Pyrimethanil Sensitivity and Resistance Mechanisms in Penicillium digitatum

Plant Disease ◽  
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.

scholarly journals Mutations that increase expression of the EmrAB-TolC efflux pump confer increased resistance to nitroxoline in Escherichia coli

2019 ◽  
Author(s):  
Fabiola Puértolas-Balint ◽  
Omar Warsi ◽  
Marius Linkevicius ◽  
Po-Cheng Tang ◽  
Dan I Andersson
Keyword(s):  
Escherichia Coli ◽  
Target Genes ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Cross Resistance ◽  
Resistance Mutations ◽  
Double Knockout ◽  
Low Level ◽  
Resistant Mutants ◽  
Level Resistance

Abstract Objectives To determine the mechanism of resistance to the antibiotic nitroxoline in Escherichia coli. Methods Spontaneous nitroxoline-resistant mutants were selected at different concentrations of nitroxoline. WGS and strain reconstruction were used to define the genetic basis for the resistance. The mechanistic basis of resistance was determined by quantitative PCR (qPCR) and by overexpression of target genes. Fitness costs of the resistance mutations and cross-resistance to other antibiotics were also determined. Results Mutations in the transcriptional repressor emrR conferred low-level resistance to nitroxoline [nitroxoline MIC (MICNOX) = 16 mg/L] by increasing the expression of the emrA and emrB genes of the EmrAB-TolC efflux pump. These resistant mutants showed no fitness reduction and displayed cross-resistance to nalidixic acid. Second-step mutants with higher-level resistance (MICNOX = 32–64 mg/L) had mutations in the emrR gene, together with either a 50 kb amplification, a mutation in the gene marA, or an IS upstream of the lon gene. The latter mutations resulted in higher-level nitroxoline resistance due to increased expression of the tolC gene, which was confirmed by overexpressing tolC from an inducible plasmid in a low-level resistance mutant. Furthermore, the emrR mutations conferred a small increase in resistance to nitrofurantoin only when combined with an nfsAB double-knockout mutation. However, nitrofurantoin-resistant nfsAB mutants showed no cross-resistance to nitroxoline. Conclusions Mutations in different genes causing increased expression of the EmrAB-TolC pump lead to an increased resistance to nitroxoline. The structurally similar antibiotics nitroxoline and nitrofurantoin appear to have different modes of action and resistance mechanisms.

Fungicidal Actions and Resistance Mechanisms of Prochloraz to Penicillium digitatum

Plant Disease ◽  
2020 ◽  
pp. PDIS-05-20-1128
Author(s):  
Yuchao Zhang ◽  
Bao Zhang ◽  
Chaoxi Luo ◽  
Yanping Fu ◽  
Fuxing Zhu
Keyword(s):  
Cell Membrane ◽  
Target Genes ◽  
Membrane Integrity ◽  
Point Mutations ◽  
Resistance Mechanisms ◽  
Penicillium Digitatum ◽  
Cell Membrane Permeability ◽  
Baseline Sensitivity ◽  
Cell Membrane Integrity ◽  
Citrus Groves

The demethylation inhibitor (DMI) fungicide prochloraz has been widely used in China to control citrus green mold, which is caused by Penicillium digitatum. The 50% effective concentration (EC50) values of prochloraz for 129 isolates of P. digitatum collected in 2017 from citrus groves of four provinces of China ranged from 0.0032 to 0.4582 mg/liter. Analysis of the distribution of natural logarithms of EC50 values indicated that 111 isolates with EC50 values lower than 0.05 mg/liter could be considered sensitive to prochloraz. Relative baseline sensitivity was established based on the 111 sensitive isolates, and the mean EC50 value was 0.0090 ± 0.0054 mg/liter (SD). Prochloraz at 60, 100, and 140 mg/liter provided preventive efficacies of 67.8, 93.0, and 96.4%, respectively. Prochloraz at 0.005 and 0.01 mg/liter disrupted cell membrane integrity of conidia but reduced cell membrane permeability of mycelia. Prochloraz at 0.01 mg/liter reduced ergosterol content in mycelia by 41.8%. Two prochloraz-resistant isolates with EC50 values of 3.97 and 5.68 mg/liter were attained by consecutive subculturing on prochloraz-amended PDA. Studies on the expression levels of three potential target genes, CYP51A, CYP51B, and CYP51C, demonstrated that whether in the absence or presence of prochloraz, only CYP51B in the resistant isolates was overexpressed at least 10-fold higher than that of the sensitive ones. Sequencing of the three genes showed that only CYP51B in the resistant isolates had a 199-bp insertion in the promoter region. In addition, only CYP51B displayed point mutations of G405S, G389C, and Y390S in the coding regions in the resistant isolates. These results were important for understanding the resistance mechanisms of P. digitatum to prochloraz.

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

2019 ◽  
Vol 109 (12) ◽  
pp. 2096-2106 ◽  
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.

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

2011 ◽  
Vol 101 (9) ◽  
pp. 1104-1111 ◽  
Author(s):  
Yang Bi ◽  
Xiaolan Cui ◽  
Xiaohong Lu ◽  
Meng Cai ◽  
Xili Liu ◽  
...  
Keyword(s):  
Ultraviolet Irradiation ◽  
Mycelial Growth ◽  
Laboratory Experiments ◽  
Phytophthora Capsici ◽  
Cross Resistance ◽  
Wild Type ◽  
Baseline Sensitivity ◽  
Mycelial Culture ◽  
Resistance Selection ◽  
Resistant Mutants

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.

scholarly journals Study on the Infection Mechanism of Penicillium Digitatum on Postharvest Citrus (Citrus Reticulata Blanco) Based on Transcriptomics

2019 ◽  
Vol 7 (12) ◽  
pp. 672 ◽  
Author(s):  
Qiya Yang ◽  
Xin Qian ◽  
Solairaj Dhanasekaran ◽  
Nana Adwoa Serwah Boateng ◽  
Xueli Yan ◽  
...  
Keyword(s):  
Iron Transport ◽  
Bioinformatics Analysis ◽  
Control Strategies ◽  
Penicillium Digitatum ◽  
Citrus Reticulata ◽  
Postharvest Diseases ◽  
Cell Wall Degrading Enzymes ◽  
Infection Mechanism ◽  
Citrus Reticulata Blanco ◽  
Pathogenic Factors

Penicillium digitatum is one of the most important pathogens known widely to cause postharvest losses of citrus. It is significant to explore its infection mechanism to improve the control technology of postharvest diseases of citrus. This research aimed to study the changes in gene expression of P. digitatum at its early stages of citrus infection by transcriptomics sequencing and bioinformatics analysis in order to explore the molecular mechanism of its infection. The results showed that genes associated with pathogenic factors, such as cell wall degrading enzymes, ethylene, organic acids, and effectors, were significantly up-regulated. Concurrently, genes related to anti-oxidation and iron transport were equally up-regulated at varying degrees. From this study, we demonstrated a simple blueprint for the infection mechanism of P. digitatum in Citrus reticulata Blanco, which provided a new direction for subsequent pathological research and paves the way for developing new control strategies.

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

Plant Disease ◽  
2012 ◽  
Vol 96 (3) ◽  
pp. 416-422 ◽  
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.

Investigating the potential mechanism of pydiflumetofen resistance in Sclerotinia sclerotiorum

Plant Disease ◽  
2021 ◽  
Author(s):  
Feng Zhou ◽  
Ye-Xian Cui ◽  
Ya-hui Ma ◽  
Jin-Ye Wang ◽  
Hai-Yan Hu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sclerotinia Sclerotiorum ◽  
Plant Pathogens ◽  
Target Genes ◽  
Crop Protection ◽  
Amino Acid Sequences ◽  
Cross Resistance ◽  
Potential Mechanism ◽  
Sclerotinia Stem Rot ◽  
Resistant Mutants

The necrotrophic pathogen Sclerotinia sclerotiorum is one of the most damaging and economically important plant pathogens. Pydiflumetofen, which was recently developed by Syngenta Crop Protection, has already been registered in China for the management of Sclerotinia stem rot (SSR) which was caused by S. sclerotiorum in oilseed rape. In an attempt to preempt and forestall the development of resistance to this useful fungicide, the current study was initiated to investigate potential mechanism of resistance in laboratory mutants. Five pydiflumetofen-resistant S. sclerotiorum mutants were successfully generated by repeated exposure to the fungicide under laboratory conditions. Although the mutants had greatly reduced sensitivity to pydiflumetofen, they were also found to have significantly (p < 0.05) reduced fitness exhibiting reduced mycelial growth and sclerotia formation on PDA medium. However, three of the four mutants had significantly (p < 0.05) increased pathogenicity on detached soybean leaves compared to their respective parental isolates, indicating a moderate to high level of fungicides resistance risk according to the criteria of Fungicide Resistance Action Committee (FRAC). Sequence analysis of four succinate dehydrogenase (Sdh) target genes identified several nucleotide changes in the sequences of the pydiflumetofen-resistant mutants, most of which were synonymous and caused no changes to the predicted amino acid sequences. However, all of the pydiflumetofen-resistant mutants had two amino acid point mutations (A11V and V162A) in their predicted SsSdhB sequence. No similar changes were found in the SsSdhA, SsSdhC, and SsSdhD genes of any of the mutants tested. In addition, there was a positive cross-resistance between pydiflumetofen and boscalid, and no cross-resistance between pydiflumetofen and other commonly used fungicides, including tebuconazole, fludioxonil, cyprodinil, dimethachlone, prochloraz, pyraclostrobin, fluazinam, procymidone, and carbendazim. These results indicate that pydiflumetofen has great potential as an alternative fungicide for the control of S. sclerotiorum, especially where resistance to other fungicides has already emerged. Mixing or alternate application with fludioxonil, prochloraz, and fluazinam could be used to limit the risk of resistance to pydiflumetofen.

scholarly journals Biological Characteristics and Molecular Mechanisms of Fludioxonil Resistance in Fusarium graminearum in China

Plant Disease ◽  
2020 ◽  
Vol 104 (9) ◽  
pp. 2426-2433
Author(s):  
F. Zhou ◽  
D. X. Li ◽  
H. Y. Hu ◽  
Y. L. Song ◽  
Y. C. Fan ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Stop Codon ◽  
Point Mutations ◽  
Premature Stop Codon ◽  
Cross Resistance ◽  
Alternative Mechanism ◽  
Head Blight ◽  
Resistant Mutants

Fusarium graminearum is the primary causal agent of Fusarium head blight (FHB) of wheat. The phenylpyrrole fungicide fludioxonil is not currently registered for the management of FHB in China. The current study assessed the fludioxonil sensitivity of a total of 53 F. graminearum isolates collected from the six most important wheat-growing provinces of China during 2018 and 2019. The baseline fludioxonil sensitivity distribution indicated that all of the isolates were sensitive, exhibiting a unimodal cure with a mean effective concentration for 50% inhibition value of 0.13 ± 0.12 μg/ml (standard deviation). Five fludioxonil-resistant mutants were subsequently induced by exposure to fludioxonil under laboratory conditions. Ten successive rounds of subculture in the absence of the selection pressure indicated that the mutation was stably inherited. However, the fludioxonil-resistant mutants were found to have reduced pathogenicity, higher glycerol accumulation, and higher osmotic sensitivity than the parental wild-type isolates, indicating that there was a fitness cost associated with fludioxonil resistance. In addition, the study also found a positive cross resistance between fludioxonil, procymidone, and iprodione, but not with other fungicides such as boscalid, carbendazim, tebuconazole, and fluazinam. Sequence analysis of four candidate target genes (FgOs1, FgOs2, FgOs4, and FgOs5) revealed that the HBXT2R mutant contained two point mutations that resulted in amino acid changes at K223T and K415R in its FgOs1 protein, and one point mutation at residue 520 of its FgOs5 protein that resulted in a premature stop codon. Similarly, the three other mutants contained point mutations that resulted in changes at the K192R, K293R, and K411R residues of the FgOs5 protein but none in the FgOs2 and FgOs4 genes. However, it is important to point out that the FgOs2 and FgOs4 expression of all the fludioxonil-resistant mutants was significantly (P < 0.05) downregulated compared with the sensitive isolates (except for the SQ1-2 isolate). It was also found that one of the resistant mutants did not have changes in any of the sequenced target genes, indicating that an alternative mechanism could also lead to fludioxonil resistance.

scholarly journals Multiple mutations in the EPSPS and ALS genes of Amaranthus hybridus underlie resistance to glyphosate and ALS inhibitors

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Maria J. García ◽  
Candelario Palma-Bautista ◽  
José G. Vazquez-Garcia ◽  
Antonia M. Rojano-Delgado ◽  
María D. Osuna ◽  
...  
Keyword(s):  
Amino Acid ◽  
Target Genes ◽  
Acetolactate Synthase ◽  
Resistance Mechanisms ◽  
Activity Level ◽  
Cross Resistance ◽  
Weed Species ◽  
Amaranthus Hybridus ◽  
Als Inhibitors ◽  
Moderate Resistance

Abstract Amaranthus hybridus is one of the main weed species in Córdoba, Argentina. Until recently, this weed was effectively controlled with recurrent use of glyphosate. However, a population exhibiting multiple resistance (MR2) to glyphosate and imazamox appeared in a glyphosate resistant (GR) soybean field, with levels of resistance up to 93 and 38-fold higher to glyphosate and imazamox, respectively compared to the susceptible (S) population. In addition to imidazolinones, MR2 plants showed high resistance levels to sulfonylamino-carbonyl (thio) benzoates and moderate resistance to sulfonylureas and triazolopyrimidines. Multiple amino acid substitutions were found in both target genes, acetolactate synthase (ALS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), responsible for conferring high herbicides resistance levels in this A. hybridus population. In the case of EPSPS, the triple amino acid substitution TAP-IVS was found. In addition, MR2 plants also showed increased EPSPS gene expression compared to susceptible plants. A Ser653Asn substitution was found in the ALS sequence of MR2, explaining the pattern of cross-resistance to the ALS-inhibitor herbicide families found at the ALS enzyme activity level. No other mutations were found in other conserved domains of the ALS gene. This is the first report worldwide of the target site resistance mechanisms to glyphosate and ALS inhibitors in multiple herbicide resistance Amaranthus hybridus.

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

Plant Disease ◽  
2011 ◽  
Vol 95 (12) ◽  
pp. 1535-1541 ◽  
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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