simple, bioelement-free colorimetric TM detection method based on Ag NPs/Co3O4 nanozyme via analyte-enhanced sensing strategy was successfully established with high sensitivity and selectivity. Our study demonstrated that hybrid noble metal NPs/MOF-based nanozyme can be a class of promising artificial nanozyme in environmental and food safety applications.

Sensitive colorimetric and fluorescence dual-mode detection of thiophanate-methyl based on spherical Fe3O4/GONRs composite nanozyme.

Pesticide detection based on nanozyme is largely limited in terms of the variety of pesticides. Herein, a spherical and well-dispersed Fe3O4/graphene oxide nanoribbons (Fe3O4/GONRs) composite nanozyme was applied to firstly develop an enzyme-free and sensitive colorimetric and fluorescence dual-mode detection of thiophanate-methyl (TM). The synthesized Fe3O4/GONRs possess excellent dual enzyme-like activities (peroxidase and catalase) and can catalyze H2O2 to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) into oxidized TMB (oxTMB). We found that Fe3O4/GONRs can adsorb TM through the synergistic effect of multiple forces, thereby inhibiting the catalytic activities of nanozyme. This inhibition can modulate the transformation of TMB to oxTMB, producing dual responses of absorbance decrease (oxTMB) and fluorescence enhancement (TMB). The limits of detection (LODs) of TM were 28.1 ng/mL (colorimetric) and 8.81 ng/mL (fluorescence), respectively. Moreover, the developed method with the recoveries of 94.8-100.8% also exhibited a good potential application in the detection of pesticides residues in water and food samples.

Updated reasoned opinion on the toxicological properties and maximum residue levels (MRLs) for the benzimidazole substances carbendazim and thiophanate-methyl.

In compliance with Article 43 of Regulation (EC) No 396/2005, EFSA received from the European Commission in 2020 a mandate to provide its reasoned opinion on the toxicological properties and maximum residue levels (MRLs) for the benzimidazole substances carbendazim and thiophanate-methyl. Specifically, EFSA was asked to assess whether thiophanate-methyl or carbendazim has clastogenic potential and, in case clastogenic potential can be excluded, to derive toxicological reference values necessary for consumer risk assessment and assessment of maximum residue levels (MRLs). Although these active substances are no longer authorised within the European Union, MRLs were established by the Codex Alimentarius Commission (codex maximum residue limits; CXLs), and import tolerances are in place. Based on the assessment of the available data, toxicological reference values and MRL proposals were derived and a consumer risk assessment was carried out. Some information required by the regulatory framework was found to be missing and a possible acute risk to consumers was identified. Hence, the consumer risk assessment was considered indicative only and all MRL proposals derived by EFSA still require further consideration by risk managers. In October 2022, to ensure that MRLs derived by EFSA in its assessment of 2021 are safe for consumers also in view of endocrine-disrupting properties, EFSA was requested to carry out a follow-up assessment taking into account the scientific criteria for identifying endocrine disruptors (ED). Based on the outcome of the assessment, the experts agreed that the reference values are also covering the concern related to the identified hazards indicative of endocrine disruption for thiophanate-methyl. No further considerations on the impact of the ED assessment on the current reference values were needed for carbendazim since the ED criteria are not met for this substance. Therefore, the risk assessment and the MRL recommendations derived in 2021 are confirmed.

Smartphone-assisted carbon dots fluorescent sensing platform for visual detection of Thiophanate-methyl in fruits and vegetables.

Trimesic acid and o-phenylenediamine (OPD) were employed as precursors to synthesize yellow-green fluorescent carbon dots (Y-G-CDs) by solvothermal synthesis for the sensitive detection of Thiophanate-methyl (TM) in real agricultural products. The Y-G-CDs probe could specifically recognize the TM primarily through π-π stacking. Moreover, the fluorescence quenching of the probe was ultimately dominated by the PET effect, based on the interaction between the abundant carboxyl groups on the surface of the Y-G-CDs and the amino group of TM. A strong linear relationship between the fluorescence quenching of the probe and TM concentration in the range of 0-10 µmol/L was observed and the limit of detection (LOD) was calculated to be 50.7 nmol/L. Compared to the interference pesticides, the Y-G-CDs probe demonstrated exceptional selectivity toward TM, with satisfactory recoveries of 96.3 % - 104.2 % in spiked food samples. The Y-G-CDs probe enables simple pretreatment, cost-effective, and on-site detection of TM in fruits and vegetables with visual detection of the TM employing a smartphone-assisted sensing platform.

Fungicide Sensitivity of Fusarium oxysporum f. sp. lentis and Fusarium acuminatum Affecting Lentil in the Northern Great Plains.

Fusarium oxysporum f. sp. lentis and F. acuminatum cause wilting and root rot in pulse crops including lentil. Fungicide seed treatments are widely used, but information about Fusarium spp. sensitivity in lentils is limited. Here, 30 F. oxysporum f. sp. lentis and 30 F. acuminatum isolates from Montana, southern Canada, North Dakota, and Washington were identified, tested for pathogenicity, and assayed for in vitro sensitivity to pyraclostrobin, prothioconazole, ipconazole, and thiophanate-methyl. F. oxysporum f. sp. lentis and F. acuminatum differed in their sensitivity to all fungicides. No resistant isolates were identified, but F. oxysporum f. sp. lentis had lower EC50 values in pyraclostrobin (averaging 0.47 µg a.i./ml) than F. acuminatum (averaging 0.89 µg a.i./ml) for mycelia assays. Both species had lower EC50 values in prothioconazole, averaging EC50 0.23 in F. oxysporum f. sp. lentis and 0.53 µg a.i./ml in F. acuminatum. F. oxysporum f. sp. lentis isolates had the lowest EC50 values on ipconazole compared to F. acuminatum (0.78 and 1.49 µg a.i./ml). The pathogens were least sensitive to thiophanate-methyl (1.74 µg a.i./ml for F. oxysporum f. sp. lentis and 1.91 µg a.i./ml for F. acuminatum). Overall sensitivity to the fungicides was higher in F. oxysporum f. sp. lentis than F. acuminatum. This study provides reference EC50 values while pointing to the possibility of differential fungicide efficacies on Fusarium spp. This will be helpful to monitor shifts in sensitivity of Fusarium spp. and devise robust root rot/wilt management approaches.

Low frequency of resistance to thiophanate-methyl in Monilinia fructicola populations from southeastern United States peach orchards.

Methyl benzimidazole carbamate (MBC) fungicides were once widely used for brown rot (Monilinia fructicola) control of peach (Prunus persica (L.) Batsch) in the southeastern US, but their use was substantially reduced and often eliminated due to widespread resistance. In this study, 233 M. fructicola isolates were collected from major peach production areas in Alabama, Georgia, and South Carolina, and sensitivity to thiophanate-methyl was examined. Isolates were also collected from one organic and two experimental peach orchards. A discriminatory dose of 1 µg/ml was used to distinguish sensitive (S) and moderately sensitive (S-LR) isolates from low resistant phenotypes, while 50 and 500 µg/ml thiophanate-methyl concentrations were used to determine high resistant (HR) phenotypes. Sequence analyses were performed to identify mutations in the ß-tubulin target gene and detached fruit assays were performed to determine the efficacy of a commercial product against isolates representing each phenotype. Results indicated 55.7%, 63.5%, and 75.9% of isolates from Alabama, Georgia, and South Carolina, respectively, were S to thiophanate-methyl; 44.3%, 36.5%, and 21.4% were S-LR; no isolates were LR; and only 3 isolates (1.3%) from South Carolina were HR. No mutations in S or S-LR isolates were found, but HR isolates revealed the E198A mutation, an amino acid change of glutamic acid to alanine conferring high resistance. The high label rate of a commercial product containing thiophanate-methyl controlled brown rot caused by S and S-LR isolates in detached fruit studies but was ineffective against HR isolates. The combinations of thiophanate-methyl with azoxystrobin or isofetamid, when mixed together and applied in an experimental orchard 14 days preharvest, significantly reduced brown rot incidence on pre and postharvest commercially ripe fruit and efficacy was comparable to that of a grower standard fungicide. These results indicate that thiophanate-methyl may again be useful to peach growers in the southeastern US for brown rot and fungicide resistance management.

Unveiling the Impact of Thiophanate-Methyl on Arthrospira platensis: Growth, Photosynthetic Pigments, Biomolecules, and Detoxification Enzyme Activities.

BACKGROUND: The intensive and injudicious use of pesticides in agriculture has emerged as a major concern due to its detrimental impact on aquatic ecosystems. However, the specific impact of broad-spectrum fungicides, such as Thiophanate-methyl (TM), on cyanobacteria remains a subject of ongoing research and debate. METHODS: In order to fill this knowledge gap, The present study aimed to comprehensively investigate the toxicological effects of TM (10-30 µg/L) on the growth, photosynthetic pigments, oxidative stress, and biochemical composition of the non-nitrogen-fixing cyanobacterium Arthrospira platensis. RESULTS: Our findings unequivocally demonstrated that TM exposure significantly inhibited the growth of A. platensis. Moreover, the decrease in chlorophyll content indicated a pronounced negative impact on the photosynthetic system of A. platensis caused by TM exposure. Notably, TM induced oxidative stress in A. platensis, as substantiated by a significant increase in lipid peroxidation (MDA) within the culture. Furthermore, the intracellular generation of hydrogen peroxide (H2O2) exhibited a positive correlation with higher TM dosages, while the levels of vital antioxidant enzymes, such as catalase (CAT) and ascorbate peroxidase (APX), exhibited a discernible decrease. This suggests that TM compromises the antioxidant defense mechanisms of A. platensis. Additionally, TM was found to enhance the activity of a plethora of enzymes involved in the detoxification of pesticides, including peroxidase (POD) and glutathione-S-transferase (GST), thereby indicating a robust detoxification response by A. platensis. Interestingly, exposure to TM resulted in a general suppression of biocomponent production, such as total proteins and total carbohydrates, which exhibited a diminishing trend with increasing TM concentration. Conversely, the lipid content witnessed a significant increase, possibly as an adaptive response to TM-induced stress. CONCLUSIONS: These findings contribute to a deeper understanding of the ecological implications of pesticide usage and emphasize the urgent need for the adoption of sustainable and environmentally-friendly agricultural practices to safeguard aquatic ecosystems.

Sensitivity of Clarireedia spp. to benzimidazoles and dimethyl inhibitors fungicides and efficacy of biofungicides on dollar spot of warm season turfgrass.

Dollar spot caused by Clarireedia spp. (formerly Sclerotinia homoeocarpa) is an economically destructive fungal disease of turfgrass that can significantly compromise turf quality, playability, and aesthetic value. Fungicides are frequently used to manage the disease but are costly and potentially unfavorable to the environment. Repeated use of some active ingredients has resulted in reduced efficacy on C. jacksonii causing dollar spot in cool-season turfgrasses in the US. Experiments were conducted to study fungicide sensitivity of Clarireedia spp. as well as to develop alternatives to fungicides against dollar spot on warm-season turfgrass in Georgia. First, 79 isolates of Clarireedia spp. collected across the state were tested on fungicide-amended agar plates for their sensitivity to thiophanate-methyl (benzimidazole) and propiconazole (dimethyl inhibitor). Seventy-seven isolates (97.5%) were sensitive (0.001 to 0.654 µg/mL) and two isolates (2.5%) were found resistant (>1000 µg/mL) to thiophanate-methyl. However, in the case of propiconazole, 27 isolates (34.2%) were sensitive (0.005 to 0.098 µg/mL) while 52 isolates (65.8%) were resistant (0.101 to 3.820 µg/mL). Next, the efficacy of three bio- and six synthetic fungicides and ten different combinations were tested in vitro against C. monteithiana. Seven bio- and synthetic fungicide spray programs comprising Bacillus subtilis QST713 and propiconazole were further tested, either alone or in a tank mix in a reduced rate, on dollar spot infected bermudagrass 'TifTuf' in growth chamber and field environments. These fungicides were selected as they were found to significantly reduce pathogen growth up to 100% on in vitro assays. The most effective spray program in growth chamber assays was 100% B. subtilis QST713 in rotation with 75% B. subtilis QST713 + 25% propiconazole tank mix applied every 14 days. However, the stand-alone application of the biofungicide B. subtilis QST713 every seven days was an effective alternative and equally efficacious as propiconazole, suppressing dollar spot severity and AUDPC up to 75%, while resulting in acceptable turf quality (>7.0) in field experiments. Our study suggests that increased resistance of Clarireedia spp. to benzimidazoles and dimethyl inhibitors warrants continuous surveillance and that biofungicides hold promise to complement synthetic fungicides in an efficacious and environmentally friendly disease management program.

Risk assessment of predatory lady beetle Propylea japonica's multi-generational exposure to three non-insecticidal agrochemicals.

The effects of non-insecticidal agrochemicals on pest natural predators remain largely unexplored except bees and silkworm. The herbicide quizalofop-p-ethyl (QpE), fungicide thiophanate-methyl (TM), and plant growth regulator mepiquat chloride (MC) have been extensively applied as non-insecticidal agrochemicals. Here, we systematically evaluated multiple effects of these 3 non-insecticidal agrochemicals on three generations of Propylea japonica, an important agroforestry predatory beetle, including the effects on its development, reproduction, enterobacteria, and transcriptomic response. The results showed that QpE exhibited a hormetic effect on P. japonica, thus significantly increasing the survival rate of generation 2 (F2) females, generation 3 (F3) females, and F3 males and body weight of F3 males. However, three successive generations exposed to TM and MC had no significant effect on longevity, body weight, survival rate, pre-oviposition period, and fecundity of P. japonica. Additionally, we investigated the effects of MC, TM, and QpE exposure on gene expression and gut bacterial community of F3 P. japonica. Under MC, TM, and QpE exposure, the overwhelming genes of P. japonica (99.90 %, 99.45 %, and 99.7 %) remained unaffected, respectively. Under TM and MC exposure, differentially expressed genes (DEGs) were not significantly enriched in any KEGG pathway, indicating TM and MC did not significantly affect functions of P. japonica, but under QpE exposure, the expression levels of drug metabolism-related genes were down-regulated. Although QpE treatment did not affect gut dominant bacterial community composition, it significantly increased relative abundances of detoxification metabolism-related bacteria such as Wolbachia, Pseudomonas and Burkholderia in P. japonica. However, TM and MC had no significant effect on the gut bacterial community composition and relative abundance in P. japonica. This study revealed for the first time the mechanism by which P. japonica might compensate for gene downregulation-induced detoxification metabolism decline through altering symbiotic bacteria under QpE exposure. Our findings provide reference for the rational application of non-insecticidal agrochemicals.

Self-assembled thiophanate-methyl/star polycation complex prevents plant cell-wall penetration and fungal carbon utilization during cotton infection by Verticillium dahliae.

No effective fungicides are available for the management of Verticillium dahliae, which causes vascular wilt disease. In this study, a star polycation (SPc)-based nanodelivery system was used for the first time to develop a thiophanate-methyl (TM) nanoagent for the management of V. dahliae. SPc spontaneously assembled with TM through hydrogen bonding and Van der Waals forces to decrease the particle size of TM from 834 to 86 nm. Compared to TM alone, the SPc-loaded TM further reduced the colony diameter of V. dahliae to 1.12 and 0.64 cm, and the spore number to 1.13 × 108 and 0.72 × 108 cfu/mL at the concentrations of 3.77 and 4.71 mg/L, respectively. The TM nanoagents disturbed the expression of various crucial genes in V. dahliae, and contributed to preventing plant cell-wall degradation and carbon utilization by V. dahliae, which mainly impaired the infective interaction between pathogens and plants. TM nanoagents remarkably decreased the plant disease index and the fungal biomass in the root compared to TM alone, and its control efficacy was the best (61.20 %) among the various formulations tested in the field. Furthermore, SPc showed negligible acute toxicity toward cotton seeds. To the best of our knowledge, this study is the first to design a self-assembled nanofungicide that efficiently inhibits V. dahliae growth and protects cotton from the destructive Verticillium wilt.

Preventive and curative effect of difenoconazole + azoxytrobin and thiophanate-methyl against lucky bamboo anthracnose disease caused by Colletotrichum dracaenophilum.

In Egypt, Dracaena sanderiana (lucky bamboo) is an ornamental plant imported from several countries. Two weeks after they arrived at the nurseries, anthracnose indications were detected on the shoots of imported D. sanderiana samples. Four Colletotrichum spp. isolates were obtained from the symptomatic lucky bamboo plants. The obtained isolates belonged to the species of C. gloeosporioides or C. dracaenophilum based on their morphological characteristics and molecular biology analyses. Pathogenicity tests reveal that C. dracaenophilum isolate 4 was found to be more pathogenic than the other isolates. The in vitro investigation was conducted with the objectives of evaluating six systemic fungicides for their inhibitory effect against C. dracaenophilum. Data reveal that, thiophanate-methyl and difenoconazole + azoxytrobin at ≥15 ppm completely inhibited the pathogen growth. Tebuconazole and flusllazole inhibited growth completely at ≥20 ppm, whereas iprodione and cyprodinil + fludioxonil had a lower effect (56.6 and 54.4% reduction, respectively) at this dose. The in vivo investigation was conducted with the objectives of evaluating the preventive and curative effects of the most effective fungicides against anthracnose disease. Lucky bamboo plants were treated with fungicide and either inoculated or not with C. dracaenophilum before being left for 25 or 60 days. On both insidiously infected and vaccinated lucky bamboo plants, the combination of difenoconazole, azoxytrobin, and thiophanate-methyl at 20 ppm greatly reduced the development of anthracnose. Tebuconazole and flusllazole were found to be phytotoxic.

Fungicide Sensitivity of Sclerotinia sclerotiorum from U.S. Soybean and Dry Bean, Compared to Different Regions and Climates.

Fungicide use is integral to reduce yield loss from Sclerotinia sclerotiorum on dry bean and soybean. Increasing fungicide use against this fungus may lead to resistance to the most common fungicides. Resistance has been reported in Brazil (Glycine max) and China (Brassica napus subsp. napus), however, few studies have investigated fungicide sensitivity of S. sclerotiorum in the United States. This work was conducted to determine if there was a difference in fungicide sensitivity of S. sclerotiorum isolates in the United States from: (i) dry bean versus soybean and (ii) fields with different frequencies of fungicide application. We further hypothesized that isolates with fungicide applications of a single active ingredient from tropical Brazil and subtropical Mexico were less sensitive than temperate U.S. isolates due to different management practices and climates. The EC50(D) fungicide sensitivity of 512 S. sclerotiorum isolates from the United States (443), Brazil (36), and Mexico (33) was determined using a discriminatory concentration (DC) previously identified for tetraconazole (2.0 ppm; EC50(D) range of 0.197 to 2.27 ppm), boscalid (0.2; 0.042 to 0.222), picoxystrobin (0.01; 0.006 to 0.027), and thiophanate-methyl, which had a qualitative DC of 10 ppm. Among the 10 least sensitive isolates to boscalid and picoxystrobin, 2 presented mutations known to confer resistance in the SdhB (qualitative) and SdhC (quantitative) genes; however, no strong resistance was found. This study established novel DCs that can be used for further resistance monitoring and baseline sensitivity of S. sclerotiorum to tetraconazole worldwide plus baseline sensitivity to boscalid in the United States.

Thiophanate-methyl induces notochord toxicity by activating the PI3K-mTOR pathway in zebrafish (Danio rerio) embryos.

Thiophanate-methyl (TM), a typical pesticide widely used worldwide, was detected in rivers, soil, fruits, and vegetables. Thus, it is urgent to identify the potential harm of TM residual to non-target organisms and its molecular mechanisms. We used zebrafish (Danio rerio) in this study to evaluate TM toxicity. TM exposure induced developmental toxicity, including inhibited hatchability, reduced heart rates, restrained spontaneous locomotion, and decreased body length. Furthermore, we observed obvious toxicity in the notochord and detected increased expression levels of notochord-related genes (shha, col2a, and tbxta) by in situ hybridization in zebrafish larvae. In addition, calcein staining, alkaline phosphatase (ALP) activity analysis, and anatomic analysis indicated that TM induced notochord toxicity. We used rescue experiments to verify whether the PI3K-mTOR pathway involved in the notochord development was the cause of notochord abnormalities. Rapamycin and LY294002 (an inhibitor of PI3K) relieve notochord toxicity caused by TM, including morphological abnormalities. In summary, TM might induce notochord toxicity by activating the PI3K-mTOR pathway in zebrafish.

Copper-Modified Double-Emission Carbon Dots for Rapid Detection of Thiophanate Methyl in Food.

The detection of food safety and quality is very significant throughout the food supply. Stable dual-emission copper-modified fluorescent carbon dots (Cu-CDs) were successfully synthesized by a simple and environment-friendly hydrothermal, which was used for the real-time detection of pesticide residues in agricultural products. By optimizing the reaction conditions, Cu-CDs showed two emission peaks, with the highest fluorescence intensities at 375 and 450 nm. The structure, chemical composition and optical properties of Cu-CDs were investigated by XRD, TEM and IR. The results showed that thiophanate methyl (TM) could induce fluorescence quenching of Cu-CDs with no other ligands by the electron transfer through π-π stacking. The synchronous response of the dual-emission sensor enhanced the specificity of TM, which showed remarkable anti-interference capability. The fluorescence quenching degree of Cu-CDs had a good linear relationship with the TM concentration; the low detection limit for a pear was 0.75 µM, and for an apple, 0.78 µM. The recoveries in the fruit samples were 79.70-91.15% and 81.20-93.55%, respectively, and the relative standard deviations (RSDs) were less than 4.23% for the pear and less than 3.78% for the apple. Thus, our results indicate the feasibility and reliability of our methods in detecting pesticide residues in agricultural products.

Effect of ozonated water, mancozeb, and thiophanate-methyl on the phyllosphere microbial diversity of strawberry.

Phyllosphere microorganisms are closely linked to plant health. This study investigated the effect of ozonated water, mancozeb, and thiophanate-methyl on phyllosphere microorganisms in strawberry plants of the "Hongyan" variety. Sequencing analysis of the phyllosphere bacterial and fungal communities was performed using 16S rRNA gene fragment and ITS1 region high-throughput sequencing after spraying ozonated water, mancozeb, thiophanate-methyl, and clear water. Proteobacteria, Actinobacteria, and Firmicutes were the dominant bacterial phyla in strawberry. The relative abundance of Proteobacteria (82.71%) was higher in the ozonated water treatment group than in the other treatment groups, while the relative abundance of Actinobacteria (9.38%) was lower than in the other treatment groups. The strawberry phyllosphere fungal communities were mainly found in the phyla Basidiomycota and Ascomycota. The relative abundance of Basidiomycota was highest in the ozonated water treatment group (81.13%), followed by the mancozeb treatment group (76.01%), while the CK group only had an abundance of 43.38%. The relative abundance of Ascomycota was lowest in the ozonated water treatment group (17.98%), 23.12% in the mancozeb treatment group, 43.39% in the thiophanate-methyl treatment group, and 55.47% in the CK group. Pseudomonas, Halomonas, and Nesterenkonia were the dominant bacterial genera on strawberry surfaces, while Moesziomyces, Aspergillus, and Dirkmeia were the dominant fungal genera. Ozonated water was able to significantly increase the richness of bacteria and fungi and decrease fungal diversity. However, bacterial diversity was not significantly altered. Ozonated water effectively reduced the relative abundance of harmful fungi, such as Aspergillus, and Penicillium, and enriched beneficial bacteria, such as Pseudomonas and Actinomycetospora, more effectively than mancozeb and thiophanate-methyl. The results of the study show that ozonated water has potential as a biocide and may be able to replace traditional agents in the future to reduce environmental pollution.

Effect of fungicides on spike characteristics in winter wheat inoculated with Fusarium culmorum.

The impact of fungicides on the head blight (FHB) development disease index, percent spike harvest index (SHI), and deoxynivalenol (DON) accumulation in wheat kernels under field conditions was evaluated after artificial spike inoculation with Fusarium culmorum (S-14). The trial was carried out using commercially available fungicides and a sensitive cultivar of bread wheat (Flamura-85) in a field of a wheat producer located in the Trakya region of Turkey. Fungicides were applied at the beginning of anthesis (ZGS 61), 48 hours after the inoculation with the pathogen. Disease index was determined 10 days and 14 days post-inoculation. The application of fungicides containing tebuconazole, thiophanate methyl plus tetraconazole and prothioconazole plus trifloxystrobin reduced the FHB disease and increased kernel number, spike weight, and kernel weight, as compared to the inoculated/non-fungicide control. The efficacy of tebuconazole and of prothioconazole plus trifloxystrobin was higher than that of thiophanate methyl plus tetraconazole on FHB disease severity, percent spike harvest index (SHI), and DON accumulation in wheat kernels.

Fungicide Sensitivity of Colletotrichum Species Causing Bitter Rot of Apple in the Mid-Atlantic U.S.A.

Apple growers in the Mid-Atlantic region of the U.S.A. have reported increased losses to bitter rot of apple. We tested the hypothesis that this increase is because the Colletotrichum population has developed resistance to commonly used single-mode-of-action (single-MoA) fungicides. We screened 220 Colletotrichum isolates obtained from 38 apple orchards in the Mid-Atlantic region for resistance to 11 fungicides in Fungicide Resistance Action Committee (FRAC) groups 1, 7, 9, 11, 12, and 29. Eleven (5%) of these isolates were resistant to FRAC group 1 with confirmed ß-tubulin E198A mutations, and two (<1%) were also resistant to FRAC group 11 with confirmed cytochrome-b G143A mutations. Such low frequencies of resistant isolates indicate that fungicide resistance is unlikely to be the cause of any regional increase in bitter rot. A subsample of isolates was subsequently tested in vitro for sensitivity to every single-MoA fungicide registered for apple in the Mid-Atlantic U.S.A. (22 fungicides; FRAC groups 1, 3, 7, 9, 11, 12, and 29), and 13 fungicides were tested in field trials. These fungicides varied widely in efficacy both within and between FRAC groups. Comparisons of results from our in vitro tests with results from our field trials and other field trials conducted across the eastern U.S.A. suggested that EC25 values (concentrations that reduce growth by 25%) are better predictors of fungicide efficacy in normal field conditions than EC50 values. We present these results as a guideline for choosing single-MoA fungicides for bitter rot control in the Mid-Atlantic U.S.A.

Molecular Diagnosis of Thiophanate-Methyl-Resistant Strains of Fusarium fujikuroi in Japan.

Fusarium fujikuroi is the pathogen of rice bakanae disease and is subclassified into gibberellin and fumonisin groups (G and F groups). Thiophanate-methyl (TM), a benzimidazole fungicide, has been used extensively to control F. fujikuroi. Previous investigation showed that F-group strains are TM sensitive (TMS), whereas most G-group strains are TM resistant (TMR) in Japan. The minimum inhibitory concentration in TMS strains was 1 to 10 µg ml-1, whereas that in TMR strains was >100 µg ml-1. E198K and F200Y mutations in ß2-tubulin were detected in TMR strains. A loop-mediated isothermal amplification-fluorescent loop primer method was developed for diagnosis of these mutations and applied to 37 TMR strains and 56 TMS strains. The results indicated that 100% of TMR strains were identified as having either the E198K mutation (41%) or the F200Y mutation (59%), whereas none of the TMS strains tested showed either mutation. We found one remarkable TMR strain in the F group that had an F200Y mutation. These results suggest that E198K and F200Y mutations in ß2-tubulin contribute to TM resistance in F. fujikuroi.

Reasoned opinion on the toxicological properties and maximum residue levels (MRLs) for the benzimidazole substances carbendazim and thiophanate-methyl.

In compliance with Article 43 of Regulation (EC) No 396/2005, EFSA received from the European Commission a mandate to provide its reasoned opinion on the toxicological properties and maximum residue levels (MRLs) for the benzimidazole substances carbendazim and thiophanate-methyl. Specifically, EFSA was asked to assess whether thiophanate-methyl or carbendazim have clastogenic potential and, in case clastogenic potential can be excluded, to derive toxicological reference values necessary for consumer risk assessment and assessment of maximum residue levels (MRLs). Although these active substances are no longer authorised within the European Union, MRLs were established by the Codex Alimentarius Commission (codex maximum residue limits; CXLs), and import tolerances are in place. Based on the assessment of the available data, toxicological reference values and MRL proposals were derived and a consumer risk assessment was carried out. Some information required by the regulatory framework was found to be missing and a possible acute risk to consumers was identified. Hence, the consumer risk assessment is considered indicative only, all MRL proposals derived by EFSA still require further consideration by risk managers and measures for reduction of the consumer exposure should also be considered.