Baseline Sensitivity of Natural Population and Resistance of Mutants in Phytophthora capsici to Zoxamide

Laboratory experiments were conducted to determine the baseline sensitivity of Phytophthora capsici and its risk for developing resistance to zoxamide. In total, 158 P. capsici isolates were collected from China. All 158 isolates were sensitive to zoxamide, with effective concentrations for 50% inhibition of mycelial growth of 0.023 to 0.383 μg/ml and a mean of 0.114 μg/ml, which showed a skewed unimodal distribution. Zoxamide-resistant mutants of P. capsici were obtained by either treating mycelial culture and zoospores with ultraviolet irradiation or adapting a culture on zoxamide-amended plates. The frequency of resistance selection averaged 1.8 × 10–7. Resistant isolates were also derived by selfing or crossing two sexually compatible isolates, resulting in a mean selection frequency of 0.47. The resistance factor (RF) for zoxamide was 25 to 100 in P. capsici mutants. Through 10 culture transfers, the mutants maintained high levels of RF (between 14 and 134) and had almost equal fitness as their wild-type parents in mycelial growth, sporulation, and virulence. There was no cross resistance between zoxamide and either flumorph, metalaxyl, azoxystrobin, or etridiazole. Based on the results above, P. capsici can develop resistance to zoxamide, and the risk is predicted to be moderate in nature.

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Wild Type Sensitivity and Mutation Analysis for Resistance Risk to Fluopicolide in Phytophthora capsici

Plant Disease ◽

10.1094/pdis-05-11-0372 ◽

2011 ◽

Vol 95 (12) ◽

pp. 1535-1541 ◽

Cited By ~ 20

Author(s):

Xiao Hong Lu ◽

Mary K. Hausbeck ◽

Xi Li Liu ◽

Jianjun J. Hao

Keyword(s):

Phytophthora Capsici ◽

The United States ◽

Fruit Rot ◽

Cross Resistance ◽

Wild Type ◽

Baseline Sensitivity ◽

Resistance Risk ◽

Crown Root ◽

Zoospore Production ◽

Resistant Mutants

Crown, root, and fruit rot caused by Phytophthora capsici is an increasing problem for vegetable growers in Michigan and the United States. The newly registered fungicide fluopicolide is effective to limit crop loss but the potential for P. capsici to develop resistance is not well known. A laboratory study assessed the risk of P. capsici developing resistance to fluopicolide. Baseline sensitivity to fluopicolide was determined using 126 P. capsici Michigan isolates. Values of effective concentrations for 50% inhibition of mycelial growth ranged from 0.08 to 0.24 μg/ml and were distributed as a unimodal curve, indicating that all isolates were sensitive to fluopicolide. Mutants resistant to fluopicolide were obtained from five isolates by screening zoospores on fluopicolide-amended (5 μg/ml) media at a mutation frequency above 1.0 × 10–7. The mutant isolates were clustered with either intermediate (resistance factor [RF] = 3.53 to 77.91) or high (RF = 2481.40 to 7034.79) resistance. Resistance was stable through 10 mycelial transfers on fungicide-free medium. All resistant mutants showed similar fitness in zoospore production, cyst germination, and virulence compared with their sensitive parents, with few exceptions. No cross-resistance was detected between fluopicolide and five other fungicides. There could be a moderately high risk of field populations of P. capsici developing resistance to fluopicolide, and populations should be monitored.

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Baseline Sensitivity and Resistance-Risk Assessment of Phytophthora capsici to Iprovalicarb

Phytopathology ◽

10.1094/phyto-12-09-0351 ◽

2010 ◽

Vol 100 (11) ◽

pp. 1162-1168 ◽

Cited By ~ 23

Author(s):

Xiao Hong Lu ◽

Shu Sheng Zhu ◽

Yang Bi ◽

Xi Li Liu ◽

Jianjun J. Hao

Keyword(s):

Mycelial Growth ◽

Phytophthora Capsici ◽

Acid Amide ◽

Resistance Management ◽

Cross Resistance ◽

Baseline Sensitivity ◽

Resistance Risk ◽

Zoospore Production ◽

Caa Fungicides ◽

Growth Characters

Iprovalicarb has been used to control Phytophthora capsici, a devastating pathogen of many economically important crops. To evaluate the risk of fungicide resistance, 158 isolates of P. capsici were examined for sensitivity to iprovalicarb by measuring mycelial growth. Values of effective concentrations for 50% mycelial growth inhibition varied from 0.2042 to 0.5540 μg/ml and averaged 0.3923 (±0.0552) μg/ml, with a unimodal distribution. This is the first report of P. capsici isolates highly resistant to iprovalicarb (resistance factor >100). Resistance of the isolates was stable through 10 transfers on iprovalicarb-free medium, and most resistant isolates had the same level of fitness (mycelial growth, zoospore production, and virulence) as their corresponding parents, indicating that iprovalicarb resistance was independent from other general growth characters. There was cross-resistance among all tested carboxylic acid amide (CAA) fungicides, including iprovalicarb, flumorph, dimethomorph, and mandipropamid, but not with non-CAA fungicides, including azoxystrobin, chlorothalonil, cymoxanil, etridiazole, metalaxyl, and zoxamide. Based on the present results, resistance risk of P. capsici to CAAs could be moderate and resistance management should be considered.

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Point Mutations in the β-Tubulin of Phytophthora sojae Confer Resistance to Ethaboxam

Phytopathology ◽

10.1094/phyto-01-19-0032-r ◽

2019 ◽

Vol 109 (12) ◽

pp. 2096-2106 ◽

Cited By ~ 3

Author(s):

Qin Peng ◽

Zhiwen Wang ◽

Yuan Fang ◽

Weizhen Wang ◽

Xingkai Cheng ◽

...

Keyword(s):

Fungicide Resistance ◽

Germination Rate ◽

Management Strategies ◽

Resistance Management ◽

Point Mutations ◽

Phytophthora Sojae ◽

Cross Resistance ◽

Baseline Sensitivity ◽

Resistant Mutants ◽

Β Tubulin

Ethaboxam is a β-tubulin inhibitor registered for the control of oomycete pathogens. The current study was established to determine the ethaboxam sensitivity of the plant pathogen Phytophthora sojae and investigate the potential for the emergence of fungicide resistance. The effective concentration for 50% inhibition (EC50) of 112 Phytophthora sojae isolates exhibited a unimodal distribution with a mean EC50 for ethaboxam of 0.033 µg/ml. Establishing this baseline sensitivity provided critical data for monitoring changes in ethaboxam-sensitivity in field populations. The potential for fungicide resistance was investigated using adaptation on ethaboxam-amended V8 agar, which resulted in the isolation of 20 resistant mutants. An assessment of the biological characteristics of the mutants including mycelial growth, sporulation, germination rate and pathogenicity indicated that the resistance risk in Phytophthora sojae was low to medium with no cross-resistance between ethaboxam and cymoxanil, metalaxyl, flumorph, and oxathiapiprolin being detected. However, positive cross-resistance was found between ethaboxam and zoxamide for Q8L and I258V but negative cross-resistance for C165Y. Further investigation revealed that the ethaboxam-resistant mutants had point mutations at amino acids Q8L, C165Y, or I258V of their β-tubulin protein sequences. CRISPR/Cas9-mediated transformation experiments confirmed that the Q8L, C165Y, or I258V mutations could confer ethaboxam resistance in Phytophthora sojae and that the C165Y mutation induces high levels of resistance. Taken together, the results of the study provide essential data for monitoring the emergence of resistance and resistance management strategies for ethaboxam, as well as for improving the design of novel β-tubulin inhibitors for future development.

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Peptide Deformylase in Staphylococcus aureus: Resistance to Inhibition Is Mediated by Mutations in the Formyltransferase Gene

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.7.1825-1831.2000 ◽

2000 ◽

Vol 44 (7) ◽

pp. 1825-1831 ◽

Cited By ~ 92

Author(s):

Peter S. Margolis ◽

Corinne J. Hackbarth ◽

Dennis C. Young ◽

Wen Wang ◽

Dawn Chen ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Genomic Library ◽

Specific Inhibitor ◽

Peptide Deformylase ◽

Cross Resistance ◽

Loss Of Function ◽

Wild Type ◽

Chromosomal Dna ◽

Resistant Mutants

ABSTRACT Peptide deformylase, a bacterial enzyme, represents a novel target for antibiotic discovery. Two deformylase homologs, defA and defB, were identified inStaphylococcus aureus. The defA homolog, located upstream of the transformylase gene, was identified by genomic analysis and was cloned from chromosomal DNA by PCR. A distinct homolog, defB, was cloned from an S. aureus genomic library by complementation of the arabinose-dependent phenotype of a P BAD -def Escherichia coli strain grown under arabinose-limiting conditions. Overexpression in E. coli of defB, but not defA, correlated to increased deformylase activity and decreased susceptibility to actinonin, a deformylase-specific inhibitor. ThedefB gene could not be disrupted in wild-type S. aureus, suggesting that this gene, which encodes a functional deformylase, is essential. In contrast, thedefA gene could be inactivated; the function of this gene is unknown. Actinonin-resistant mutants grew slowly in vitro and did not show cross-resistance to other classes of antibiotics. When compared to the parent, an actinonin-resistant strain produced an attenuated infection in a murine abscess model, indicating that this strain also has a growth disadvantage in vivo. Sequence analysis of the actinonin-resistant mutants revealed that each harbors a loss-of-function mutation in the fmt gene. Susceptibility to actinonin was restored when the wild-type fmt gene was introduced into these mutant strains. An S. aureusΔfmt strain was also resistant to actinonin, suggesting that a functional deformylase activity is not required in a strain that lacks formyltransferase activity. Accordingly, thedefB gene could be disrupted in an fmt mutant.

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Pyrimethanil Sensitivity and Resistance Mechanisms in Penicillium digitatum

Plant Disease ◽

10.1094/pdis-08-20-1739-re ◽

2020 ◽

Author(s):

Yuchao Zhang ◽

Yanping Fu ◽

Chaoxi Luo ◽

Fuxing Zhu

Keyword(s):

Target Genes ◽

Sodium Dodecyl ◽

Resistance Mechanisms ◽

Penicillium Digitatum ◽

Cross Resistance ◽

Postharvest Diseases ◽

Cell Wall Degrading Enzymes ◽

Baseline Sensitivity ◽

Nucleotide Mutation ◽

Resistant Mutants

Pyrimethanil is an anilinopyrimidines (AP) fungicide and highly effective in controlling green mold caused by Penicillium digitatum but has not yet been registered in China to control postharvest diseases of citrus. In the present study, baseline sensitivity of P. digitatum to pyrimethanil was established based on the effective concentrations for 50% inhibition (EC50) values of 127 isolates collected from five major citrus-growing regions of China. The distribution of these EC50 values was unimodal but with a long right tail. The mean EC50 value was 0.137 ± 0.046 μg/mL (SD), and the minimum and maximum were 0.073 and 0.436 μg/mL, respectively. Pyrimethanil in potato dextrose agar (PDA) at 0.20 μg/mL decreased methionine production in the mycelia by 21.6% and reduced the activities of cell wall-degrading enzymes cellulase and pectinase by 9.1 and 32.8%, respectively. Twelve pyrimethanil-resistant mutants were obtained by consecutive sub-culturing of 12 arbitrarily selected sensitive isolates on pyrimethanil-amended PDA for 4 generations, and the resistance factors ranged from 69 to 3421. There was no cross-resistance between pyrimethanil and prochloraz (r = 0.377, P = 0.123). Compared with their parental isolates, pyrimethanil-resistant mutants had reduced pathogenicity to citrus fruit but higher tolerance to hydrogen peroxide. No differences were detected in tolerance to NaCl, CaCl2, Congo red, or sodium dodecyl sulfate (SDS). Exogenous addition of methionine into PDA partially alleviated the toxicity of pyrimethanil to the sensitive isolates but had no significant effect on toxicity to the resistant mutants. Sequencing of cystathionine γ-synthase encoding genes CGS1 and CGS2, the potential target genes for pyrimethanil, showed that there was no nucleotide mutation in the coding region of CGS of the pyrimethanil-resistant mutants. However, the relative expression of CGS1 and CGS2 genes of the pyrimethanil-resistant mutants was reduced by 42.5 and 57.4%, respectively. These results have important implications for applications of pyrimethanil to control P. digitatum and for understanding the modes of action and resistance mechanisms of pyrimethanil.

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Baseline Sensitivity and Control Efficacy of Tetramycin Against Phytophthora capsici Isolates in China

Plant Disease ◽

10.1094/pdis-09-17-1396-re ◽

2018 ◽

Vol 102 (5) ◽

pp. 863-868 ◽

Cited By ~ 4

Author(s):

Dicheng Ma ◽

Jiamei Zhu ◽

Leiming He ◽

Kaidi Cui ◽

Wei Mu ◽

...

Keyword(s):

Mycelial Growth ◽

Southern China ◽

Phytophthora Capsici ◽

Environmental Safety ◽

Phytophthora Blight ◽

Baseline Sensitivity ◽

Control Efficacy ◽

Mean Values ◽

Greenhouse Experiments

Tetramycin is a new biopesticide that combines high-level and broad-spectrum fungicidal activity, low toxicity, and environmental safety. In this study, 90 Phytophthora capsici isolates obtained from various regions in southern China were characterized for their baseline sensitivity to tetramycin. The protective and curative activities of tetramycin against P. capsici were determined on leaves of pepper, and the control efficacy of tetramycin in greenhouse experiments was also determined. Compared with mycelial growth, the formation of sporangia and the discharge of zoospores were inhibited by lower concentrations of tetramycin, approximately 5 µg ml−1 on V8 media. The frequency distribution curves for the tetramycin sensitivity were unimodal, with mean values for the fungicide concentration that reduced mycelial growth, sporangia formation, and zoospore discharge by 50% compared with the control of 1.18 ± 0.91, 0.64 ± 0.42, and 0.63 ± 0.30 µg ml−1, respectively. In addition, no correlation was observed between tetramycin and other fungicides tested, including mandipropamid, azoxystrobin, mefenoxam, fluazinam, fluopicolide, and famoxadone. Tetramycin exhibited both protective and curative effects against P. capsici in vitro, and its protective activity was better than its curative activity. In greenhouse experiments, tetramycin concentration of 60 and 90 µg ml−1 provided a protective control efficacy of 47.1 to 56.4% and curative efficacy of 43.3 to 52.7%. These results demonstrated that tetramycin could serve as an excellent alternative fungicide to control Phytophthora blight of pepper.

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Baseline Sensitivity and Cross-Resistance to Succinate-Dehydrogenase-Inhibiting and Demethylation-Inhibiting Fungicides in Didymella bryoniae

Plant Disease ◽

10.1094/pdis-09-11-0744-re ◽

2012 ◽

Vol 96 (7) ◽

pp. 979-984 ◽

Cited By ~ 37

Author(s):

A. Thomas ◽

D. B. Langston ◽

K. L. Stevenson

Keyword(s):

Succinate Dehydrogenase ◽

Fungicide Resistance ◽

Mycelial Growth ◽

Effective Concentration ◽

Cross Resistance ◽

Didymella Bryoniae ◽

Baseline Sensitivity ◽

History Of ◽

Dmi Fungicides ◽

Positive Correlations

Didymella bryoniae, which causes gummy stem blight (GSB) of watermelon, has a history of developing resistance to fungicides, most recently the succinate-dehydrogenase-inhibiting (SDHI) fungicide boscalid. To facilitate fungicide resistance monitoring, baseline sensitivity distributions were established for demethylation-inhibiting (DMI) fungicides tebuconazole and difenoconazole and the SDHI fungicide penthiopyrad, and reestablished for the SDHI fungicide boscalid. In all, 71 isolates with no known prior exposure to SDHIs or DMIs were used to determine the effective concentration at which mycelial growth was inhibited by 50% (EC50). EC50 values for boscalid, penthiopyrad, tebuconazole, and difenoconazole were 0.018 to 0.064, 0.015 to 0.057, 0.062 to 0.385, and 0.018 to 0.048 μg/ml, with median values of 0.032, 0.026, 0.118, and 0.031 μg/ml, respectively. Significant positive correlations between the sensitivity to penthiopyrad and boscalid (P < 0.0001, r = 0.75) and between tebuconazole and difenoconazole (P < 0.0001, r = 0.59) indicate a potential for cross-resistance between chemically related fungicides. In 2009, 103 isolates from fungicidetreated watermelon fields were tested for sensitivity to boscalid and penthiopyrad using a discriminatory concentration of 3.0 μg/ml. Of the isolates tested, 82 were insensitive and 14 were sensitive to both fungicides. Because of the significant potential for cross-resistance between closely related fungicides, growers will be advised not to use both SDHIs or both DMIs successively in the same fungicide spray program.

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Baseline Sensitivity of Monilinia fructicola from China to the DMI Fungicide SYP-Z048 and Analysis of DMI-Resistant Mutants

Plant Disease ◽

10.1094/pdis-06-11-0495 ◽

2012 ◽

Vol 96 (3) ◽

pp. 416-422 ◽

Cited By ~ 41

Author(s):

F. P. Chen ◽

J. R. Fan ◽

T. Zhou ◽

X. L. Liu ◽

J. L. Liu ◽

...

Keyword(s):

Brown Rot ◽

Cross Resistance ◽

Monilinia Fructicola ◽

Lesion Area ◽

Site Mutation ◽

Baseline Sensitivity ◽

Dmi Fungicides ◽

Resistant Mutants ◽

Beijing China

Sterol 14α-demethylase inhibitors (DMIs) continue to be important in the management of brown rot of Monilinia spp. worldwide. In this study, the sensitivity of 100 Monilinia fructicola isolates from four unsprayed orchards and two packinghouses in Beijing, China, to the new DMI fungicide SYP-Z048 was evaluated and ranged from 0.003 to 0.039 and 0.016 to 0.047 μg/ml, respectively. Laboratory mutants resistant to SYP-Z048 were generated using UV irradiation but no mutants occurred spontaneously. Resistance was stable after 10 weekly consecutive transfers on fungicide-free medium. Three parameters, including growth rate, sporulation in vitro, and lesion area, were significantly different when sensitive isolates and resistant mutants were analyzed as groups. Mutants grew more slowly and developed significantly smaller lesions on detached fruit, and their sporulation ability in vitro was reduced. Cross resistance was found between SYP-Z048 and propiconazole (ρ = 0.82, P < 0.0001) but not between SYPZ048 and tridemorph, carbendazim, procymidone, azoxystrobin, or pyrimethanil. SYP-Z048 resistance in mutants exhibiting 50% mycelial growth inhibition values greater than 0.3 μg/ml was correlated with the presence of a mutation in the CYP51 gene that encodes the target protein for DMI fungicides. The mutation caused an amino acid change from tyrosine to phenylalanine at position 136 (Y136F). To our knowledge, this is the first baseline sensitivity of M. fructicola collected from China to a DMI fungicide. The inability of M. fructicola to generate spontaneous DMI-resistant mutants coupled with reduced fitness of Y136F mutants can explain why this target site mutation has not yet emerged as a DMI fungicide resistance determinant in M. fructicola field populations worldwide.

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Evaluation of the Risk of Development of Fluopicolide Resistance in Phytophthora erythroseptica

Plant Disease ◽

10.1094/pdis-02-18-0366-re ◽

2019 ◽

Vol 103 (2) ◽

pp. 284-288 ◽

Cited By ~ 1

Author(s):

Xuemei Zhang ◽

He Jiang ◽

Jianjun Hao

Keyword(s):

Agar Medium ◽

Effective Concentration ◽

Potato Tubers ◽

Wild Type ◽

Moderate Risk ◽

Baseline Sensitivity ◽

Pink Rot ◽

Phytophthora Erythroseptica ◽

Resistant Mutants ◽

Resistance Stability

Fluopicolide has shown effective pink rot (Phytophthora erythroseptica) control in potato disease management. To efficiently utilize this chemical, the risk of fluopicolide resistance in P. erythroseptica needs to be assessed. In this study, 34 isolates of P. erythroseptica were obtained from symptomatic potato tubers with pink rot in Maine. The sensitivity of these wild-type isolates to fluopicolide was assessed by culturing them on agar medium amended with fluopicolide at various concentrations. The 50% effective concentration (EC50) of fluopicolide for the inhibition of mycelial growth was determined and used to establish a baseline sensitivity of these P. erythroseptica isolates to fluopicolide. The wild-type isolates were sensitive to fluopicolide, with EC50 values ranging from 0.08 to 0.35 μg/ml. By exposing P. erythroseptica zoospores to agar medium containing 100 μg/ml fluopicolide, 6 out of the 34 wild-type isolates produced fluopicolide-resistant mutants. The mutants were transferred to fungicide-free V8 medium consecutively for 10 times, and the 10th transfer of mutants was examined for resistance stability and biological fitness. In general, the mutants had similar or slower growth rates compared with their wild-type parents, and the virulence of some mutants was significantly reduced. The results indicated a low to moderate risk of P. erythroseptica developing resistance to fluopicolide, and suggested a trade-off between fluopicolide resistance and biological fitness in P. erythroseptica.

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Resistance to Pyraclostrobin and Boscalid in Populations of Botrytis cinerea from Stored Apples in Washington State

Plant Disease ◽

10.1094/pdis-94-5-0604 ◽

2010 ◽

Vol 94 (5) ◽

pp. 604-612 ◽

Cited By ~ 43

Author(s):

Y. K. Kim ◽

C. L. Xiao

Keyword(s):

Botrytis Cinerea ◽

Fungicide Resistance ◽

Mycelial Growth ◽

Gray Mold ◽

Apple Fruit ◽

Conidial Germination ◽

Postharvest Disease ◽

Cross Resistance ◽

Baseline Sensitivity

Gray mold caused by Botrytis cinerea is a major postharvest disease of apple. Pristine, a formulated mixture of pyraclostrobin and boscalid, was recently registered for use on apple. Pristine applied within 2 weeks before harvest is effective in controlling gray mold in stored apple fruit. To determine the baseline sensitivity of B. cinerea populations to these fungicides, 40 isolates from organic and 80 from conventional apple orchards where Pristine had not been used were tested for mycelial growth or conidial germination on fungicide-amended media. To monitor fungicide resistance, gray-mold-decayed apple fruit originating from orchards in which Pristine had been used were sampled from a fruit packinghouse. Isolates of B. cinerea recovered from the fruit were tested for resistance to the two fungicides. In the in vivo study in the orchards, Pristine was applied to fruit 1 day before harvest. Fruit were then harvested, wounded, and inoculated with isolates exhibiting different fungicide-resistance phenotypes. Fruit were stored at 0°C for 8 weeks for decay development. The effective concentration that inhibits mycelial growth by 50% relative to the control (EC50) values for sensitive isolates ranged from 0.008 to 0.132 μg/ml (mean = 0.043, n = 116) for pyraclostrobin and from 0.003 to 0.183 μg/ml (mean = 0.075, n = 117) for Pristine in a mycelial growth assay on potato dextrose agar. The EC50 values of boscalid for sensitive isolates ranged from 0.065 to 1.538 μg/ml (mean = 0.631, n = 29) in a conidial germination assay on water agar. Four isolates were resistant to pyraclostrobin, with resistance factors (RFs) ranging from 12 to 4,193. Of the four pyraclostrobin-resistant isolates, one also was resistant to boscalid (RF = 14) and Pristine (RF = 373), and two exhibited reduced sensitivity to Pristine (RF = 16 and 17). The minimum inhibitory concentration for conidial germination (for boscalid) or mycelial growth (for pyraclostrobin and Pristine) of sensitive isolates was 5 μg/ml, which is thus recommended as a discriminatory concentration for phenotyping isolates for resistance to these fungicides. Of the 56 isolates obtained from decayed apple fruit that had been exposed to Pristine, 11 (approximately 20%) were resistant to both pyraclostrobin and boscalid and 1 was resistant only to pyraclostrobin. Of the additional 43 isolates obtained from decayed apple fruit originating from an organic orchard, 3 were resistant only to pyraclostrobin, 2 were resistant only to boscalid, and 2 were resistant to both fungicides. It appeared that there was no cross resistance between pyraclostrobin and boscalid because of the existence of isolates resistant only to either pyraclostrobin or boscalid. Pristine applied at label rate in the orchard failed to control gray mold on apple fruit inoculated with the Pristine-resistant isolates. This is the first report of multiple resistance to pyraclostrobin, boscalid, and Pristine in field populations of B. cinerea. Our results suggest that the development of dual resistance to pyraclostrobin and boscalid in B. cinerea populations could result in the failure to control gray mold with Pristine.

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