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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.

Garlic leaf blight caused by Stemphylium eturmiunum was first reported in Jiangsu Province in China. The dicarboximide fungicide (DCF) procymidone is reported to possess broad-spectrum action in inhibiting filamentous fungi and is widely used to control leaf disease of various plants. Of 41 Stemphylium eturmiunum isolates collected in this study from commercial garlic farms in Pizhou and Dafeng counties of Jiangsu Province, eight isolates were resistant to procymidone. The following three phenotypes were categorized according to in vitro responses to DCFs: sensitive, low resistance to iprodione and procymidone, and high resistance to all iprodione and procymidone. The fitness of all resistant isolates was decreased in accordance with data on mycelial growth, conidiation, and virulence. After treatment with 10 µg/ml of procymidone for 4 h, mycelial intracellular glycerol concentrations of resistant isolates were significantly lower than those of sensitive isolates. Positive cross-resistance was observed between dicarboximides and phenylpyrroles, but there was no cross-resistance between dicarboximides and fluazinam or difenoconazole in the two resistant phenotypes. Nucleotide sequence alignment of two-component histidine kinase genes from sensitive and resistant isolates indicated that amino acid mutations were located at the histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein and at the phosphatase domain of the N-terminal region and the response regulator domain of the C-terminal region. To our knowledge, this is the first report of DCF resistance in Stemphylium eturmiunum, and these findings will help establish a rational strategy to manage DCF-resistant populations of Stemphylium eturmiunum in the field.

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.

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.

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.

Botrytis leaf blight, caused by Botrytis squamosa, is an important disease of onion, mainly in the Southern region of Brazil. Studies on disease management that include host resistance, chemical, biological and alternative methods are scarce in the region. This work aimed to evaluate the effect of control treatments and 40 onion genotypes with varying resistance levels on disease management. Control treatments included seven fungicides, one biocontrol agent, and a calcium phosphite treatment. Among the genotypes, Onix and Liborio cv. exhibited the lowest levels of disease severity in the 2015 experiment, while the Bola Agroecologica, Preciosa, Onix, and Super Agroecologica cv. were least affected in the 2016 experiment. Trifloxistrobin (75 g/ha) + tebuconazole (150 g/ha); boscalid (200 g/ha) + kresoxim-methyl (100 g/ha); and fluazinam (398.4 g/ha) best controlled the disease, while dicarboximides, Bacillus subtilis and phosphorus pentoxide (600 g/ha) + calcium (120 g/ha) did not differ from the control. We updated the data on host resistance to Botrytis squamosa and report for the first time the inefficacy of iprodione and procymidone for controlling Botrytis leaf blight in Brazil.

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

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.

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.

International audience; Iprodione is a fungicide widely used in viticulture in most agricultural countries. It was banned recently in the European community because of its carcinogenic and endocrine disrupting characters. In this work, a cheap analytical method able to monitor iprodione in a white wine was developed. Molecularly imprinted sol-gel polymers (MIS) specific to iprodione and using green solvents were synthesized. An experimental design having the following factors (solvent volume and crosslinker quantity) was used to prepare an optimal MIS. In terms of selectivity, the optimal MIS showed the best partition coefficient towards iprodione in a white wine containing four other competing fungicides (procymidone, pyrimethanil, azoxystrobin and iprovalicarb). A solid phase extraction method using the optimal MIS was optimized and applied to analyse iprodione in a white wine. Low detection and quantification limits were reached 11.7 and 39.1 µg/L respectively.

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.

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.

This study reports the optimization of a binary dispersive liquid-liquid microextraction method for the determination of iprodione, procymidone, and chlorflurenol by gas chromatography mass spectrometry. The study was aimed at using two extraction solvents to increase the extraction efficiency of all analytes. The binary solvents recorded results higher than the mono-solvents. After examining the effects of main experimental parameters and their interactions by analysis of variance, 200 µL of binary mixture (dichloromethane and 1,2-dichloroethane), 2.5 mL of ethanol, and 15 s vortex were obtained as optimum parameters. The detection and quantification limits calculated for the analytes were found to be between 0.30–1.6 and 1.0–5.3 ng/mL, respectively. Enhancement in detection power calculated as a ratio of the binary extraction detection limit to the detection limit of direct GC-MS analysis was 105-, 214-, and 233-fold for chlorflurenol, iprodione, and procymidone, respectively. In order to check the accuracy of the developed method, recovery study was performed. Water sampled from a lake and two wastewater samples from treatment facilities were spiked at two concentrations, and the percent recovery calculated for the samples ranged between 87 and 116%. These results confirmed the suitability of the method to real samples for accurate determination of the analytes at trace levels. © 2018, Springer Nature Switzerland AG.

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.

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.

Ultrasound-assisted extraction (UAE) and pressurized liquid extraction (PLE) followed by gas chromatography-triple quadrupole-mass spectrometry (GC TQ-MS) were used for the rapid determination of 11 fungicides (metalaxyl, cyprodinil, procymidone, iprovalicarb, myclobutanyl, kresoxim-methyl, benalaxyl, fenhexamide, tebuconazole, iprodione and dimethomorph) in white grape bagasse. The extractions were optimized on real non-spiked samples by means of experimental design and the optimal conditions were selected to achieve the method validation. The PLE procedure showed much higher efficiency than UAE for the target fungicides. Under the selected extraction conditions, PLE showed satisfactory linearity, repeatability and reproducibility. Recoveries for the majority of studied fungicides were higher than 80% with relative standard deviations (RSD) lower than 12%. Limits of detection (LODs) for GC TQ-MS were very low, at the sub ngg(-1) for the majority of the target fungicides, well below the European maximum residue limits (MRLs) for wine and table grapes, and vine leaves. Eighteen white grape bagasse samples were analyzed and nine out of eleven targets were detected in the samples. Seven of them were detected in more than 50% of the samples and most samples contained at least four of the target analytes. The most frequently found compounds were tebuconazole and dimethomorph with concentrations between 1.6-130 and 2.0-1788ngg(-1), respectively. Some samples showed high levels of many of the studied fungicides (high ngg(-1), even μgg(-1) for cyprodinil, fenhexamide, iprodione and dimethomorph), but all of them below the European maximum residue limits (MRLs) for wine grapes.

In order to preconcentrate iprodione fungicide in white wine samples, molecularly imprinted polymers (MIPs) specific for iprodione were synthesized using two polymerization approaches: precipitation (MIPp) and bulk polymerization (MIPb). A comparison of the performance of the MIPs and the corresponding non-imprinted polymers (NIPs) was conducted in batch studies. In this case, the MIPp revealed better recognition properties toward iprodione in wine samples than the MIPb. The MIPp and MIPb were then used as sorbents on solid phase extraction cartridges (MISPEp and MISPEb consecutively) in order to pre-concentrate iprodione from white wine samples. The optimization of the MISPE elution step was done using MIPp and acetonitrile was shown to be the best eluting solvent. MISPEp showed better iprodione recovery and pre-concentration factor than MISPEb. The selectivity of the MISPE method for iprodione was evaluated in white wine samples in the presence of two other fungicides, pyrimethanil and procymidone. MISPEp was very selective for iprodione compared with the two other fungicides. However, MISPEb was able to preconcentrate iprodione as well as its analogues.

A novel approach for the determination of six fungicides (triadimefon, procymidone, hexaconazole, myclobutanil, diniconazole and iprodione) in fruit samples is presented. Analytes were extracted using the dispersive liquid–liquid microextraction technique and determined by GC–ECD. Parameters affecting the dispersive liquid–liquid microextraction performance, such as the kind and volume of extraction and dispersive solvents, extraction time and salt concentration, were studied and optimized. Under the optimum extraction conditions, the linearities of the method were obtained in the range of 0.5–20.0 μg kg−1 for triadimefon, hexaconazole, diniconazole and procymidone, and 1.0–40.0 μg kg−1 for myclobutanil and iprodione, with the correlation coefficients ranging from 0.9902 to 0.9995. The enrichment factors ranged from 685 to 820 and the extraction recoveries ranged from 81.3 to 98.4%. The relative standard deviations varied from 3.1 to 7.8%. The limits of detection of the method were in the range of 0.02–0.12 μg kg−1. Results showed that the method we proposed can meet the requirements for the determination of target fungicides in fruit samples. Several compounds considered in this study were found in fruit samples.

During a 15-year monitoring survey (April 1995–March 2010) of pesticide residues in agricultural products, 1542 domestic samples (cereals, vegetables, fruits, etc.) collected in Hyogo prefecture, Japan, were analyzed. The number of pesticides tested increased annually from 110 to 615, and 2198 pesticides, including trace levels, were detected in 844 samples. Annual detection rates of positive samples ranged from 36 to 77% during 15 years, and the average rate was 65% in the past 5 years. With the increase in pesticides tested, total detections in FY 2005–2009 markedly increased, trace level detections composed more than half of all detections, and the numbers of samples containing 4 or more residues increased two-fold. Procymidone and iprodione were frequently found in vegetables, and kresoxim-methyl, acetamiprid, iprodione and captan in fruits. In addition, the detection of carbendazim, dinotefuran and imidacloprid increased in the past 5 years. Violations of MRLs or the uniform limit (0.01 μg/g) were observed in 9 samples (0.6%), among which pesticides unregistered for application were detected in 8 samples. The residue levels of unregistered pesticides should be assessed, as the uniform limit was applied for their regulation in many cases.

The baseline sensitivity of Botrytis cinerea to propamidine and assessment of the risk of propamidine resistance in vitro are presented in this article. The baseline sensitivities of 41 wild-type strains were distributed as a unimodal curve with EC50 values of mycelial growth ranging from 0.182 to 1.460 μg ml−1, with a mean of 0.79 ± 0.27 μg ml−1. A total of 10 resistant mutants, obtained from one parental strain, were induced by UV irradiation and selected for resistance to propamidine with an average frequency of 1.98 × 10−9 and 0.025 respectively. These mutants were divided into three classes of resistant phenotypes with low (LR), moderate (MR) and high (HR) levels of resistance, determined by the EC50 values of 5.0–15.0 μg ml−1, 15.1–75.0 μg ml−1 and more than 75.0 μg ml−1 respectively. Neither positive cross-resistance nor negative cross-resistance was detected between propamidine and the fungicides, benzimidazole carbendazim, anilino-pyrimidine pyrimethanil, dicarboximide iprodione or procymidone. All 10 propamidine-resistant mutants showed reduced mycelial growth in vitro, sporulation, spore germination and pathogenicity when compared with the parental strain. These studies demonstrated that propamidine possesses a low risk of resistance developing. However, as B. cinerea is a high-risk pathogen, appropriate precautions against resistance development should be taken.

Pesticides applied to grape vines before harvest may concentrate in the grape seed due to their high oil solubility. Twenty-four samples of grape marc, representing a range of red and white wine grape varieties, were collected and analysed for selected fungicides and insecticides. Fifteen of the 24 samples were matched with insecticide and fungicide application diaries. Residue concentrations of the fungicides, procymidone, iprodione, cyprodinil, fenhexamid, fludioxinil, pyrimethanil and trifloxystrobin, and the insecticides, indoxacarb and tebufenozide, were higher in grape seed oil and grape seed meal than in the fruit and the marc. The relative concentrations were approximately proportional to the octanol to water partition coefficients, log Kow. A range of other fungicides and insecticides were detected but were not significantly concentrated in the oil and seed meal relative to fruit and marc. The presence of pesticide residues in grape seed oil and grape seed meal will impact on the possibility of producing these wine by-products.

Carbendazim (MBC) was widely used to control Sclerotinia stem rot routinely during the 1980s in China, but development of MBC resistance in the causal agent Sclerotinia sclerotiorum led to control failures of this disease. In this study it was found that the MBC resistance in S. sclerotiorum populations was widespread throughout Jiangsu Province with a resistance frequency of 29.54% in the 1786 collected isolates during the growing seasons of 2006 to 2008. The resistance frequencies differed among sampled cities, ranging from 3.1% to 54.9%. The field MBC-resistant isolates showed comparable mycelial growth, sclerotia production and pathogenicity to the wild-type sensitive isolates, which suggested that the field MBC-resistant isolates might have sufficient parasitic fitness to compete with the field MBC-sensitive isolates in the field. In the in vitro sensitivity test, boscalid showed greater activity against S. sclerotiorum than dicarboximide fungicides (dimethachlon, iprodione and procymidone). The treatment 50% boscalid (WG) 125 g a.i. ha−1 was comparable in efficacy to the treatment 50% iprodione (WP) 600 g a.i. ha−1, and better than other treatments of 6% dimethachlon (WP) 690 g a.i. ha−1 and 50% procymidone (WP) 337.5 g a.i. ha−1, whereas MBC failed to control Sclerotinia stem rot (control efficacy only 16.0%). The most active agent for controlling Sclerotinia stem rot was boscalid in our study.

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is the main fungal disease of oilseed rape (Brassica napus) in China. Numerous fungicide applications are required for control. Dimethachlon, one of the dicarboximide fungicides, has been the major fungicide for disease control after benzimidazole resistance became widespread. Fungal populations were collected throughout Jiangsu Province between 2006 and 2007 in order to determine their sensitivity to dimethachlon. A total of 1,066 single-sclerotium isolates of S. sclerotiorum were collected, and most of the isolates were considered sensitive to dimethachlon. Five isolates collected in Yancheng and Changzhou showed normal growth at 5 μg/ml dimethachlon with the resistance factor ≈10 (resistance factor was estimated as ratios between the EC50 values of resistant isolates and the average EC50 values of sensitive ones) compared to the sensitive isolates (EC50 is the concentration of fungicide causing 50% reduction in growth). Through in vitro selection for resistance to the fungicide, 25 dimethachlon-resistant mutants were derived from 10 wild-type isolates of S. sclerotiorum. The resistance factors for the isolates ranged from 198 to 484, and the isolates were considered highly resistant to dimethachlon. Therefore, at least two different mechanisms of resistance seem to be involved: one that may provide a moderate resistance (insensitivity) and a second that may give a high resistance level under laboratory conditions. There was positive cross-resistance between dimethachlon and other dicarboximide fungicides, such as iprodione and procymidone, in these S. sclerotiorum isolates. The field dimethachlon-insensitive and the laboratory-induced dimethachlon-resistant isolates appeared to have mycelial growth, sclerotial production, and pathogenicity comparable to their wild-type parental isolates. Also, results of osmotic tests showed that there were no significant difference in mycelial radial growth between the field dimethachlon-sensitive and field dimethachlon-insensitive isolates on potato dextrose agar plates amended with 2, 4, 6, or 8% (wt/vol) NaCl, but the laboratory-induced dimethachlon-resistant isolates grew significantly more slowly than their wild-type sensitive parents under all concentrations of NaCl. Because these studies yielded a high frequency of laboratory resistance in S. sclerotiorum, together with the occurrence of field insensitivity, appropriate precautions against resistance development in natural populations should be taken.

Four hundred and sixty six isolates of B. cinerea were obtained from infected leaves, stems and fruits of paprika grown in greenhouses or plastic film houses in Gangwon province, Korea, between August and November in 2006 and 2007. These isolates were classified into five representative phenotypes of resistant (R) and sensitive (S) reactions as SSR, SRR, RSS, RRS and RSR according to the responses of isolates against benzimidazole, dicarboximide and N-phenylcarbamate fungicide in order. The percentage of five phenotypes were 51.3, 2.4, 35.6, 8.1 and 2.6%, respectively. The SSR phenotype (51.3%) was the most common. Among the nineteen fungicide products evaluated to compare their efficacy against gray mold pathogen on the paprika fruit inoculated with fungal mycelia, the mixture of diethofencarb and carbendazim was the most effective followed by iprodione, boscalid, the mixture of iprodione and thiophanate-methyl, fludioxonil, polyoxin-B, fluazinam, the mixture of tebuconazole and tolyfluanid and procymidone; while in the assay methods inoculated with fungal spores, the mixture of tebuconazole and tolyfluanid was the most effective in controlling gray mold followed by boscalid, fludioxonil, the mixture of diethofencarb and carbendazim and the mixture of pyrimethanil and chlorothalonil.

The aim of this work was to report on a total of 23 fungicides and insecticides residues in 75 green and leafy vegetables (Swiss chards, Spinaches and Lettuces) collected from Ourense (NW Spain) by Spring 2007. The pesticides in the study samples were extracted with acetonitrile; the extracts were then cleaned-up by solid-phase extraction and concentrated before determination by PTV (Programmable Temperature Vaporization Injector) – GC-ITMS. Use of analyte protectants mixtures provided the best results in terms of effective compensation for matrix-induced enhancement effect. Pesticide residues were determined above the maxima residue limits (MRL) in 15 of the 75 analyzed samples, with a total of 18 violations of the MRL (three of the samples did not fulfil with two different pesticide MRL). The highest concentrations of fungicides were found in lettuce (procymidone, 12 mg/kg) and the highest concentrations of insecticides were found in Swiss chard (cypermethrin, 6 mg/kg). More positives for fungicides were detected and at larger concentrations than insecticides, especially for lettuces. Accumulation of pesticides in lettuces is higher than for the other leafy vegetables. The findings of this study pointed to the following recommendation: the need for a monitoring program for residues of iprodione and procymidone, together with cypermethrin, in food crops at the national level.

The differences in residue pattern between Italy and South Africa, the main exporters of table grapes to the Danish market, were investigated. The results showed no major differences with respect to the number of samples with residues, with residues being found in 54-78% of the samples. Exceedances of the European Union maximum residue limit (MRL) were found in five samples from Italy. A number of samples were rinsed to study the possible reduction of residues. For copper, iprodione, procymidone and dithiocarbamates a significant effect of rinsing was found (20-49% reduction of residues). However, no significant effect was found for organophosphorus pesticides and pyrethroids, whereas the number of samples with residues of benzilates, phenylamids and triazoles was insufficient to demonstrate any significant effects. An intake calculation showed that the average intake from Italian grapes was 3.9 microg day(-1) for pesticides and 21 microg day(-1) for copper. Correspondingly, the intakes from South African grapes were 2.6 and 5.7 microg day(-1), respectively. When the total exposure of pesticides from grapes were related to acceptable daily intake, expressed as the sum of Hazard Quotients, the exposure were approximately 0.5% for Italian samples and 1% for South African samples.

This work describes the optimization and validation of a method employing solid-liquid extraction with low temperature partitioning (SLE/LTP) together with analysis by gas chromatography with electron capture detection (GC/ECD) for the determination of nine pesticides (chlorothalonil, methyl parathion, procymidone, endosulfan, iprodione, λ-cyhalothrin, permethrin, cypermethrin, and deltamethrin) in lettuce. The method was found to be selective, accurate, and precise, with means recovery values in the range of 72.3-103.2%, coefficients of variation ⩽ 12%, and detection limits in the range 0.4-37 μg kg(-1). The matrix components significantly influence the chromatographic response of the analytes (above 10%). The optimized and validated method was applied to determine the residual concentrations of the fungicides iprodione and procymidone that had been applied to field crops of lettuce. The maximum residual concentrations of the pesticides in the lettuce samples were 13.6 ± 0.4 mg kg(-1) (iprodione) and 1.00 ± 0.01 mg kg(-1) (procymidone), on the day after application of the products.

We report the detection of several fungicide residues (Dichlofluanid, Benomyl, Iprodione, Procymidone and Vinclozolin) in red and white bottled wines from two Portuguese wine-producing zones. Studies were done in order to evaluate the active compound transfer percentage from grapes to the final product along fermentation process. We also investigated their effects in Saccharomyces and non-Saccharomyces yeasts as well as their influence on the physical, chemical and organoleptic wine properties. All the tested fungicides had a negative effect on in vitro yeast growth, with Dichlofluanid and Benomyl being the most toxic. Zygosaccharomyces rouxii and Saccharomyces cerevisiae were the most resistant yeasts while Rhodotorula glutinis was the most susceptible. Microvinification experiments carried out in the presence of fungicides produced wine samples with unaltered physical, chemical and organoleptic properties, though Benomyl, a slight delay in the initiation of fermentation process was observed.

Effects of sixteen chemical pesticides on conidial germination of C. thromboides and P. nouryi were investigated at two concentrations (R and 0.2R, where R=the lowest manufacture's recommended concentration for field use). Of the fungicides tested, propamocarb hydrochloride did not significantly affect conidial germination of C. thromboides, but at R it completely inhibited conidial germination of P. nouryi. At 0.2R of procymidone, iprodione, haloxyfop-methyl and fenoxaprop-p-ethyl, conidia of C. thromboides had some germination after 24 h, although they entirely restricted conidial germination of the fungus at the concentration of R. Tribenuron-methyl, one herbicide, had no adverse effect on conidial germination of C. thromboides and P. nouryi after 6 h at the two concentrations, whereas the other seven herbicides were strongly antagonistic to P. nouryi, except for carfentrazone-ethyl at 0.2R.

A study of the matrix-induced effect was performed for 16 common pesticides, most frequently found in monitoring studies in tomato, pepper and cucumber, using a simple multiresidue method with gas chromatography (GC) and electron-capture (ECD) or nitrogen-phosphorus (NPD) detection, without a previous cleanup step. Anomalously high gas chromatography responses and subsequently very high recoveries for several pesticides in the extracts were obtained by a conventional calibration with pesticide solution in ethyl acetate. Sample matrix enhancement varied from little to no effect for some pesticides (e.g. chlorpyrifos, pirimicarb) to >200% in the case of certain susceptible pesticides (captan, procymidone, iprodione). Pronounced matrix effects were observed at low concentration levels of analyte for all the ECD-detected pesticides. The use of matrix-standards solutions was found to reduce the recoveries of most pesticides to the levels of 70–110% acceptable for residue analysis.

In this work we analysed samples of different commercial composts to verify the presence of metabolites of dicarboximidic fungicides (Iprodione, Procymidone, Vinclozolin and Chlozolinate) widely used in agriculture as botritycides. The commercial compost extracts, obtained by sonication with acetonitrile, were analysed by HPLC-DAD-MS. The detection limits (DL) of the analytical method were appraised for all considered compounds and for both the detectors. The analysis confirmed the presence of fungicide residues (i.e. Iprodione, 48 mg/kg) in three samples of commercial compost and traces of some corresponding metabolites (i.e. Metabolite I of Procymidone, 20 mg/kg) for most examined matrices. These results underline a possible presence of these fungicides and corresponding metabolites in soil, after the addition of compost.

To evaluate the sensitivity of Monilinia fructicola to dicarboximides used in controlling brown rot of peach, the fungus was isolated from commercial peach orchards in Chochiwon (CH), Chongdo (CD), Gyeongsan (GY), and Youngduk (YO) in Korea. The population shift of dicarboximide-resistant isolates of M. fructicola was investigated for 3 years starting 1998. The frequency of procymidone-resistant isolates (PRI) was higher in CD and GY than in CH and YO. The frequency of PRI was higher in the mid season (July-August) than in the rest of the year. Cross-resistance rate of PRI to iprodione was over 87.8% during the investigation, and double-resistance to both procymidone and carbendazim was less than 10%. However, the rate of cross-resistant isolates to vinclozolin was low. In the orchards in GY and CH without any fungicide spray, the PRI population was persistent and did not vary for 3 years. The results suggest that dicarboximide resistance of M. fructicola could be a problem in controlling brown rot and blossom blight on peach trees because it may take a long time to recover the population with sensitive isolates even in the absence of these fungicides.

The reduction mechanism of two pesticides, iprodione [3-(3,5-dichlorophenyl)- N -(1-methylethyl)-2,4-dioxo-1-imidazolidinecarboxamide] and procymidone [3-(3,5-dichlorophenyl)-1,5-dimethyl-3-azabicyclo[3.1.0]hexane-2,4-dione], was studied in the absence and presence of β-cyclodextrin in a dimethylsulfoxide solvent. Distribution of the reduction products changed in the presence of β-cyclodextrin. The complex formation led to protection of the imidazolidine ring of iprodione against cleavage, with a subsequent decrease in aniline-type products. In the case of procymidone, the mechanism exclusively led to the formation of monochloro derivatives of the original pesticide. Host–guest-type interactions of these pesticides with cyclodextrin nano-cavities was unambiguously demonstrated by voltammetric measurements that were more sensitive than conventional spectroscopic methods, such as UV-VIS and NMR.

The dicarboximide fungicides vinclozolin, iprodione and procymidone were analyzed in white wines using stir bar sorptive extraction (SBSE) in combination with thermal desorption-capillary GC-MS analysis (TD-cGC-MS). The method was optimized using spiked water samples in a concentration range between 0.5 and 100 microg/l. Iprodione was measured as its degradation product 3,5-dichlorophenyl hydantoin. Limits of quantification in the full scan MS mode are 0.5 microg/l for vinclozolin and procymidone and 5 microg/l for iprodione. In the ion monitoring mode, concentrations 100 times lower can be dosed. Because of wine matrix effects on the recoveries, quantification of the target fungicides in wine had to be carried out by standard addition. For the thermolabile iprodione, the accuracy of SBSE-TD-cGC-MS was verified using SBSE followed by liquid desorption and analysis by liquid chromatography-atmospheric pressure chemical ionization mass spectroscopy. Procymidone and iprodione were detected in wines in concentrations up to 65 microg/l while the highest concentration of vinclozolin detected was smaller than 3 microg/l.

The somatic recombinogenic activity of the phenylpyr-role fungicide fludioxonil, in diploid Aspergillus nidulans was found similar to that caused by aromatic hydrocarbon and dicarboximide fungicides (AHDFs), such as iprodione, chlozolinate and tolclofos-methyl. All these fungicides not only increased the number of mitotic recombinants but also provided similar appearance, small sectors, of white and yellow mitotic recombination products. Fludioxonil highly resistant strains (resistant factor approximately 5000) of Botrytis cinerea were isolated at high frequency (1.08 × 10 -5 ). Study of cross-resistance patterns of mutant strains to other fungicides, revealed cross-resistance of fludioxonil with dicarboximides (iprodione, procymidone, and chlozolinate) and aromatic hydrocarbons, such as tolclofos-methyl, pentachloronitrobenzene (PCNB), tecnazene and chloroneb. The positive cross-resistance relationships found between phenylpyrroles and members of the AHDFs and their ability to increase mitotic instability in diploid A. nidulans, indicate that phenylpyrroles should be included with AHDFs. A study of fitness parameters in wild-type and representative fludioxonil-resistant mutants of B. cinerea, showed that the mutation(s) leading to fludioxonil resistance may or may not affect some fitness-determining characteristics, such as sensitivity to high osmolarity, growth rate, conidial germination and germ-tube elongation. Pathogenicity tests on cucumber seedlings showed that an osmosensitive representative strain of B. cinerea, resistant to fludioxonil, was as virulent as the wild-type strain. The phenylpyrrole fungicide was ineffective, even in high concentrations, to control grey mould caused by this isolate.

This paper describes the determination of 3,5-DCA in commercial composts, a common metabolite in a class of fungicides, and dicarboximides (Vinclozolin, Chlozolinate, Iprodione, Procymidone) which are commonly used in agriculture. The extracts, obtained in acetonitrile by sonication, are analysed by HPLC/DAD without clean-up. This method has shown several advantages: reduced manipulation of samples, good recovery (80-90%) and good reproducibility (RSD%7). The limit of detection (DL) of the analytical method has been estimated as 15 microg/kg for the common metabolite, and 35-145 microg/kg for the four fungicides in the matrices.