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Black spot, caused by Alternaria alternata, poses a severe threat to the industry of Dendrobium officinale, a Chinese indigenous medicinal herb. Dicarboximide fungicides (DCFs) have been intensively used to control this disease for decades in China, and offer excellent efficacy. The resistance of phytopathogenic pathogens against DCFs are reportedly selected in fields; however, the DCF resistance of A. alternata from D. officinale is not well understood. The isolates of A. alternata with low procymidone resistance (ProLR) were detected in the commercial orchards of D. officinale in China in 2018 and biochemically characterized in this study. The result showed that the ProLR isolates were selected in the commercial orchards with a resistance frequency of 100%, and no significant difference in mycelial growth, sporulation, and virulence was observed among the ProLR and procymidone-sensitive (ProS) isolates. A positive cross-resistance pattern was exhibited between procymidone and iprodione. Results of amino acid sequence alignment of AaOS-1 from the tested isolates showed that all of the ProLR genotypes could be categorized into two groups, including group I (mutations at AaOs-1) and group II (no mutation). Under procymidone (5.0 µg/ml) treatment conditions, the AaOs-1 expression levels increased in the ProS isolates and ranged from approximately 2.94- to 3.69-fold higher than those under procymidone-free conditions, while the AaOs-1 expressions of the ProLR isolates were significantly lower than those in the ProS isolates under the same conditions. The data indicated that the mutations at AaOs-1 are involved in the DCF resistance of A. alternata selected in the D. officinale orchards.

Alternaria alternata, a causal agent of leaf blights and spots on a wide range of hosts, has a high risk of developing resistance to fungicides. Procymidone, a dicarboximide fungicide (DCF), has been widely used in controlling Alternaria leaf blights in China for decades. However, the resistance of A. alternata against DCFs has rarely been reported from crucifer plants. A total of 198 A. alternata isolates were collected from commercial fields of broccoli and cabbage during 2018–2019, and their sensitivities to procymidone were determined. Biochemical and molecular characteristics were subsequently compared between the high-level procymidone-resistant (ProHR) and procymidone-sensitive (ProS) isolates, and also between ProHR isolates from broccoli and cabbage. Compared with ProS isolates, the mycelial growth rate, sporulation capacity and virulence of most ProHR isolates were reduced; ProHR isolates displayed an increased sensitivity to osmotic stresses and a reduced sensitivity to sodium dodecyl sulfate (SDS); all ProHR isolates showed a reduced sensitivity to hydrogen peroxide (H2O2) except for the isolate B102. Correlation analysis revealed a positive cross-resistance between procymidone and iprodione, or fludioxonil. When treated with 10 μg/mL of procymidone, both mycelial intracellular glycerol accumulations (MIGAs) and relative expression of AaHK1 in ProS isolates were higher than those in ProHR isolates. Sequence alignment of AaHK1 from ten ProHR isolates demonstrated that five of them possessed a single-point mutation (P94A, V612L, E708K or Q924STOP), and four isolates had an insertion or a deletion in their coding regions. No significant difference in biochemical characteristics was observed among ProHR isolates from two different hosts, though mutations in AaHK1 of the cabbage-originated ProHR isolates were distinct from those of the broccoli-originated ProHR isolates.

Sclerotinia sclerotiorum is one of the most damaging and economically important necrotrophic plant pathogens, infecting more than 400 plant species globally. Although the phenylpyrrole fungicide fludioxonil has high activity against S. sclerotiorum, reports indicate that there is also substantial potential for the development of fungicide resistance. However, the current study investigating five fludioxonil-resistant laboratory mutants found a significant fitness cost associated with fludioxonil resistance resulting in significantly (P < 0.05) reduced mycelial growth and sclerotia formation on potato dextrose agar as well as significantly (P < 0.05) lower pathogenicity on detached tomato leaves, with one mutant, LK-1R, completely losing the capacity to cause infection. In addition, all of the fludioxonil-resistant mutants had significantly (P < 0.05) increased sensitivity to osmotic stress (0.5 M of potassium chloride and 1.0 M of glucose), which is consistent with the proposed fludioxonil target sites within the high osmolarity glycerol stress response mitogen-activated protein kinase (HOG1-MAPK) signaling transduction pathway. Sequence analysis of six genes from this two-component pathway, including SsHk, SsYpd, SsSk1, SsSk2, SsPbs, and SsHog, revealed several mutations that may be associated with fludioxonil resistance. For example, six separate point mutations were found in SsHk that led to changes in the predicted amino acid sequence, including A136G, F249V, G353A, E560K, M610K, and K727R. Similarly, SsPbs had three mutations (D34G, S46L, and L337E), SsSk1 and SsYpd had two (S53G and A795V for SsSk1, and E67G and Y141H for SsYpd), and SsHog and SsSk2 had one each (V220A and S763P, respectively). To our knowledge, these constitute the first reports of amino acid changes in proteins of the HOG1-MAPK pathway being associated with fludioxonil resistance in S. sclerotiorum. This study also showed a positive cross-resistance between fludioxonil and dimethachlone and procymidone, but none with tebuconazole or carbendazim, indicating that the inclusion of tebuconazole within an integrated pest management program could reduce the risk of fludioxonil resistance developing in field populations of S. sclerotiorum and ensure the sustainable production of soybeans in China into the future.

A new idea for the modification of a carboxylic group in phenoxyacetic herbicides is proposed. Common herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA), and their chalcogen analogs (arylthio and arylselenyl fragments instead of a phenoxy one) were used as carboxylic components in the tricomponent Passerini reaction. Cyclohexyl isocyanide and 4-isocyano-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-NC) were used as isocyanides. Acetone, acetaldehyde and formaldehyde were used as carbonyl components. Passerini adducts 4a–4L were obtained in yields of 32–59%. The structures of the Passerini adducts were determined using spectroscopic methods: MS, HR MS, 1H NMR, 13C NMR, 77Se NMR (if appropriate) and IR spectra (MS, HR MS, IR for the nitroxides 4k and 4L). The herbicidal activity of the Passerini adducts synthesized (4a–4L) was tested against ten weed species (at 2 kg/ha using 2,4-D and MCPA as a control). 100% herbicidal post-emergent activity was reported for 4i, 4j, 4k, 4L [a weed black mustard (Brassica nigra)], for 4k, 4L [weeds pale smartweed (Polygonum lapathifolium) and fathen (Chenopodium album)], as well as for 4k (weeds common poppy (Papaver rhoeas) and gallant soldier (Galinsoga parviflora)). The fungistatic activity of the Passerini adducts synthesized (4a–4L) was tested against six phytopathogenic fungi species (at 200 mg/L and procymidone as a control). The tested compounds showed a weak activity (% of inhibition) against: Alternaria alternata: 4g (47), 4i (26), 4j (26), 4k (50), Botrytis cinerea 4g (38), Fusarium culmorum 4g (44), Phytophthora cactorum 4i (35), 4j (35), 4k (30), Rhizoctonia solani 4g (30), 4i (24), Phytophthora infestans 4g (29), 4i (34), 4j (28), 4k (46).

Fungal pathogen Botrytis cinerea, the casual agent of gray mold of vegetables and fruits, has a high risk of developing resistance to fungicide. Tebuconazole, one kind of demethylation inhibitor (DMI) fungicides, has been increasingly applied for the control of tomato gray mold in China. However, very limited information is available on the resistance profile of B. cinerea to tebuconazole. In this study, the baseline sensitivity of B. cinerea to tebuconazole was determined based on 138 isolates from field sites having no history of DMI usage, with a mean EC50 value of 0.29 μg/mL. Another 159 B. cinerea isolates collected in the greenhouse and field from 2011 to 2016 were demonstrated to have a shifted sensitivity to tebuconazole, with a mean EC50 value of 0.66 μg/mL. EC50 values of 10 B. cinerea isolates with reduced sensitivity to tebuconazole were greater than 1.56 μg/mL, and these reduced-sensitive isolates had a fitness penalty in sporulation and conidial germination, but showed similar mycelial growth rate and pathogenicity with those of the sensitive isolates. Positive cross-resistance was observed only between tebuconazole and the other two DMIs difenoconazole and prochloraz, but not between tebuconazole and the non-DMIs iprodione, procymidone, or fludioxonil. In reduced-sensitive isolates, no amino acid variation was found in the BcCYP51 protein. When exposed to tebuconazole, the expression level of BcCYP51 increased in these reduced-sensitive B. cinerea isolates as compared to sensitive ones, thus contributing to the reduced sensitivity of the pathogen to tebuconazole. Additionally, the nucleotide mutation observed in the 1200 bp upstream region of BcCYP51 had no correlation with the development of fungicide resistance in B. cinerea isolates. These findings will be helpful for the understanding of DMI resistance mechnism in B. cinerea.

Gray mold caused by Botrytis cinerea is a fungal disease that critically threatens agricultural production, and carbendazim was the first fungicide used to control B. cinerea. However, B. cinerea developed serious resistance to carbendazim, and this fungicide has thus rarely been used in the past decade in China. Due to the extended discontinuation of carbendazim use, the evolution of the resistance of B. cinerea to carbendazim in recent years is unclear, and whether carbendazim can effectively control gray mold is largely unknown. Therefore, this study determined the sensitivity of 407 B. cinerea isolates collected from 2014 to 2018 to carbendazim and the ability of carbendazim to control gray mold in the field. The results showed that the frequency of B. cinerea isolates resistant to carbendazim remained above 95%. Three different mutation types responsible for the resistance of B. cinerea to carbendazim were identified at codon 198 in the β-tubulin gene sequence: E198V (changed from GAG to GTG), E198A (changed from GAG to GCG), and E198K (changed from GAG to AAG). Over the last 5 years, E198V was the major mutation. However, an analysis of its evolution revealed that the percentage of the E198V mutation declined after 2017 to 56.5% in 2018. In addition, the proportion of isolates with the E198K mutation decreased over time, and no isolates with this mutation were found in either 2017 or 2018. The proportion of the E198A mutation increased over the 5-year test period to reach 43.5% in 2018. Furthermore, three greenhouse experiments demonstrated that carbendazim has lost its ability to control gray mold. We attribute the above findings to our results showing that the carbendazim-resistant isolates had no fitness penalties compared with the carbendazim-sensitive isolates for sporulation and mycelial growth. In particular, the E198A mutant isolates exhibited a strong ability to sporulate, suggesting that the E198A mutation might become dominant in the future. Interestingly, the results showed that carbendazim-sensitive isolates could be easily controlled by four conventional fungicides, namely boscalid, procymidone, iprodione, and pyrimethanil, with mean EC50 values of 0.71 ± 0.2 mg liter−1, 1.33 ± 0.39 mg liter−1, 0.59 ± 0.33 mg liter−1, and 6.02 ± 3.02 mg liter−1, respectively. In conclusion, carbendazim has lost its application value and is ineffective for the control of gray mold.

The gray mold caused by Botrytis cinerea has a significant impact on tomato production throughout the world. Although the synthetic fungicide fludioxonil can effectively control B. cinerea, there have been several reports of resistance to this fungicide. This study indicated that all of the fludioxonil-resistant strains tested, including one field-resistant isolate and four laboratory strains, had reduced fitness relative to sensitive isolates. In addition to having reduced growth, sporulation, and pathogenicity, the resistant strains were more sensitive to osmotic stress and had significantly (P < 0.05) higher peroxidase activity. BOs1, a kinase in the high-osmolarity glycerol stress response signal transduction pathway, is believed to harbor mutations related to fludioxonil resistance. Sequence analysis of their BOs1 sequences indicated that the fludioxonil-resistant field isolate, XXtom1806, had four point mutations resulting in four amino acid changes (I365S, S531G, T565N, and T1267A) and three amino acids (I365S, S531G, and T565N) in the histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis receptors, and phosphatases domain, which associated with fludioxonil binding. Similarly, two of the laboratory strains, XXtom-Lab1 and XXtom-Lab4, had three (Q846S, I1126S, and G415D) and two (P1051S and V1241M) point mutations, respectively. A third strain, XXtom-lab3, had a 52-bp insertion that included a stop codon at amino acid 256. Interestingly, the BOs1 sequence of the fourth laboratory strain, XXtom-lab5, was identical to those of the sensitive isolates, indicating that an alternative resistance mechanism exists. The study also found evidence of positive cross-resistance between fludioxonil and the dicarboximide fungicides procymidone and iprodione, but no cross-resistance was detected with any other fungicides tested, including boscalid, carbendazim, tebuconazole, and fluazinam.

In this study the phytotoxic, cytotoxic, genotoxic and mutagenic effects of two commercial fungicide-active compounds, procymidone (PR) and iprodione (IP), were determined. The parameters evaluated were germination and root growth, mitotic index, chromosomal and nuclear aberrations, and molecular analyses were also performed in the model plant Allium cepa L. The results demonstrated that the active compounds PR and IP were phytotoxic, delaying germination and slowing the development of A. cepa seedlings. Moreover, PR and IP showed cytogenotoxicity towards A. cepa meristematic cells, inducing chromosomal changes and cell death. The mutagenic activity of the active compounds was demonstrated by the detection of DNA changes in simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers in the treated cells compared to the negative control. Together, these results contribute to a better understanding of the damage caused by these substances in living organisms and reveal a promising strategy for prospective studies of the toxic effects of environmental pollutants.

Made available in DSpace on 2021-06-25T11:10:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) In Brazil, Sclerotinia stem rot (SSR) control in soybean depends primarily on application of the fungicides carbendazim, fluazinam, and procymidone. While these fungicides can provide effective control of the disease, their protection period, which is crucial for timing their optimal application, is poorly understood. In the present study, the protection period of carbendazim, fluazinam and procymidone was determined using a detached leaf bioassay (DLB) and chromatography. For the DLB, soybean plants were treated twice with each fungicide with or without mineral oil as adjuvant; leaves were collected at two or three-day intervals from 11 to 23 days, inoculated with Sclerotinia sclerotiorum mycelium, and the necrotic area was assessed 48 h after inoculation. For the chromatography test, each fungicide was applied and the leaves were collected every two days after treatment for 14 days. Fungicide residue concentration in tissue was determined according to a modified version of the quick, easy, cheap, effective, rugged and safe (QuEChERS) method. At 5, 7, and 9 days after the first application, fluazinam, carbendazim and procymidone, respectively, had no protective effect even when applied with mineral oil. Greater control efficacy was achieved after a second application. Fluazinam showed protective activity up to 4 days while carbendazim and procymidone showed about 10 days. The mineral oil only enhanced protection activity of procymidone in the second application. The analytical chemical separation indicated that the relative concentration of fluazinam decreased at a faster rate than that of carbendazim and procymidone. At day 14, the concentration of carbendazim and procymidone decreased from 34.97 to 9.39 mg kg−1, and 58.98 to 11.22 mg kg−1, respectively, suggesting that 10–14 days represent the approximate timing in spacing two consecutive applications. The knowledge of the protection period of these fungicides sets the basis to improving their timing of applications. Carrington Research Extension Center North Dakota State University Department of Plant Protection School of Agriculture São Paulo State University (UNESP) School of Agricultural Sciences Northwest Missouri State University Department of Plant Protection School of Agriculture São Paulo State University (UNESP) CAPES: 001

Leafy vegetables are widely consumed in South Korea, especially in the form of kimchi and namul (seasoned vegetables) and are used for wrapping meat. Therefore, the management of pesticide residues in leafy vegetables is very important. A total of 17,977 samples (49 leafy vegetables) were mainly collected in the largest production area of leafy vegetables (Gwangju Metropolitan City and Chonnam Province) in South Korea. They were analyzed within the government’s monitoring programs (Gwangju Metropolitan City) of pesticide residues between 2005 and 2019. Pesticide residues were found in 2815 samples (15.7%), and 426 samples (2.4%) from among these exceeded the specified maximum residue limits (MRLs). Samples exceeding the MRLs were mostly detected in spinach, ssamchoo (brassica lee ssp. namai), crown daisy, lettuce, and perilla leaves. Azoxystrobin, dimethomorph, and procymidone were the most frequently detected pesticides. However, procymidone, diniconazole, and lufenuron were found to most frequently exceed the MRLs. The rate of MRLs exceeding has been managed below the average (2.4%) more recently than in the past in this area. Further, leafy vegetables with the most violations of the MRLs in our study in South Korea were not harmful to health by a risk assessment (the range of the hazard index was 0.001–7.6%).

Dicarboximide fungicides mainly including procymidone, iprodione, vinclozolin, and dimethachlon are often applied as protective fungicides, 3,5-dichloroaniline (3,5-DCA) is their common metabolite in plant and environment. In this study, the acute toxicity of procymidone, iprodione and their metabolite of 3,5-DCA toward zebrafish was evaluated by semi-static method. The enrichment and metabolism of procymidone and iprodione in zebrafish were also clarified. The results indicated that procymidone and iprodione exhibited moderately toxic to adult zebrafish with the LC50 of 2.00 mg/L, 5.70 mg/L at 96 h. Both procymidone and iprodione could be metabolized to 3,5-DCA in zebrafish, which showed higher toxic to adult zebrafish with the LC50 of 1.64 mg/L at 96 h. From the perspective of histomorphology, for all treatment groups, the brain of the zebrafish was significantly damaged, while the damage to gut and gills was lighter. For procymidone, the biological concentration factor (BCF8d) were 236 and 246 at the exposure concentration of 0.2 mg/L and 0.04 mg/L, and the BCF8d were 3.2 and 2.4 for iprodione at the exposure concentration of 0.5 mg/L and 0.1 mg/L. Therefore, the procymidone and iprodione were moderate-enriched and low-enriched in zebrafish, respectively.

Botrytis cinerea is a destructive plant pathogenic ascomycete that causes serious pre- and post-harvest losses worldwide. The novel sterol 14α-demethylase inhibitor (DMI) pyrisoxazole was recently registered for the control of tomato gray mold caused by B. cinerea in China. Baseline sensitivity of 110 B. cinerea isolates collected from nine provinces in China to pyrisoxazole was demonstrated, with a mean EC50 of 0.057 ± 0.029 μg/ml. Eleven stable mutants resistant to pyrisoxazole were generated via UV irradiation (RU-mutants) and spontaneous selection (RS-mutants) of conidia. The efficacy of pyrisoxazole against the resistant mutants was significantly lower than that of the sensitive isolates. Most of the pyrisoxazole- resistant mutants were less fit than the sensitive isolates, with reduced sporulation, conidia germination, sclerotium production, and pathogenicity, which was confirmed by the competitive ability test. Positive cross-resistance was only observed between pyrisoxazole and the DMIs tebuconazole and prochloraz, but not between pyrisoxazole and non-DMIs iprodione, procymidone, diethofencarb, fluazinam, pyrimethanil, or fludioxonil. A two-point mutation, at G476S and K104E in the RU-mutants, and a one point mutation, M231T, in the RS-mutants, were detected in the CYP51 protein of the resistant mutants. When exposed to pyrisoxazole, the induced expression level of CYP51 increased in the resistant isolates as compared to sensitive ones. Molecular docking suggested that G476S and M231T mutations both led to the loss of electrostatic interactions between CYP51 and pyrisoxazole, while no change was found with the K104E mutation. Thus, two point mutations on CYP51 protein combined with induced expression of its target gene appeared to mediate the pyrisoxazole resistance of B cinerea.

In 2014 and 2015, a total of 151 tobacco brown spot (Alternaria alternata) isolates were collected from Guizhou Province in China to evaluate their resistance to the benzimidazole thiophanate-methyl, the carbamate diethofencarb, and the dicarboximide procymidone. Resistance to thiophanate-methyl and diethofencarb was observed in all isolates. Resistance to all the three fungicides, thiophanate-methyl, diethofencarb, and procymidone was detected at a frequency of 6.0%. The F167Y single mutation in the β-tubulin gene was found to be associated with resistance to thiophanate-methyl,but no mutation was found in the coiled-coil region of the histidine kinase-encoding gene OS1, a fungal gene for dicarboximide resistance. Procymidone applied at the rate of 20 mg l−1 inhibited spot lesion formation on tobacco leaves with an efficacy of 51.7% for the low resistance (LR) isolates and 74.2% for the procymidone-sensitive isolates. Thiophanate-methyl applied at 100 mg l−1, however, slightly promoted the expansion of disease lesions with an efficacy of −7.7%. Azoxystrobin applied at 10 and 20 mg l−1 provided efficacies of 91.1 and 100%, respectively, regardless of whether the isolates were thiophanate-methyl resistant or procymidone-LR. Further studies suggested that azoxystrobin exhibited excellent protective activity and good curative activity against A. alternata in plants. The baseline sensitivity to azoxystrobin was then determined. In the presence SHAM, the mean EC50 values for conidial germination inhibition were 0.49 ± 0.22 (Mean ± SD) mg l−1. Interestingly, no resistance was recovered through UV irradiation or Agrobacterium tumefaciens-mediated mutagenesis. This research indicated widespread resistance to thiophanate-methyl and diethofencarb, low frequency of (6.0%) resistance to procymidone in A. alternata populations from tobacco, and suggested that azoxystrobin could potentially constitute a good alternative for the management of tobacco brown spot disease.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most important diseases in oilseed rape‐growing areas of China. To determine the frequency of resistance of field isolates of S. sclerotiorum to carbendazim and dimethachlone, a total of 556 isolates from 10 different regions of Henan Province were obtained between 2015 and 2016. The frequency of isolates with a high‐resistance phenotype and a moderate‐resistance phenotype to carbendazim was 69.2% and 10.8%, respectively. However, S. sclerotiorum isolates resistant to dimethachlone were not detected. The baseline sensitivity of S. sclerotiorum to dimethachlone was distributed as a unimodal curve with a mean EC₅₀ value of 0.39 ± 0.09 μg ml⁻¹ for the inhibition of mycelial growth. Four dimethachlone‐resistant mutants were obtained from 20 wild‐type isolates induced by exposure to increasing concentrations of the fungicide in vitro. The mutants showed high levels of resistance to dimethachlone, with resistance factors that ranged from 179 to 323. Positive cross‐resistance occurred between dimethachlone and procymidone, iprodione, and fludioxonil; however, no cross‐resistance was observed for carbendazim and boscalid. The fitness of the dimethachlone‐resistant mutants was significantly lower than that of the wild‐type isolates, as measured by mycelial growth, hyphal dry weight, sclerotium number and dry weight, and pathogenicity. Additionally, based on osmotic tests, the inhibition of mycelial growth caused by NaCl applied at different concentrations was significantly higher for the dimethachlone‐resistant mutants than for their wild‐type parents.

In the current study, sensitivity distribution of Sclerotinia sclerotiorum populations to fluazinam was determined using 103 strains collected from the fields of Jiangsu Province of China in 2016-2017 and the resistance risk of fluazinam was assessed. The average EC50 (50% effective concentration) values and MIC (minimum inhibitory concentration) values of 103 S. sclerotiorum strains against fluazinam were 0.0073±0.0045μg/ml and

As a broad-spectrum with low toxicity, procymidone (PCM), is widely used in agriculture and frequently observed in aquatic system, which may cause some impacts on aquatic organisms. Here, to determine the developmental toxicity of PCM, embryonic and larval zebrafish were exposed to PCM at 0, 1, 10, 100 μg/L in dehydrogenated natural water containing 0.01% acetone for 7 days. The results showed that high concentration of PCM could cause the pericardial edema and increase the heart rates in larval zebrafish, suggesting that PCM had developmental toxicity to zebrafish. We also observed that PCM exposure not only changed the physiological parameters including TBA, GLU and pyruvic acid, but also changed the transcriptional levels of glycolipid metabolism related genes. In addition, after transcriptomics analysis, a total of 1065 differentially expressed genes, including 456 up-regulated genes and 609 down-regulated genes, changed significantly in 100 μg/L PCM treated larval zebrafish. Interestingly, after GO (Gene Ontology) analysis, the different expression genes (DEGs) were mainly enriched to the three different biology processes including GABA-nervous, lipid Metabolism and response to drug. We also observed that the levels of GABA receptor related genes including gabrg2, gabbr1α, gabbr1 and gabra6α were inhibited by PCM exposure. Interestingly, the swimming distance of larval zebrafish had the tendency to decrease after PCM exposure, indicating that the nervous system was affected by PCM. Taken together, the results confirmed that the fungicide PCM could cause developmental toxicity by influencing the lipid metabolism and GABA mediated nervous system and behavior in larval zebrafish. We believed that the results could provide an important data for the influence of PCM on aquatic animals.

Anthracnose, which is caused by Colletotrichum acutatum, is a destructive disease of pepper. A preliminary study demonstrated that fludioxonil (a phenylpyrrole fungicide) has good activity against C. acutatum and thus has potential to be used as an alternative fungicide for the management of pepper anthracnose. However, there is no information regarding the baseline sensitivity and resistance risk of C. acutatum to fludioxonil. Thus, the sensitivities of 205 isolates of C. acutatum to fludioxonil were determined. The results showed that the frequency distributions of the EC50 values were unimodal, and the mean EC50 values for the inhibition of mycelial growth and spore germination were 0.031 μg/mL and 0.035 μg/mL, respectively. Three stable mutants with high resistance to fludioxonil were obtained in the laboratory. Two parameters, namely in vitro sporulation and the in vitro and in vivo germination of spores, showed significant difference (P

Bipolaris maydis (anamorph: Cochliobolus heterostrophus) is the causal agent of Southern Corn Leaf Blight (SCLB), leading to huge annually losses worldwide. Although fludioxonil, a phenylpyrrole fungicide with a broad spectrum of activity, was introduced in the 1990s, no baseline sensitivity has been established for B. maydis. One hundred field isolates were used to establish a baseline sensitivity of B. maydis against fludioxonil during 2015-2016. The results showed that the baseline sensitivity was distributed as a unimodal curve with a mean EC50 value of 0.044±0.022μgmL-1. With repeated exposure to fludioxonil, a total of five fludioxonil-resistant mutants (RF>100, RF=Resistance factor) were obtained in the laboratory. Compared with the parental isolates, the five fludioxonil-resistant mutants showed decreased fitness in sporulation and virulence, and exhibited different features of sensitivity to various stresses (oxidation and osmotic pressure, cell membrane and cell wall inhibitors), but not in mycelial growth on PDA without stress amendation. The five fludioxonil-resistant mutants showed a positive cross-resistance between fludioxonil and the dicarboximide fungicide procymidone, but not between fludioxonil and boscalid or fluazinam. All mutants exhibited stable resistance to fludioxonil after 10 transfers, as indicated by resistance factor values that ranged from 116.82 to 445.59. When treated with 1.0 M NaCl, all the fludioxonil-resistant mutants showed greater mycelial glycerol content than corresponding parental isolates. Sequencing alignment results of Bmos1 indicated that mutant R27-5 had a single point mutation (Z1125K), while the mutant R104 had a 34-bp deletion fragment between the codons of amino acid residues 1125 to 1236 and encodes a putative attenuated 1133-AA protein. The 34-bp deletion fragment led to not only a 11-AA deletion(DNAVNQKLAVR), but also the resulting frameshift mutation and early stop. The mutations of R27-5 and R104 were located in the Rec domain of the Bmos1 gene. No mutations at the Bmos1 were detected in the other three resistant mutants R27-1, R27-2 and R32.

This study assessed the fenhexamid sensitivity of 143 Botrytis cinerea isolates collected from greenhouse strawberries in five regions of China between 2012 and 2013, and identified four isolates with moderate levels of resistance: two from the Xinjiang Uygur Autonomous Region and two from Hebei Province. The baseline fenhexamid sensitivity of B. cinerea exhibited a unimodal distribution with a mean EC50 value of 0.20 ± 0.10 μg/ml (SD). The EC50 values of the fenhexamid-resistant isolates ranged from 0.05 to 0.40 μg/ml. Molecular analysis of the fenhexamid target gene erg27 revealed that the resistant isolates collected from Xinjiang (163-6 and 163-22) contained three mutations that led to amino acid changes (V365A, E368D and A378T) known to be associated with fenhexamid resistance, but that the isolates from Hebei lacked any mutations, indicating that an alternative mechanism could be responsible for their resistance. Most of the biological characteristics of the fenhexamid-resistant isolates, such as mycelial growth, sclerotia production and pathogenicity, did not significantly differ from those of the sensitive ones (p ≤ .05), but it was noted that some of the resistant isolates exhibited reduced rates of sporulation and spore germination. In addition, the resistant isolates exhibited lower osmotic sensitivity than the sensitive ones. The study found no evidence of cross-resistance with other fungicides, but that there was negative cross-resistance with procymidone, iprodione, carbendazim and pyraclostrobin, which indicates that the inclusion of these fungicides within an integrated pest management (IPM) programme could help to minimize the risk of fenhexamid resistance developing in B. cinerea.

The aim of this study was to evaluate the resistance of Botrytis cinerea to the fungicides currently used for its control in Brazil. Isolates of the fungus were collected from different strawberry-producing fields in the states of Espírito Santo, Minas Gerais, and São Paulo, Brazil. First, a total of 183 isolates were identified at the species level using specific primers for the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene. The isolates were grown on potato dextrose agar (PDA) containing the fungicides procymidone, iprodione, and thiophanate-methyl in different concentrations: 0.0 (control), 0.1; 1.0; 10.0; 100.0 and 1,000.0 μg∙mL−1. The percentage of mycelial growth inhibition was used to determine the effective concentration of the fungicide that was able to inhibit colony growth by 50% (EC50). Approximately 25.7% of the isolates were resistant to iprodione, 53.0% were resistant to procymidone, and 93.0% were resistant to thiophanate-methyl. Moreover, crossresistance and multiple resistance were verified, with 19.7% of the isolates showing resistance to 3 fungicides simultaneously. This finding explains the ineffectiveness of fungicides application to control gray mold in strawberry fields in Brazil and highlights the need for new strategies to manage this disease in the culture.

Two experiments were conducted at different locations, the first in the 2012-13 season, and the second in the 2013-14 season, to evaluate the effectiveness of fluazinam and procymidone fungicides applied at volumes of 100 and 200 L ha −1 , using three types of nozzles (hollow cone, flat fan, and air induction flat fan) in soybean, for the control of white mold ( Sclerotinia sclerotiorum Lib. de Bary). This study assessed white mold incidence, severity, sclerotia production and yield. In the 2012–13 crop season experiment, there was no difference between the spray volumes of 100 and 200 L ha −1 for any of the white mold assessments. With regard to the nozzles, the use of the air induction flat fan nozzle resulted in lower incidence and production of sclerotia. In the 2013-14 season experiment, the use of the flat fan nozzles, and the air induction flat fan nozzles resulted in the lowest rates of white mold incidence. Procymidone promoted a reduction in sclerotia production in comparison with fluazinam. No differences in yield were detected between the studied factors. Therefore, it was concluded that the spray volume did not influence the control of white mold and the use of air induction nozzle was efficient in controlling white mold in soybeans.

Incorrect and excess usage of pesticides during crop cultivation poses a serious threat to human health and ecosystems. In this study, we tested for the presence of 201 pesticide residues in 90 batches of Panax notoginseng (P. notoginseng) and 10 batches of planting soil. Pesticide residue characteristics and the relationship between pesticides present in P. notoginseng and the soil were discussed. Twenty-nine pesticides were detected in P. notoginseng samples and 15 pesticides were found in the soil samples. In P. notoginseng samples, the 68.9% of the identified pesticides were fungicides, and six fungicides (procymidone, iprodione, pyrimethanil, propiconazole, dimethomorph and tebuconazole) were found in >90% of the samples. Nine insecticides were found, with one insecticide, chlorpyrifos, detected in 93.3% of the P. notoginseng samples. The residual concentrations of 17 pesticides were found at levels exceeded the “non-Chinese” maximum residue levels (MRLs) for Ginseng and 17 pesticides were found at levels exceeding the MRLs set by China for “pollution-free” P. notoginseng. We observed no significant differences in pesticide residues were found on P. notoginseng from different cultivation areas. We also analyzed the degradation kinetics of pesticides in the soil, as well as their bioconcentration factors (BCFs), and found that the fungicides iprodione and myclobutanil displayed strong uptake from the soil to the root of P. notoginseng. Together, our data suggest that fungicides should be considered as key monitoring substances in P. notoginseng and planting soil.

Thirty-four novel compounds were synthesized using chesulfamide (N-(2-trifluoromethyl-4-chlorophenyl)-2-oxocyclohexyl sulfonamide), a high-profile fungicide, as the lead compound, and their structures were characterized by 1H NMR, 13C NMR, MS and elemental analysis. Additionally, the structure of (1S,2R)-2-((3-bromophenethyl)amino)-N-(4-chloro-2-trifluoromethylphenyl)cyclohexane-1-sulfonamide (IV-9) was confirmed by X-ray single crystal diffraction. The mycelium inhibition tests, spore germination inhibition tests, tomato pot tests and field trials were performed against strains of B. cinerea. Bioassay results showed that most of target compounds had good fungicidal activity against B. cinerea, in particular, IV-9 exhibited similar or superior effects to procymidone, boscalid and pyrisoxazole in all in vitro and in vivo tests. Moreover, there was no positive cross-resistance found between the compound IV-9 and eight commercial fungicides (azoxystrobin, boscalid, chlorothalonil, diethofencarb, fludioxonil, procymidone, pyrimethanil and pyrisoxazole) in the cross-resistance validation test performed by an innovative method.

White mold, caused by Sclerotinia sclerotiorum, is a yield-limiting disease of soybean in Brazil. Uniform fungicide trials have been conducted annually since 2009. Data from 74 cooperative field trials conducted over a 10-year period were assembled. We selected five fungicides applied two times around flowering: dimoxystrobin plus boscalid (DIMO+BOSC), carbendazim plus procymidone (CARB+PROC), fluazinam (FLUZ), fluopyram (FLUO), and procymidone (PROC). For comparison, thiophanate-methyl (TMET) applied four times was also included as a low-cost treatment. Network models were fitted to the log of white mold incidence (percentages) and log of sclerotia mass data (grams/hectare) and to the nontransformed yield data (kilograms/hectare) for each treatment, including the untreated check. Back-transformation of the meta-analytic estimates indicated that the lowest and highest mean (95% confidence interval [CI]) percent reductions in incidence and sclerotia mass were 54.2 (49.3 to 58.7) and 51.6% (43.7 to 58.3) for TMET and 83.8 (79.1 to 87.5) and 87% (81.9 to 91.6) for CARB+PROC, respectively. The overall mean (95% CI) yield responses ranged from 323 kg/ha (247.4 to 400.3) for TMET to 626 kg/ha (521.7 to 731.7) for DIMO+BOSC, but the variance was significantly reduced by a binary variable (30% threshold) describing disease incidence in the untreated check. On average, an increment of 352 kg/ha was estimated for trials where the incidence was >30% compared with the low-disease scenario. Hence, the probability of breaking even on fungicide costs for the high-disease scenario was >65% for the more effective, but more expensive fungicide (FLUZ) than TMET. For the low-disease scenario, profitability was less likely and depended more on variations in fungicide cost and soybean price.

Gray mold, caused by Botrytis cinerea, is one of the most destructive diseases of strawberry in China. For resistance monitoring, 198 B. cinerea isolates were collected from strawberry greenhouses at 10 locations in Hubei Province. The isolates were screened for resistance to fungicides procymidone and zoxamide. In mycelium growth assays for procymidone, the mean values of effective concentration at which mycelium growth is inhibited by 50% (EC50) for sensitive (ProS) and resistant (ProR) isolates were 0.25 μg/mL and 2.21 μg/mL, respectively. The frequency of ProR isolates was 14%, and the highest frequency (48%) was observed in Yichang. Positive cross-resistance was detected for ProR isolates to other dicarboximide fungicides, but not to phenylpyrroles. Comparative analysis of fitness parameters revealed increased osmotic sensitivity of ProR isolates compared to ProS ones. Sequence analysis of the dicarboximide target gene BcOs1 revealed that ProR isolates carried either a single point mutation at codon 365 (I365S) or a pair of point mutations (Q369P and N373S). The mean EC50 values for zoxamide sensitive (ZoxS) and resistant (ZoxR) isolates were 0.31 μg/mL and 7.76 μg/mL, respectively. Only six (3%) isolates from three locations were found to be resistant to zoxamide. All ZoxR isolates were found resistant to carbendazim. Fitness parameters did not show significant difference between ZoxR and ZoxS isolates. Sequence analysis of the beta-tubulin gene in resistant isolates revealed four previously reported point mutations (E198A, E198K, F200Y and T351I). The mutation T351I was detected only in the isolates possessing E198K point mutation. Mutation F200Y was detected in a highly resistant isolate. Results of this study will be helpful for the management of fungicide resistance in B. cinerea.

A forecasting model (BSPcast) developed for prediction of brown spot (Stemphylium vesicarium) of pear was evaluated as an advisory system for reduced fungicide use in disease management programs. Eleven trials were performed during 1995, 1996, and 1997 in five orchards in two different climatic areas in Catalunya (Spain) and Emilia-Romagna (Italy). Values of 3-day cumulative daily infection risk (CR) provided by the model were used to determine risk periods during the growing season of pear and were taken as thresholds to schedule fungicide sprays. The fungicide application programs tested using the model consisted of guided schedules with CR action thresholds of 0.4, 0.5, or 0.6, and fixed sprays following a standard commercial schedule. In nine out of 10 trials, no significant differences were observed in disease incidence on fruit at harvest between the fixed-spray commercial schedule and guided sprays using thresholds of 0.4 or 0.5. The average savings in number of fungicide sprays applied using BSPcast compared with the fixed-spray schedule were 20 to 70% when using fungicides with a 15-day protection period (kresoxim-methyl or procymidone) and ranged from 20 to 50% when using a fungicide with a 7-day protection period (thiram).

The objectives of this work were to investigate the Botrytis species that cause gray mold in strawberries in Parana and to study their sensitivity to fungicides: procymidone (P), iprodione (I), boscalid (B), thiophanate-methyl (T), fluazinam (F), azoxystrobin (A) and difenoconazole (D). The identification of 150 isolates of Botrytis sp. was carried out by amplifying the DNA by PCR using specific primers. The sensitivity to fungicides was determined by in vitro assays with discriminatory doses and EC50 (effective concentration to inhibit 50% of the growth of the fungus), molecular characterization of mutations and ex vivo assays for disease control efficacy. Thirty phenotypes were selected from combinations (S-sensitive and R-resistant) for the EC50 studies. Molecular characterization was performed using specific primers for mutations in the cyt b, bos1 and β-tubulin genes to confirm the resistance genotype. Tests were carried out with fruit treated with the fungicides (P), (B), (F) and (A) x the different phenotypes. All isolates were confirmed as Botrytis cinerea. Forty-one isolates were classified as fungicide sensitive phenotypes and of these, six isolates showed resistance to all seven tested fungicides. The percentage of isolates resistant to the fungicides azoxystrobin, boscalid, difenoconazole, fluazinam, iprodione and procymidone was 90.7; 45.3; 33.3; 32.7; 44.0 and 43.3%, respectively. A total of 94.6% isolates were classified as highly resistant to thiophanate-methyl. The EC50 for the fungicides procymidone, fluazinam and boscalid ranged from less than 0.1 to greater than 100 μg/ml. For azoxystrobin, most isolates had an EC50 greater than 100 μg/ml. Isolates with more than one mutation for different fungicides were found. For the assay in fruits with infections from the field, only the fungicide fluazinam differed from the control treatment, showing lower final incidence of 33.3% and control efficiency of 55.6%. For the assay with inoculated fruits, the fungicide fluazinam best controlled the disease for the three isolates presenting an average final incidence of 33.3% and an average control efficiency of 61.9%. Therefore, disease management in the state of Parana should be reviewed in terms of registering new fungicides to gray mold and the use of integrated management measures to reduce the reliance on fungicides.

Botrytis cinerea , the fungal pathogen responsible for causing gray mold in tomatoes, frequently exhibits multiple fungicide resistance. In this study, the sensitivity to carbendazim (Car), diethofencarb (Die), procymidone (Prc), and pyrimethanil (Pyr) was determined for 263 isolates of B. cinerea from diseased fruit and leaves of tomato plants from 87 commercial greenhouses in Henan Province of China during 2013 and 2014. Of the isolates tested, 89%, 93%, 85%, and 86% were resistant to Car, Die, Prc, and Pyr, respectively. Twelve phenotypes were found having different sensitivity patterns, and multi-fungicide resistance was found to one, two, three or four fungicides. The percentage of multiple fungicide resistant isolates (Car R Die R Prc R Pyr R ) was 68%. Mycelial growth, spore production, and mycelial dry weight were not significantly different between resistant and sensitive phenotypes of the B. cinerea isolates. However, the virulence of Car S Die R Prc S Pyr S isolates was higher than that of the other fungicide-resistance phenotypes. All of the carbendazim and diethofencarb resistant phenotypes carried a point mutation at codon 198 (E198A or E198K) or 200 (F200Y) in the β-tubulin gene, and all of the procymidone resistant phenotypes carried point mutations at codons 369 (Q369P) and 373 (N373S) in the Bos1 gene. These results provided important reference data for assessment of resistance risk of B. cinerea isolates from commercial tomato transplants in Henan Province, indicating the existence of a high frequency of resistance to carbendazim, diethofencarb, procymidone, and pyrimethanil revealing the necessity of a more integrated control of gray mold.

Botrytis cinerea is an important airborne plant pathogen with a wide host range. Gray mold caused by B. cinerea usually leads to both yield and quality reduction and causes severe losses. In China, the percentage of yield loss caused by B. cinerea gray mold in vegetable production is generally 10 to 20 %, sometimes up to over 60 % in some severely infected regions. The novel fungicide pyrisoxazole was registered for the control of tomato gray mold in China and has not been put into widespread use. In this study, baseline sensitivity of B. cinerea to pyrisoxazole was established based on EC50 values of 165 isolates sampled from tomato from Liaoning Province of China in 2012. Results showed that the frequency distribution of pyrisoxazole EC50 values was a unimodal curve with a right-hand tail. The mean EC50 value was 0.0676 ± 0.0409 (SD) μg/ml and the range of individual EC50 values was 0.0128 to 0.1987 μg/ml. There was no cross-resistance between pyrisoxazole, carbendazim and procymidone. The control efficacy of pyrisoxazole was assessed on detached leaves and in pot experiments. Pyrisoxazole provided over 80 % preventive control efficacy and over 50 % curative control efficacy at 100, 200, 400 μg/ml on detached leaves. It also provided over 80 % preventive control efficacy and over 60 % curative control efficacy at 100, 200, 400 μg/ml in pot experiments. These results indicate that pyrisoxazole is a highly effective fungicide with both preventive and curative effect for the control of B. cinerea.

In this study, 73 samples from soils planted with Panax notoginseng and six P. notoginseng samples were collected in Yunnan Province to investigate the residual levels of six pesticides and their relationships with P. notoginseng and soil. All six pesticides were detected in the soils planted with P. notoginseng located in three regions of Shilin, Kaiyuan, and Yanshan. The detection frequencies of the pesticides in the soils followed the order: quintozene (100%) iprodione (96%) procymidone (69%) chlorothalonil (51%) pyrimethanil (49%) pyraclostrobin (29%). The median concentrations of iprodione, pyraclostrobin, pyrimethanil, quintozene, procymidone, and chlorothalonil were 46.40, 6.4, 3.1, 2.86, 2.69, and 0.24 μg/kg, respectively. The mean concentrations of pesticides in the three regions followed the order: Kaiyuan Shilin Yanshan, except for iprodione. Furthermore, the concentrations of pesticide residues in soils in each region followed the order: soils never planted with P. notoginseng soils previously planted with P. notoginseng soils currently planted with P. notoginseng. The concentration of chlorothalonil in P. notoginseng followed the order: root stem leaf, whereas those of the other five pesticides followed the opposite order: root stem leaf. There were significant positive correlations between the mean concentrations of pesticides in P. notoginseng and those in the corresponding soils. These results indicate that the rational application of pesticides in P. notoginseng cultivation would be effective for reducing the accumulation of pesticides in P. notoginseng to protect people from the harmful effects of residual pesticides.

Altogether, 192 Botrytis cinerea isolates collected from tomato greenhouses at different locations in Hubei Province were evaluated for their sensitivity to fungicides procymidone and zoxamide. The mean effective concentration to cause 50% growth inhibition (EC50) values of procymidone for sensitive and resistant isolates were 0.25 and 3.60 μg/ml, respectively. The frequency of procymidone-resistant (ProR) isolates was 18%, and the highest frequency was recorded in Jingmen. Positive cross-resistance was observed for ProR isolates to other dicarboximide fungicides but not to phenylpyrroles. Significant differences were observed for fitness parameters (i.e., mycelial growth, osmotic sensitivity, and virulence between sensitive and resistant isolates). Amino acid sequence of the Bos1 gene revealed that ProR isolates carried either point mutations at codon 365 (I365S) or a pair of point mutations at codons 369 (Q369P) and 373 (N373S). For zoxamide, the mean EC50 values for sensitive and resistant isolates were 0.22 and 5.32 μg/ml, respectively. Approximately 14% of the isolates were found to be resistant to zoxamide, and the highest frequency of resistance was also observed in Jingmen. There was positive cross-resistance for zoxamide-resistant (ZoxR) isolates to carbendazim. No significant differences were observed for fitness parameters between zoxamide-sensitive and ZoxR isolates. Sequence analysis of the β-tubulin gene of Botrytis cinerea revealed two previously reported point mutations (E198A and E198K) and one new point mutation (T351I). This new mutation was detected in only those isolates which possessed the E198K but not E198A substitution. This study allows for a better understanding of the resistance development profile in Hubei Province. Results will be useful for the improvement of fungicide resistance management strategies.

Botrytis Cinerea is a plant pathogen that affect a large number of plant species like tomatoes, Lettuce, Grapes, and Strawberries among others. Sulfonamides are widely used in pharmaceutical industries as anti-cancer, anti-inflammatory and anti-viral agents. To complement our previous QSAR study, a ligand-based design and ADME/T study were carried out on these sulfonamides compounds for their fungicidal activity toward “Botrytis Cinerea”. With the help of AutoDock Vina version 4.0 in Pyrex software, the docking analysis was performed after optimization of the compounds at DFT/B3LYP/6-31G∗ quantum mechanical method using Spartan 14 softwar. Using the model generated in the previous QSAR work, the descriptors of the chosen model were considered in modifying the most promising compound ‘9’ in which twelve (12) derivatives were designed and found to have better activity than the template (compound 9). With compound 9j having the highest activity that turns out to be about 14 and 15 times more potent than the commercial fungicides “procymidone and chlorothalonil”. Furthermore, ADME/T properties of the designed compounds were calculated using the SwissADME online tool in which all the compounds were found to have good pharmacokinetic profile. Moreover, a molecular docking study on selected compounds of the dataset (compound 8, 13, 14, 19, 20, 21, 22 and 29) revealed that compound ‘20’ turned out to have the highest docking score of -8.5 kJ/mol. This compound has a strong affinity with the macromolecular target point (PDB ID: 3wh1) producing H-bond and hydrophobic interaction at the target point of amino acid residue. The molecular docking analysis gave an insight on the structure-based design of the new compounds with better activity against B. cinerea., Organic chemistry; Pharmaceutical chemistry; Theoretical chemistry; Ligand-based design; ADME/T; Molecular docking; Anti-fungal; Botrytis cinerea

Botrytis cinerea is an economically important fungal pathogen with a host range of over 200 plant species. Unfortunately, gray mold disease caused by B. cinerea has not been effectively controlled because of its high risk for fungicide resistance development. As a part of our ongoing efforts to develop novel sulfonamides as agricultural fungicides against Botrytis cinerea, we introduced 2-aminoethanesulfonic acid (taurine) substructure, designed and synthesized a series of novel 2-substituted acylaminoethylsulfonamides. The newly synthesized sulfonamides were evaluated in vitro and in vivo for their fungicidal activity against Botrytis cinerea, of which the 2-ethoxyacetylamide derivative (V-A-12, EC50 = 0.66 mg·L−1) exhibited the highest potency in vitro and superior fungicidal activity compared with procymidone (EC50 = 1.06 mg·L−1). In vivo bioassay indicated that compound V-A-12 could be effective for the control of tomato gray mold. Moreover, the structure-activity relationship of these sulfonamides was analyzed by establishing a three-dimensional quantitative structure-activity relationship (3D-QSAR) model, which can provide guidance for the development of sulfonamides as fungicides. Finally, the effeicacy of sulfonamide derivatives was again verified in the activity evaluation against resistant Botrytis cinerea strains. These results further enhance the development value of 2-substituted acylaminoethylsulfonamides to control the tomato gray mold.

The dissipation kinetics of a combined use of procymidone and thiamethoxam, and their impact on the biodiversity and pathogen on surface of tomatoes were studied. The half-lives of procymidone and thiamethoxam, used either on their own or in combination with each other, were 2.94 or 3.26 days and 2.41 or 2.67 days, respectively. The residues dropped below the maximum residue limit (MRL) after 7 or 10 days (MRL 2 mg·kg−1 for procymidone), and 10 or 14 days (MRL 0.2 mg·kg−1 for thiamethoxam), respectively. The phylum Bacteroidetes, Cyanobacteria, and Proteobacteria, were dominantly present in all studied samples. The genus Escherichia-Shigella was found and exposed to the dissipation of procymidone (r = −0.9209 for procymidone on its own, and r = −0.8611 for procymidone in combination with thiamethoxam). These results will contribute to establish adequate monitoring of pesticides residues and their incorporation in surface ecology and pathogen management strategies in tomatoes.

Thiophanate-methyl (TM), fluazinam, and procymidone are fungicides extensively used for white mold control of common bean in Brazil. We assessed the sensitivity of Brazilian isolates of Sclerotinia sclerotiorum to these three fungicides using discriminatory doses and concentration that results in 50% mycelial growth inhibition (EC50) values. In total, 282 isolates from the most important production areas were screened and none was resistant to fluazinam or procymidone. The EC50 values varied from 0.003 to 0.007 and from 0.11 to 0.72 μg/ml for fluazinam and procymidone, respectively. One isolate was resistant to TM. The EC50 of the TM-resistant isolate was greater than 100 μg/ml, whereas the EC50 of the sensitive isolates varied from 0.38 to 2.23 μg/ml. The TM-resistant isolate had a L240F mutation in the β-tubulin gene. This is the first report of mutation at codon 240 causing resistance to a benzimidazole fungicide in S. sclerotiorum. The high-resolution melting analysis allowed the distinction of TM-sensitive and -resistant isolates by specific melting peaks and curves. The TM-resistant isolate had mycelial growth, sclerotia production, and aggressiveness comparable with that of the sensitive isolates, indicating that this genotype will likely compete well against sensitive isolates in the field. This study demonstrates that resistance to TM, fluazinam, and procymidone is nonexistent or rare. Resistance management practices should be implemented, however, to delay the spread of TM-resistant genotypes.

After four years of cold storage, dimethachlon resistance of two laboratory-induced resistant Sclerotinia sclerotiorum isolates SCG7 and LA50 declined by 99.5% and 98.9%, respectively, and cross resistance to iprodione and procymidone also declined dramatically. Along with the decline of fungicide resistance, osmotic sensitivity to sodium chloride and glucose decreased tremendously; mycelial growth rate, sclerotia number and weight per potato dextrose agar (PDA) plate increased on average by 118.6%, 85. 5% and 64.5%, respectively; and virulence to detached leaves of oilseed rape increased by 72.7% on average. Significant negative correlations were detected between dimethachlon resistance levels and mycelial growth rate on PDA (r = -0.980, P = 0.021), and between resistance levels and lesion diameters on detached leaves of oilseed rape plants (r = -0.997, P = 0.002). These results have profound implications for assessing the potential risk for resistance development to dicarboximide fungicides in S. sclerotiorum.

BACKGROUND The dicarboximide fungicide dimethachlone has been widely used in China for more than 12 years to control the Sclerotinia stem rot caused by Sclerotinia sclerotiorum disease. First signs of resistance in the field are reported at low frequency. In this study, four resistant isolate/mutants were used to explore still unknown mechanisms leading to dimethachlone resistance. RESULTS The resistant isolate/mutants had significantly higher EC50 values compared with the sensitive control isolates. Cross-resistance was confirmed between dimethachlone and procymidone, iprodione and fludioxonil. The resistant isolate/mutants revealed a decreased mycelial growth rate, were less pathogenic on leaves of oilseed rape, were more sensitive to osmotic pressure and oxidative stress and released more electrolytes compared with the sensitive isolates. Only in one lab mutant did we find a point mutation (V238A) in the SsOs1 gene of the high-osmolarity glycerol (HOG) signalling pathway. The expression of this gene was lost in the field resistant isolate HN456-1-JBJ and decreased in mycelium that was subjected to either high osmotic pressure or dimethachlone; however, another key gene in the HOG pathway, SsHog1, could be induced in the resistant isolate and mutants with NaCl treatment. CONCLUSION This study demonstrates that resistance to dicarboximide fungicide dimethachlone in S. sclerotiorum is emerging in China. Several fitness parameters, including mycelial growth rate, sclerotia formed in vitro, aggressiveness on leaves and osmotic and H2O2 sensitivity, indicate that the resistant strains may not effectively compete with sensitive isolates in the field in the absence of selection pressure. Lost expression or the V238A point mutation in the SsOs1 gene may confer resistance to dicarboximide fungicide dimethachlone in S. sclerotiorum, but this study illustrates that other, yet unknown mechanisms also exist. © 2015 Society of Chemical Industry

An outbreak of grey leaf spot caused by Stemphylium solani was observed on tomato in Shandong Province of China in recent years and brought huge economical losses. Fludioxonil is a phenylpyrrole fungicide with strong antifungal activity against S. solani. To evaluate the risk of S. solani developing fludioxonil resistance, a total of 145 field isolates were examined for sensitivity to fludioxonil by measuring mycelial growth. The baseline sensitivity was distributed as a unimodal curve with a mean EC50 value of 0.0659 (±0.0170) µg mL−1. Five mutants with high resistance to fludioxonil (RF > 1000) were obtained by successively selecting on fludioxonil-amended plates in the laboratory. All the resistant mutants associated with strongly reduced fitness in mycelial growth, sporulation and pathogenicity. Fludioxonil had positive cross-resistance with procymidone and iprodione, but there was no cross-resistance with other fungicides including boscalid, fluazinam, azoxystrobin and flusilazole. Based on the current results, resistance risk of S. solani to fludioxonil could be moderate. This is the first report of baseline sensitivity of S. solani to fludioxonil and its risk assessment. In order to delay the resistance development, it is recommended that fludioxonil can be used as one component of the mixture or fungicides with different modes of action should be alternatively used for this disease management.

A series of novel 3-(2-(substituted phenyl)hydrazinylmethylidene)furan-2,4(3H,5H)-diones were designed and synthesized with ethyl 4-chloroacetoacetate as the starting material. Their structures were confirmed by FT-IR, 1H NMR, 13C NMR, EI-MS and elemental analysis. Bioassay data demonstrated that these compounds exhibited remarkable antifungal activity against Fusarium graminearum, Botrytis cinerea, Rhizoctonia cerealis and Colletotrichum capsici. Compound 3-(2-(4-bromophenyl)hydrazinylmethylidene)furan-2,4(3H,5H)-dione (5g) had excellent bioactivity against Botrytis cinerea with an EC50 value of 0.18 μg/mL - markedly lower than the 0.24 μg/mL of the commercial fungicide procymidone. The result revealed that introducing the halogenated phenylhydrazine at the 3-position of furan-2,4(3H, 5H)-dione was an effective way to design new tetronic acid derivatives as new fungicides.

Cyprodinil belongs to the chemical class of anilinopyrimidines fungicides. In this study, baseline sensitivity of Sclerotinia sclerotiorum (Lib.) de Bary to cyprodinil was determined using 100 strains collected from the fields in Jiangsu Province of China. The EC 50 (50% effective concentration) values ranged from 0.0636–0.8163 μg/ml with a mean value of 0.1869 (±0.1118) ug/ml for mycelial growth. Nine cyprodinil-resistant mutants (Range of resistance factor: 20.22–271.59) were obtained from sensitive strains exposed on PDA medium amended with cyprodinil and the resistance was stable after their ten transfers on PDA without the fungicide or stored at 4 °C for two months. There was positive cross-resistance between cyprodinil and pyrimethanil but not to fludioxonil, dimetachlone, procymidone, carbendazim and boscalid in S. sclerotiorum . Compared with the parental strains, all of the nine cyprodinil-resistant mutants decreased in sclerotial production. The dry weight of mycelia, pathogenicity and cell membrane permeability of most resistant mutants decreased. The mycelial growth, oxalic acid content, and the response to various stress for resistant mutants were almost the same as the sensitive parental strains. Sequencing alignment results showed that there was no alteration of amino acid in cystathionine γ-synthase (MetB) and cystathionine β-lyase (MetC) between cyprodinil-resistant mutants and their sensitive parental strains, which indicated that MetB or MetC was not the molecular target of cyprodinil in S. sclerotiorum . The addition of amino acids L-methionine, L-cystine or L-cysteine decreased the inhibition of cyprodinil against mycelial growth of S. sclerotiorum , which indicated that cyprodinil could not only inhibited methionine biosynthesis but also suppressed cystine and cysteine biosynthesis. These results will contribute to evaluating the resistance risk of cyprodinil for management of the plant diseases of Sclerotinia stem rot caused by S. sclerotiorum and further increase our understanding about the mode of action of cyprodinil.

La lechuga (Lactuca sativa L. Var L. crispa L.) es de las hortalizas más cultivadas en el municipio de Mosquera, Cundinamarca, Colombia. En esta investigación se evaluaron dos productos biológicos (Enterobacter sp., TVL-1 y TVL-2) con capacidad de suplir las necesidades de fosforo de la planta y un producto biológico (Trichoderma koningiopsis TH003) con capacidad de controlar Sclerotinia sp., estos se compararon con los métodos tradicionales de fertilización y control químico (Fungicida a base del ingrediente activo procimidona), el diseño estadístico fue 14 tratamientos, evaluados en un ensayo bajo el diseño de bloques completos al azar, con tres repeticiones. La adición de las bacterias mejoró el crecimiento de las plantas, sin que se demostrara diferencia por efecto de los Enterobacter TVL-1 y TVL-2. El efecto de aplicar las Enterobacter sp., es mayor cuando se aplican solas, que cuando se aplican acompañadas de T. koningiopsis TH003. Es decir, cuando se observan los tratamientos sin T. koningiopsis TH003, el peso fresco aéreo aumentó en un 32,3% (0,15 g/planta) con la aplicación de Enterobacter sp., en semillero; en campo el aumento del peso fresco aéreo fue de 16,9% respecto al testigo sin Enterobacter sp. El tratamiento con el producto biológico Trichoderma koningiopsis TH003, presentó incidencia de Sclerotinia sp., inferior al 0,35% y una severidad inferior al 15%; el testigo absoluto con fertilización presentó incidencia de 10,71% y una severidad del 30%, por lo que se concluye que Trichoderma koningiopsis TH003 tiene un buen control de las enfermedades que se presentan en el cultivo.

In order to discover new antifungal agrochemicals that could have highly active and novel motifs, thirty-six new 2-acylaminocycloalkylsulfonamides (IV) were synthesized. Their structures were characterized and confirmed by 1H NMR, 13C NMR, IR, MS, elemental analysis and X-ray single crystal diffraction. In vitro and in vivo activities against various Botrytis cinerea strains were evaluated. Bioassay results revealed that most of the title compounds exhibited excellent in vitro fungicidal activity, in which compound IV-26 showed the highest activity against sensitive, low-resistant, moderate-resistant and high-resistant strains of B. cinerea compared with the positive fungicide procymidone. Meanwhile in vivo fungicidal activity of compound IV-31 was better than the commercial fungicides procymidone and chesulfamide in greenhouse trial. The structure activity relationship (SAR) was also discussed and the results were of importance to the structural optimization and development of more potent sulfonamides antifungal agents.

Y5247 (development code no.), a quinone-outsider-inhibitor (QoI), is a novel fungicide with a broad-spectrum of fungicidal activity against plant filamentous fungi, including B. cinerea. Compared to azoxystrobin, Y5247 had a stronger inhibitory effect on mycelial growth of B. cinerea. The baseline sensitivity of 100 strains showed the EC50 values for Y5247 inhibiting mycelial growth of B. cinerea ranged from 0.0033 to 0.3921 μg/mL with a mean of 0.0859 (±0.0788) μg/mL and the frequency distribution of EC50 values was unimodal. Mutants with stable resistance to Y5247 were not obtained following exposure to ultraviolet light or chemical mutagenesis. However, strains resistant to Y5247 were collected from greenhouses where other QoI fungicides had frequently been sprayed to control gray mold of strawberry. Among the Y5247-resistant strains, EC50 values for Y5247 and other QoIs, including azoxystrobin, kresoxim-methyl and pyraclostrobin, were positively correlated. No cross-resistance was detected between Y5247 and the other fungicides with different modes of action such as carbendazim, diethofencarb, boscalid, fluazinam, procymidone, and fludioxonil. The Y5247-resistant strains showed comparable fitness in mycelial growth, sporulation and pathogenicity to that of Y5247-sensitive strains. The risk of B. cinerea developing resistance to Y5247 was assessed as high. Analysis of cytb in B. cinerea showed that all the Y5247-sensitive and resistant strains carried the type II cytb (which lacks the intron Bcbi-143/144) and the point mutation G143A occurred in all the resistant strains tested.

The dicarboximide fungicide dimethachlon has been widely used for controlling Sclerotinia sclerotiorum in China for more than a decade. To assess the current status of dimethachlon resistance in S. sclerotiorum in China, 2,424 isolates were collected from disease-infected oilseed rape and soybean plants in five provinces of China in 2011 and 2012, and dimethachlon resistance was monitored by mycelial growth inhibition method on potato dextrose agar (PDA) media. Dimethachlon at 5 μg/ml was used as a discriminatory dose to detect resistance in all isolates, and 50% effective concentration values were determined for all dimethachlon-resistant isolates and some sensitive isolates. No dimethachlon resistance was detected in isolates from Anhui province (eastern China), Gansu province (northwestern China), and Qinghai province (western China). In Hunan province (central China), 3 of 268 (1.12%) isolates collected from oilseed rape plants in 2012 were resistant to dimethachlon, and the resistance ratios for the three resistant isolates were 4.56, 32.70, and 105.53, respectively. In Heilongjiang province (northeastern China), 8 of 243 (3.29%) isolates collected from soybean plants in 2011 were resistant to dimethachlon, with resistance ratios of 5.57 to 94.80; 11 of 409 (2.69%) isolates collected in 2012 were resistant to dimethachlon, with resistance ratios of 3.21 to 9.69. Cross-resistance studies showed that there was positive cross-resistance between dimethachlon and iprodione, procymidone, and the N-phenyl carbamate fungicide diethofencarb. No cross-resistance was found between dimethachlon and carbendazim, tebuconazole, kresoxim-methyl, thiram, and boscalid. Compared with the sensitive isolates of S. sclerotiorum, the field-dimethachlon-resistant isolates were more sensitive to osmotic pressure, grew more slowly on PDA media, and were less pathogenic on leaves of oilseed rape.

Botrytis cinerea is a necrotrophic plant pathogen that causes gray mold: a disease inflicting significant damage and crop losses in a wide range of cultivated plant species in Argentina and elsewhere. In Argentina, control of gray mold with the two mainstay fungicides, Carbendazim (Car) and Procymidone (Prc), has become ineffective, suggesting that this pathogen has developed resistance to these fungicides active ingredients. However, the actual sensitivity of this pathogen to these fungicides and the genetic basis for resistance in the region are unknown. In this study, we report the sensitivity to Car and Prc of 12 isolates of B. cinerea collected in Argentina. The fungus was recovered from diseased strawberry and blueberry fruits and tomato leaves from four commercial locations in 2017. Seven isolates were resistant to Car (Car-R), two to Prc (Prc-R), and two had both Car and Prc resistance (CarPrc-R). All the Car-R phenotypes carried the point mutation E198A in the β-tubulin gene (tub) and all the Prc-R phenotypes carried the point mutations Q369P and N373S in the two-component histidine kinase gene (Bos1). To our knowledge, this is the first report of resistance to Car and Prc in B. cinerea from strawberry, blueberry and tomato in Argentina. A comprehensive survey to document the regional occurrence of resistance in the country is underway.

Eighteen N-substituted phenyl-2-acyloxycyclohexylsulfonamides (III) were designed and synthesized by the reaction of N-substituted phenyl-2-hydroxyl- cycloalkylsulfonamides (I, R(1)) with acyl chloride (II, R(2)) in dichloromethane under the catalysis of TMEDA and molecular sieve. High fungicidal active compound N-(2,4,5-trichlorophenyl)-2-(2-ethoxyacetoxy) cyclohexylsulfonamide (III-18) was screened out. Mycelia growth assay against the Botrytis cinerea exhibited that EC50 and EC80 of compound III-18 were 4.17 and 17.15 μg mL(-1) respectively, which was better than the commercial fungicide procymidone (EC50 = 4.46 μg mL(-1) and EC80 = 35.02 μg mL(-1)). For in vivo activity against B. cinerea in living leaf of cucumber, the control effect of compound III-18 was better than the fungicide cyprodinil. In addition, this new compound had broader fungicidal spectra than chlorothalonil.

Sclerotinia sclerotiorum is a filamentous fungal pathogen that can infect many economically important crops and vegetables. Alternative oxidase is the terminal oxidase of the alternative respiratory pathway in fungal mitochondria. The function of alternative oxidase was investigated in the regulation of sensitivity of S. sclerotiorum to two commercial fungicides, azoxystrobin and procymidone which have different fungitoxic mechanisms. Two isolates of S. sclerotiorum were sensitive to both fungicides. Application of salicylhydroxamic acid, a specific inhibitor of alternative oxidase, significantly increased the values of effective concentration causing 50% mycelial growth inhibition (EC50) of azoxystrobin to both S. sclerotiorum isolates, whereas notably decreased the EC50 values of procymidone. In mycelial respiration assay azoxystrobin displayed immediate inhibitory effect on cytochrome pathway capacity, but had no immediate effect on alternative pathway capacity. In contrast, procymidone showed no immediate impact on capacities of both cytochrome and alternative pathways in the mycelia. However, alternative oxidase encoding gene (aox) transcript and protein levels, alternative respiration pathway capacity of the mycelia were obviously increased by pre-treatment for 24 h with both azoxystrobin and procymidone. These results indicate that alternative oxidase was involved in the regulation of sensitivity of S. sclerotiorum to the fungicides azoxystrobin and procymidone, and that both fungicides could affect aox gene expression and the alternative respiration pathway capacity development in mycelia of this fungal pathogen.

A series of N-(2,4,5-trichlorophenyl)-2-Oxo- and 2-Hydroxycycloalkyl- sulfonamides (series II and III) were designed and synthesized based on the leading compound chesulfamide (code name: CAUWL-2004-L-13). Their struc- tures were confirmed by 1 H NMR, IR and elemental analysis. Compounds II and III showed excellent activities against the Botrytis cinerea both in vitro and in vivo. Mycelia growth and conidial germination assays exhibited that EC50 of compound IId were 0.64 μg mL -1 and 0.34 μg mL -1 respectively. For in vivo control of B. cinerea in cucumber seedlings, compounds IIb and IId showed better control effect than the commercial fungicides procymidone and pyrimethanil. In addition, these new compounds had broader fungicidal spectra than chlorothalonil. The fungicidal activity was affected obviously by the size of cycloalkane. All the compounds with six-, seven-, and eight-membered ring showed high fungi- cidal activities.

A series of novel 2-substituted aminocycloalkylsulfonamides were designed and synthesized by highly selective N-alkylation reaction, whose structures were characterized by 1H NMR, 13C NMR and HRMS. Among them, the configuration of compounds III12 and III20 were confirmed by X-ray single crystal diffraction. Bioassays demonstrated that the title compounds had considerable effects on different strains of Botrytis cinerea and Pyricularia grisea. Comparing with positive control procymidone (EC50 = 10.31 mg/L), compounds III28, III29, III30 and III31 showed excellent fungicidal activity against a strain of B. cinerea (CY-09), with EC50 values of 3.17, 3.04, 2.54 and 1.99 mg/L respectively. Their in vivo fungicidal activities were also better than the positive controls cyprodinil, procymidone, boscalid and carbendazim in pot experiments. Moreover, the fungicidal activity of III28 (EC50 = 4.62 mg/L) against P. grisea was also better than that of the positive control isoprothiolane (EC50 = 6.11 mg/L). Compound III28 would be great promise as a hit compound for further study based on the structure-activity relationship.

The aim of this study was to evaluate the residues of 140 pesticides in raspberries from north-eastern Poland (2005-2010). Gas chromatography with electron capture detector (GC-ECD) and nitrogen phosphorous detector (GC-NPD) was used. Among the 128 samples, 66 (51.6%) were found to detect residues: 14.1% contained one pesticide and around 38% multiple pesticide residues. The most frequently detected were pyrimethanil residues (36.0%). Twenty-seven (21.1%) raspberry samples exceeded the maximum residue limits. The estimated daily intakes ranged from 0.003% to 3.183% of the acceptable daily intake (ADI) for adults 0.008% and 9.7% for toddlers, respectively. The most critical case is procymidone, the acute risk was 180.9% of acute reference dose (ARfD) for toddlers and for adults (83% of ARfD) which is high.