Resistance risk assessment of mefentrifluconazole in Corynespora cassiicola and the control of cucumber target spot by a two-way mixture of mefentrifluconazole and prochloraz.

The cucumber target spot, caused by Corynespora cassiicola, is a major cucumber disease in China. Mefentrifluconazole, a new triazole fungicide, exhibits remarkable efficacy in controlling cucumber target spot. However, the resistance risk and mechanism remain unclear. In this study, the inhibitory activity of mefentrifluconazole against 101 C. cassiicola isolates was determined, and the results indicated that the EC50 values ranged between 0.15 and 12.85 µg/mL, with a mean of 4.76 µg/mL. Fourteen mefentrifluconazole-resistant mutants of C. cassiicola were generated from six parental isolates in the laboratory through fungicide adaptation or UV irradiation. The resistance was relatively stable after ten consecutive transfers on a fungicide-free medium. No cross-resistance was observed between mefentrifluconazole and pyraclostrobin, fluopyram, prochloraz, mancozeb, or difenoconazole. Investigations into the biological characteristics of the resistant mutants revealed that six resistant mutants exhibited an enhanced compound fitness index (CFI) compared to the parental isolates, while others displayed a reduced or comparable CFI. The overexpression of CcCYP51A and CcCYP51B was detected in the resistant mutants, regardless of the presence or absence of mefentrifluconazole. Additionally, a two-way mixture of mefentrifluconazole and prochloraz at a concentration of 7:3 demonstrated superior control efficacy against the cucumber target spot, achieving a protection rate of 80%. In conclusion, this study suggests that the risk of C. cassiicola developing resistance to mefentrifluconazole is medium, and the overexpression of CcCYP51A and CcCYP51B might be associated with mefentrifluconazole resistance in C. cassiicola. The mefentrifluconazole and prochloraz two-way mixture presented promising control efficacy against the cucumber target spot.

Resistance to the DMI fungicide mefentrifluconazole in Monilinia fructicola: risk assessment and resistance basis analysis.

BACKGROUND: Brown rot disease, caused by Monilinia fructicola, poses a significant challenge to peach production in China. The efficacy of mefentrifluconazole, a new triazole fungicide, in controlling brown rot in peaches has been remarkable. However, the resistance risk and mechanism associated with this fungicide remain unclear. This study was designed to assess the resistance risk of M. fructicola to mefentrifluconazole and reveal the potential resistance mechanism. RESULTS: The mean median effective concentration (EC50 ) of 101 M. fructicola isolates to mefentrifluconazole was 0.003 µg mL-1 , and the sensitivity exhibited a unimodal distribution. Seven mefentrifluconazole-resistant mutants were generated from three parental isolates in the laboratory through fungicide adaption. The biological characteristics of the resistant mutants revealed that three of them exhibited enhanced survival fitness compared to the parental isolates, whereas the remaining four mutants displayed reduced survival fitness. Mefentrifluconazole showed strong positive cross-resistance with fenbuconazole, whereas no cross-resistance was observed with pyrimethanil, procymidone or pydiflumetofen. No overexpression of MfCYP51 gene was detected in the resistant mutants. Multiple sequence alignment revealed that three resistant mutants (MXSB2-2, Mf12-1 and Mf12-2) had a point mutation (G461S) in MfCYP51 protein. Molecular docking techniques confirmed the contribution of this point mutation to mefentrifluconazole resistance. CONCLUSION: The risk of M. fructicola developing resistance to mefentrifluconazole is relatively low-to-medium and point mutation G461S in MfCYP51 could confer mefentrifluconazole resistance in M. fructicola. This study provided essential data for monitoring the emergence of resistance and developing resistance management strategies for mefentrifluconazole. © 2023 Society of Chemical Industry.

Resistance Risk Assessment for the New OSBP Inhibitor Y18501 in Pseudoperonospora cubensis and Point Mutations (G705V, L798W, and I812F) in PscORP1 that Confer Resistance.

Y18501 is a new oxysterol-binding protein inhibitor (OSBPI) that shows strong inhibitory activity against Pseudoperonospora cubensis. In this study, the sensitivities of 159 Ps. cubensis isolates to Y18501 were determined, with EC50 values ranging from 0.001 to 11.785 µg/mL, indicating that a Y18501-resistant subpopulation has appeared in the field. Ten Y18501-resistant mutants were obtained by fungicide adaptation and displayed fitness equal to or stronger than their parental isolates, which suggests that the resistance risk of Ps. cubensis to Y18501 is high. The consecutive applications of Y18501 in the field resulted in the rapid resistance of Ps. cubensis and decreased control efficacy of cucumber downy mildew (CDM), which could be alleviated by compounding with mancozeb. A positive cross-resistance was detected between Y18501 and oxathiapiprolin. The amino acid substitutions G705V, L798W, and I812F in PscORP1 conferred resistance to Y18501 in Ps. cubensis, which was validated by molecular docking and molecular dynamics simulations.

Resistance to pydiflumetofen in Botrytis cinerea: risk assessment and detection of point mutations in sdh genes that confer resistance.

BACKGROUND: Gray mold caused by Botrytis cinerea Pers. is one of the most significant airborne diseases. It can infest a wide range of crops, causing significant losses in yield and quality worldwide. Pydiflumetofen, a new generation succinate dehydrogenase inhibitor (SDHI), is currently being registered in China to control gray mold in a variety of crops. The baseline sensitivity, resistance risk, and resistance mechanism of Botrytis cinerea to pydiflumetofen were assessed in this study. RESULTS: A total of 138 strains of B. cinerea from 10 different regions were tested for their sensitivity to pydiflumetofen, and the mean EC50 value was 0.0056 µg mL-1 . Eight mutants were obtained by fungicide adaption from five sensitive parental isolates, and the resistance factor (RF) ranged from 51 to 135. The mutants exhibited strong adaptive traits in conidial production, conidial germination, and pathogenicity. Positive cross-resistance was only observed between other SDHIs (i.e. boscalid, fluopyram, and isopyrazam). Two different types of pydiflumetofen-resistant mutants were identified: point mutation P225L in sdhB and double mutation G85A and I93V in sdhC. The in vivo control efficacy of pydiflumetofen on the resistant mutants carrying P225L in sdhB as well as G85A and I93V in sdhC was significantly decreased to 52.62% and 32.27%, respectively. CONCLUSION: The fitness was significantly higher for all pydiflumetofen-resistant mutants than the corresponding parental. Two types of point mutations, sdhB-P225L and sdhC-G85A and I93V, might confer resistance to pydiflumetofen in B. cinerea. A precautionary resistance management strategy should be implemented. © 2021 Society of Chemical Industry.

Resistance assessment of pyraoxystrobin in Magnaporthe oryzae and the detection of a point mutation in cyt b that confers resistance.

Pyraoxystrobin is a new QoI fungicide developed in China. The present study was aimed at determining the baseline sensitivity of M. oryzae to pyraoxystrobin and investigating the potential resistance risk and resistance mechanism of pyraoxystrobin in M. oryzae. The results showed that the mean EC50 of 109 M. oryzae isolates to pyraoxystrobin was 0.0094 µg/mL and the sensitivity exhibited a unimodal distribution. The established baseline sensitivity could provide critical data for monitoring sensitivity changes of M. oryzae to pyraoxystrobin in rice fields. The potential resistance risk was assessed by investigating the biological characteristics of the resistant mutants obtained by fungicide adaptation. The results indicated that the resistance risk of pyraoxystrobin in M. oryzae was medium to high with positive cross-resistance between pyraoxystrobin and azoxystrobin, but without cross resistance between pyraoxystrobin and carbendazim, isoprothiolane, and prochloraz. Further investigation revealed that the pyraoxystrobin-resistant mutants had a G143S mutation in the cyt b protein. Molecular docking confirmed that the G143S substitution conferred high resistance to pyraoxystrobin in M. oryzae. Collectively, the results of this study provided essential data for monitoring the emergence of resistance and developing resistance management strategies for pyraoxystrobin.

Tracking pesticide exposure to operating workers for risk assessment in seed coating with tebuconazole and carbofuran.

BACKGROUND: Coating seed with pesticides is an effective way to control plant pests, however, factory-based coating processes may carry a potential risk to operational workers of chemical exposure. To study the risk, carbofuran and tebuconazole were used to coat corn seed and their subsequent distribution on the bodies of workers was measured at manufacturers XFS and LS (Shanxi, China). Clothing was collected from workers during operations and analyzed using high-performance liquid chromatography. RESULTS: At XFS, dermal exposure to carbofuran was 4.83, 3.31 and 1.48 mg kg-1 , and exposure to tebuconazole was 6.88, 5.16 and 1.72 mg kg-1 for coating, packing and transport workers, respectively. At LS, dermal exposure to carbofuran was 2.32, 0.46 and 0.55 mg kg-1 , and exposure to tebuconazole was 1.69, 0.46 and 0.70 mg kg-1 , for coating, packing and transport workers, respectively. The level of pesticide exposure was significantly higher for seed-coating workers than for packing and transport workers. The main area of exposure was the hands for all workers and the lower limbs for packers; exposure was relatively uniform for pesticide handlers. Occupational risk was assessed based on margin of exposure (MOE). In seed-coating, the MOE was greater than 100 for tebuconazole, indicating no potential risk, but ranged from 0.25 to 2.88 for carbofuran, indicating the risk of a health impact. CONCLUSION: The level of exposure varied depending on type of operation undertaken and body parts of workers' body, but the risk of a health impact was highly associated with pesticide toxicity. This provides a guideline for workers in pesticide manufacturing to ensure safe operation of the seed-coating process. © 2021 Society of Chemical Industry.

Survival Cost and Diverse Molecular Mechanisms of Magnaporthe oryzae Isolate Resistance to Epoxiconazole.

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases on rice worldwide. Epoxiconazole is a 14α-demethylation inhibitor (DMI) with excellent control on rice blast; to date, no resistant isolates have been observed in the field. Four mutants resistant to epoxiconazole were generated from three parental isolates via fungicide adaptation. Resistance was stable after 10 weekly consecutive transfers on fungicide-free medium. Three parameters, including growth rate, sporulation in vitro, and aggressiveness, were significantly lower for mutants compared with their parental isolates, with the exception of the low-resistance isolate. Sporulation and aggressiveness were negatively correlated with effective concentration values for 50% inhibition of mycelial growth for parental isolates and mutants (P < 0.05). Cross-resistance was found between epoxiconazole and prochloraz (ρ = 0.863, P = 0.000) or difenoconazole (ρ = 0.861, P = 0.000). The resistance factor for mutants was positively correlated with the relative expression of MoCYP51A in epoxiconazole treatment (r = 0.977, P = 0.02). In addition, two putative amino acid substitutions in MoCYP51A were found in two resistant mutants: Y126F in the high-resistance mutant and I125L in the low-resistance mutant. Mutation Y126F reduced the affinity of MoCYP51A with epoxiconazole, whereas I125L was not in the binding pocket of epoxiconazole. No amino acid change or overexpression in MoCYP51B was found in any of the mutants studied. To our knowledge, this is the first study to report DMI resistance observed in M. oryzae. The survival cost of M. oryzae resistance to epoxiconazole might be the reason why DMI resistance has not yet emerged in field populations worldwide.

Activity and Resistance Assessment of a New OSBP Inhibitor, R034-1, in Phytophthora capsici and the Detection of Point Mutations in PcORP1 that Confer Resistance.

R034-1 is a new member of the piperidinyl thiazole isoxazoline class of fungicides that shows high activity against most plant-pathogenic oomycetes and could effectively inhibit several developmental stages of Phytophthora capsici. Here, the potential resistance risk for R034-1 was evaluated in P. capsici. The baseline sensitivities of 135 isolates to R034-1 showed a unimodal curve, with a mean EC50 value of 0.004 µg/mL. Twelve resistant mutants were generated by fungicide adaptation and displayed lower fitness compared to parental isolates, which suggests that the resistance risk of P. capsici to R034-1 is low. R034-1 and oxathiapiprolin are structurally related, and resistant isolates display cross-resistance to both compounds, suggesting that these fungicides may target the same oxysterol binding protein. Comparison of PcORP1 genes in the resistant mutants and their parental isolates revealed (N767S, N767I, and G700V) amino acid substitutions in the R034-1 resistant mutant. Causality was functionally validated using site-directed mutagenesis of the target gene using the CRISPR/Cas9 system.

Sensitivity of Different Developmental Stages and Resistance Risk Assessment of Phytophthora capsici to Fluopicolide in China.

Sensitivities of Phytophthora capsici to fluopicolide were investigated in vitro, with results showing that fluopicolide had strong inhibitory activities on each development stage of P. capsici, in particular on the motility of the zoospore. The potential resistance risk for fluopicolide in P. capsici was evaluated. The baseline sensitivities to fluopicolide of 146 isolates obtained from 28 provinces in China were initially determined, and the 50% inhibition of mycelial growth (EC50) distribution was a unimodal curve with a mean of 0.17 µg/ml. A series of fluopicolide-resistant mutants of P. capsici were obtained by fungicide adaptation, and their biological traits were determined. Most of the resistant mutants showed similar favorable fitness in mycelial growth, sporangium and zoospore production, cystospore germination, and pathogenicity compared with their sensitive parents, with few exceptions. Additionally, the cross-resistance result indicated that the sensitivity of fluopicolide did not correlate with other oomycete fungicides, apart from fluopimomide (LH-2010A). These results suggest a moderate to high resistance risk of P. capsici to fluopicolide in China.

Resistance assessment for SYP-14288 in Phytophthora capsici and changes in mitochondria electric potential-associated respiration and ATP production confers resistance.

BACKGROUND: Phytophthora capsici is a destructive plant oomycete pathogen that could lead to devastating losses in food production. Fungicide application is the main way to control plant disease caused by P. capsici. SYP-14288, a novel fungicide with a unique mode of action, could be used to control a broad range of plant diseases. Here, the potential for SYP-14288 resistance in P. capsici and the resistance mechanism involved were evaluated. RESULTS: Baseline sensitivities of 133 isolates to SYP-14288 were determined and found to conform to a unimodal curve with a mean half-maximal effective concentration (EC50 ) of 0.625 µg mL-1 . In total, 21 stable SYP-14288-resistant mutants were generated by fungicide adaptation in three sensitive isolates. The fitness of all the mutants was found to be lower than that of the parental isolates. Otherwise, downregulation of various ATPases may confer different resistance levels in P. capsici. Finally, multiple biochemical studies strongly suggest that both ATP content and electric potential were reduced in SYP-14288-resistant mutants, and as a compensatory mechanism, respiration was facilitated to make up for the energy defect in mutants. CONCLUSION: The low fitness of SYP-14288-resistant mutants suggests that the resistance risk of P. capsici to SYP-14288 is low. Resistance may be led by a permeability change in the mitochondrial inner membrane in SYP-14288-resistant isolates, and lower ATP consumption lifestyles may be key to the SYP-14288 resistance generated in P. capsici. The current study could benefit the registration and application of the novel fungicide SYP-14288. © 2020 Society of Chemical Industry.

Resistance risk assessment for fludioxonil in Sclerotinia homoeocarpa in China.

Sclerotinia homoeocarpa causes dollar spot disease on turfgrass and is a serious problem on many species worldwide. Fludioxonil, a phenylpyrrole fungicide, is not currently registered for dollar spot control in China. In this study, the baseline sensitivity to fludioxonil was established using an in vitro assay for 105 isolates of S. homoeocarpa collected from 10 locations in different regions of China. Results indicate that the frequency distribution of effective concentration for 50% inhibition of mycelial growth (EC50) values of the S. homoeocarpa isolates was unimodal (W = 0.9847, P = .2730). The mean EC50 value was 0.0020 ±â€¯0.0006 µg/ml with a range from 0.0003 to 0.0035 µg/ml. A total of 7 fludioxonil-resistant mutants were obtained in laboratory, the mutants were stable in fludioxonil sensitivity after the 10th transfer, with resistance factor (RF) ranging from 4.320 to >13,901.4. The mutants showed a positive cross-resistance between fludioxonil and the dicarboximide fungicide iprodione, but not propiconazole, fluazinam, and thiophanate-methyl. When mycelial growth rate, pathogenicity and osmotic sensitivity were assessed, the mutants decreased in the fitness compared with their parental isolates. Sequence alignment of the histidine kinase gene Shos1 revealed a 13-bp fragment deletion only in one mutant, no mutations were observed on Shos1 in the rest resistant mutants.

Resistance Assessment for Oxathiapiprolin in Phytophthora capsici and the Detection of a Point Mutation (G769W) in PcORP1 that Confers Resistance.

The potential for oxathiapiprolin resistance in Phytophthora capsici was evaluated. The baseline sensitivities of 175 isolates to oxathiapiprolin were initially determinated and found to conform to a unimodal curve with a mean EC50 value of 5.61 × 10(-4) µg/ml. Twelve stable oxathiapiprolin-resistant mutants were generated by fungicide adaptation in two sensitive isolates, LP3 and HNJZ10. The fitness of the LP3-mutants was found to be similar to or better than that of the parental isolate LP3, while the HNJZ10-mutants were found to have lost the capacity to produce zoospores. Taken together these results suggest that the risk of P. capsici developing resistance to oxathiapiprolin is moderate. Comparison of the PcORP1 genes in the LP3-mutants and wild-type parental isolate, which encode the target protein of oxathiapiprolin, revealed that a heterozygous mutation caused the amino acid substitution G769W. Transformation and expression of the mutated PcORP1-769W allele in the sensitive wild-type isolate BYA5 confirmed that the mutation in PcORP1 was responsible for the observed oxathiapiprolin resistance. Finally diagnostic tests including As-PCR and CAPs were developed to detect the oxathiapiprolin resistance resulting from the G769W point mutation in field populations of P. capsici.

Resistance to the novel fungicide pyrimorph in Phytophthora capsici: risk assessment and detection of point mutations in CesA3 that confer resistance.

Pyrimorph is a novel fungicide with high activity against the plant pathogen Phytophthora capsici. We investigated the risk that P. capsici can develop resistance to pyrimorph. The baseline sensitivities of 226 P. capsici isolates, tested by mycelial growth inhibition, showed a unimodal distribution with a mean EC(50) value of 1.4261 (± 0.4002) µg/ml. Twelve pyrimorph-resistant mutants were obtained by repeated exposure to pyrimorph in vitro with a frequency of approximately 1 × 10(-4). The resistance factors of the mutants ranged from 10.67 to 56.02. Pyrimorph resistance of the mutants was stable after 10 transfers on pyrimorph-free medium. Fitness in sporulation, cystospore germination, and pathogenicity in the pyrimorph-resistant mutants was similar to or less than that in the parental wild-type isolates. On detached pepper leaves and pepper plants treated with the recommended maximum dose of pyrimorph, however, virulence was greater for mutants with a high level of pyrimorph resistance than for the wild type. The results suggest that the risk of P. capsici developing resistance to pyrimorph is low to moderate. Among mutants with a high level of pyrimorph resistance, EC(50) values for pyrimorph and CAA fungicides flumorph, dimethomorph, and mandipropamid were positively correlated. This indicated that point mutations in cellulose synthase 3 (CesA3) may confer resistance to pyrimorph. Comparison of CesA3 in isolates with a high level of pyrimorph resistance and parental isolates showed that an amino acid change from glutamine to lysine at position 1077 resulted in stable, high resistance in the mutants. Based on the point mutations, an allele-specific PCR method was developed to detect pyrimorph resistance in P. capsici populations.

[Nonbiologic risk factors of pulmonary tuberculosis among adults in Henan: a case-control study].

OBJECTIVE: To study the nonbiologic risk factors of tuberculosis (TB) among adults. METHODS: 1:2 matched case-control study was conducted. 158 new smear positive pulmonary TB patients with 25 - 60 years of age were notified and registered from three county anti-TB institutions in Henan from October 2002-March 2004, were selected as cases. Two healthy persons were selected to match each case, with the same sex and age group, from the nearest neighbors of the case as controls. Interview was carried out with a uniform designed questionnaire at the residence of the object. Univariate and multivariate condition logistic regression models were used. RESULTS: The results of univariate analysis showed that marital status, education, self-employed occupation, smoking, out-migration for work away from hometown, household annual income level and household property were significantly associated with TB prevalence (P < 0.05); multivariate analysis showed that marital status, self-employed occupation, smoking, household economic condition and out-migration for work, away from home were also associated with TB (P < 0.05), and OR values were 2.826, 2.350, 1.536, 0.707, 2.096, respectively. CONCLUSION: Being single, smoking, self-employed occupation and out-migration for work were nonbiologic risk factors of TB while good household economic condition was a protective factor.

Cost of malaria control in China: Henan's consolidation programme from community and government perspectives.

OBJECTIVE: To assist with strategic planning for the eradication of malaria in Henan Province, China, which reached the consolidation phase of malaria control in 1992, when only 318 malaria cases were reported. METHODS: We conducted a prospective two-year study of the costs for Henan's malaria control programme. We used a cost model that could also be applied to other malaria programmes in mainland China, and analysed the cost of the three components of Henan's malaria programme: suspected malaria case management, vector surveillance, and population blood surveys. Primary cost data were collected from the government, and data on suspected malaria patients were collected in two malaria counties (population 2 093 100). We enlisted the help of 260 village doctors in six townships or former communes (population 247 762), and studied all 12 325 reported cases of suspected malaria in their catchment areas in 1994 and 1995. FINDINGS: The average annual government investment in malaria control was estimated to be US$ 111 516 (case-management 59%; active blood surveys 25%; vector surveillance 12%; and contingencies and special projects 4%). The average cost (direct and indirect) for patients seeking treatment for suspected malaria was US$ 3.48, equivalent to 10 days' income for rural residents. Each suspected malaria case cost the government an average of US$ 0.78. CONCLUSION: Further cuts in government funding will increase future costs when epidemic malaria returns; investment in malaria control should therefore continue at least at current levels of US$ 0.03 per person at risk.