Azoxystrobin-resistant Isolates Research Articles

Isolates of Colletotrichum truncatum with Resistance to Multiple Fungicides from Soybean in Northern Thailand.

In Thailand, four systemic fungicides-carbendazim (Car), azoxystrobin (Azo), difenoconazole (Dif), and penthiopyrad (Pen)-are commonly used to control soybean anthracnose caused by Colletotrichum truncatum; however, the pathogen has developed resistance. From 2019 to 2020, fungicide resistance in C. truncatum from fields in Chiang Rai and Chiang Mai was monitored. In tests of 85 C. truncatum isolates for resistance to multiple fungicides, 15.3% were CarRAzoR, 34.1% were triple resistant (CarRAzoRDifR or CarRAzoRPenR), and 50.6% were CarRAzoRDifRPenR. Surprisingly, all isolates tested had lost their sensitivity to one or more of the fungicides tested. The carbendazim-resistant isolates carried a point mutation in the β-tubulin gene at codon 198 (E198A) or 200 (F200Y), and all azoxystrobin-resistant isolates had a mutation in the cytochrome b gene at codon 143 (G143A) or 129 (F129L). Moreover, a novel mutation at codon 208 (S208Y) in the gene encoding succinate dehydrogenase subunit B was detected in all of the isolates highly resistant to penthiopyrad. No mutation linked with difenoconazole resistance was detected in the genes encoding cytochrome P450 sterol 14α-demethylase. To the best of our knowledge, this is the first report of C. truncatum isolates resistant to multiple fungicides and serves as a warning to take measures to prevent the occurrence and distribution of these multiple-fungicide-resistant populations in soybean fields.

Development of colorimetric loop-mediated isothermal amplification (LAMP) assay for detecting the azoxystrobin-resistant isolates in Colletotrichum truncatum

Anthracnose, mainly caused by Colletotrichum truncatum, is a serious threat to soybean production worldwide. Azoxystrobin, a quinone outside inhibitor (QoI) fungicide, has long been frequently used in Thailand. As expected, its performance has decreased as the pathogen has developed fungicide resistance, mainly due to mutations in the target cytochrome b (cytb) gene. The substitution from glycine to alanine at codon 143 (G143A) is related to the azoxystrobin-completely resistant phenotype of C. truncatum. We thus used the G143A mutation as the reaction marker to develop a loop-mediated isothermal amplification (LAMP) method combined with hydroxynaphthol blue (HNB) for visual, simple, and rapid detection of azoxystrobin-resistant isolates of C. truncatum. Among four sets of primers designed, primer sets 1, 2, and 4 distinguished the isolate carrying the G143A mutation from wild-type isolate. Optimum conditions for the LAMP assay using primer set 4 were 63 °C for 80 min with a visible detection limit of 1 ng. Moreover, the LAMP assay was highly specific and accurate in tests of 125 isolates of C. truncatum from 13 fields in northern Thailand. This LAMP assay is highly efficient, simple, accurate for rapid field monitoring of azoxystrobin resistance in C. truncatum and implementing effective control measures.

Metyltetraprole activity against plant pathogens with relatively rare cytochrome b haplotypes for azoxystrobin resistance

Metyltetraprole is a novel quinone outside inhibitor (QoI) fungicide designed to avoid cross-resistance in cytochrome b G143A-harboring QoI-resistant phytopathogenic fungi. The resistance factors of G143A-harboring fungal isolates for metyltetraprole are around 1, but > 200 for the reference QoI fungicide azoxystrobin. In this study of metyltetraprole activities against azoxystrobin-resistant isolates carrying G137R, G137S, L299F, N256S + L299F, or L275F + L299F in cytochrome b, metyltetraprole had potent activity against all isolates with these cytochrome b haplotypes. The resistance factors ranged from 0.7 to 2.9 for metyltetraprole and from 3.0 to 175.1 for azoxystrobin. We revealed unique metyltetraprole inhibitory activities against QoI-resistant plant pathogens.

In-vitro sensitivity of Alternaria solani isolates to azoxystrobin and difenoconazole fungicides in Kenya and detection of Cyt b mutations associated with azoxystrobin resistance

Early blight (EB) caused by Alternaria solani is one of the most important tomato diseases in Kenya and farmers predominantly rely on synthetic fungicides to control it. However, there have been many complaints from farmers about the declining control efficacy of many fungicides used. In this study, 96 A. solani isolates, collected from tomato fields in 3 counties of Kenya (Kirinyaga, Kajiado and Kiambu), were assessed in vitro for sensitivity to two commonly used fungicides; Azoxystrobin and Difenoconazole, at varying concentrations. Isolates were considered resistant to the fungicide whenever their Percent Mycelial Growth Inhibition (MGI) at manufacturer's recommended dosage was below 50% and sensitive if above 50%. Although Difenoconazole resistance was not detected at the tested concentrations, majority of the isolates (64%) were resistant to Azoxystrobin. To determine the genetic basis of this Azoxystrobin resistance, DNA was extracted from all isolates, the Cyt b gene amplified and analyzed for resistance-associated mutations, at positions 129, 137 and 143. Results indicated that two mutations were present in the isolates. The F129L mutation was present in all Azoxystrobin resistant isolates plus 10 sensitive ones with MGI values close to the discriminatory threshold (MGI 50%). The G143S mutation was detected in 26 isolates (27%) but did not have a significant effect on sensitivity of isolates to Azoxystrobin. These findings indicate that Kenya's A. solani populations are developing resistance to some fungicides by mutation and provide justification for deployment of anti-resistance strategies, to ensure more effective management of tomato early blight.

Improving fungicide-based management of Cercospora leaf spot in table beet in New York, USA

Cercospora leaf spot (CLS), caused by Cercospora beticola Sacc., is the dominant foliar disease affecting the profitability of table beet production in New York, USA. The disease is controlled primarily with fungicides, but sustainability is critically threatened by strobilurin resistance within the C. beticola population and a limited suite of alternatives. This study evaluated the temporal dynamics of strobilurin resistance within the C. beticola population over three years, and quantified sensitivity to selected demethylation inhibitors (DMIs; propiconazole and difenoconazole) and succinate dehydrogenase inhibitors (SDHIs; fluopyram, penthiopyrad, boscalid and benzovindiflupyr). Resistance to azoxystrobin (EC50 > 0.2 µg mL−1) was prevalent and detected in all fields sampled in 2015 and 2016, and six of 10 fields in 2017. Within fields, the incidence of azoxystrobin-resistant isolates ranged between 4.8% and 70%. No significant differences were detected in the frequency of azoxystrobin-resistant isolates within populations from fields with conventional and organic practices in 2016 and 2017. Reduced sensitivity to propiconazole and difenoconazole was detected in less than 1% of isolates. The EC50 values for the SDHIs were higher than the maximum concentrations (≥ 20 µg mL−1) evaluated. Two small-plot replicated field trials were also conducted over two years to evaluate alternatives to the commercial standards, propiconazole and azoxystrobin. Two applications of benzovindiflupyr + difenoconazole and pydiflumetofen + difenoconazole significantly reduced CLS severity and epidemic progress compared with plots that received propiconazole and non-treated controls.

Frequency of fungicide-resistant Botrytis cinerea populations isolated from ‘Thompson Seedless’ table grapes in the Central Valley of Chile

During the 2013 and 2014 growth seasons, 526 single spore isolates of Botrytis cinerea were collected from naturally infected ‘Thompson Seedless’ table grape flowers from fifteen orchards in three regions of the Central Valley of Chile. The isolates were tested for resistance to azoxystrobin, boscalid, fenhexamid, fludioxonil and pyrimethanil. Among the 526 isolates, 106 (20.15%) were sensitive to all fungicides tested; from north to south, the frequency of sensitive isolates in the regions of Valparaiso, Metropolitana and O’Higgins ranged from 48.15% to 21.1% and 5.88%, respectively. Four hundred and twenty isolates (79%) showed resistance to single or multiple fungicides, 134 (25.4%) were simultaneously resistant to azoxystrobin and pyrimethanil. No fludioxonil-resistant isolates were found, indicating that fludioxonil has great potential for gray mold control in table grapes in Chile. From sixty randomly selected B. cinerea isolates, only the azoxystrobin-resistant isolates carried the G143A point mutation; according to the cytochrome b ( cyt b ) gene structure, the third intron Bcbi-143/144 was only detected in the azoxystrobin-sensitive isolates. The H272R and H272Y point mutations in the succinate dehydrogenase subunit B ( sdhB ) gene were associated only with the boscalid-resistant isolates. The F412S and F412V point mutations were found in the sequenced erg27 gene of randomly selected fenhexamid-resistant isolates. These results contribute to the knowledge of B. cinerea fungicide resistance for table grape vine crops in Central Chile, particularly for the development of multiple-resistance and the associated resistance mechanisms of azoxystrobin, boscalid and fenhexamid-resistant isolate populations. Antiresistance strategies are discussed in a general manner.

Temporal Dynamics and Spatial Variation of Azoxystrobin and Propiconazole Resistance in Zymoseptoria tritici: A Hierarchical Survey of Commercial Winter Wheat Fields in the Willamette Valley, Oregon.

Fungicide resistance can cause disease control failure in agricultural systems, and is particularly concerning with Zymoseptoria tritici, the causal agent of Septoria tritici blotch of wheat. In North America, the first quinone outside inhibitor resistance in Z. tritici was discovered in the Willamette Valley of Oregon in 2012, which prompted this hierarchical survey of commercial winter wheat fields to monitor azoxystrobin- and propiconazole-resistant Z. tritici. Surveys were conducted in June 2014, January 2015, May 2015, and January 2016. The survey was organized in a hierarchical scheme: regions within the Willamette Valley, fields within the region, transects within the field, and samples within the transect. Overall, frequency of azoxystrobin-resistant isolates increased from 63 to 93% from June 2014 to January 2016. Resistance to azoxystrobin increased over time even within fields receiving no strobilurin applications. Propiconazole sensitivity varied over the course of the study but, overall, did not significantly change. Sensitivity to both fungicides showed no regional aggregation within the Willamette Valley. Greater than 80% of spatial variation in fungicide sensitivity was at the smallest hierarchical scale (within the transect) of the survey for both fungicides, and the resistance phenotypes were randomly distributed within sampled fields. Results suggest a need for a better understanding of the dynamics of fungicide resistance at the landscape level.

Characterization of resistance to multiple fungicides in Botrytis cinerea populations from Asian ginseng in northeastern China

Botrytis cinerea infects ginseng throughout the whole growing season and results in significant economic losses. Fungicides have been the primary strategy in disease control, but frequent applications can increase the risk of fungicide resistance. To determine the resistance of field populations, 76 B. cinerea isolates were collected from 10 ginseng fields in Northeastern China in 2012 and 2013. The sensitivities of these isolates were examined to carbendazim, iprodione, pyrimethanil and azoxystrobin by analyzing mycelial growth inhibition. Almost all test isolates were resistant to at least one fungicide. For example, 27.6 %, 49.9 % and 21.1 % of the population was resistant to one, two or three of four test fungicides, respectively. There was only one isolate that was sensitive to all four fungicides. However, none were simultaneously resistant to all of the four fungicides. Mutations for E198A in the β-tubulin gene and G143A in the cyt b gene were found in all carbendazim- and azoxystrobin-resistant isolates, respectively, indicating a high level of resistance. Various mutations in the bos1 gene were associated with iprodione resistance, among which two combined point mutations, D757N and E369P, could be a novel genotype. Gene structure analysis showed that 50 % of the isolates carried the Bcbi143/144 intron in cyt b, indicating a high inherent risk for the development of G143A-associated resistance to quinone outside inhibitor fungicides. Most resistant isolates did not show reduced compound fitness in mycelial growth, sclerotial and conidial production, aggressiveness and sensitivity to osmotic stress. In conclusion, multi-fungicide resistance of B. cinerea contributed to the inefficiency of current fungicides.

Shift of sensitivity of Botrytis cinerea to azoxystrobin in greenhouse vegetables before and after exposure to the fungicide

From 2004 to 2006, 213 isolates of Botrytis cinerea never exposed to QO center inhibitors (QOIs) were collected to determine the baseline sensitivity to azoxystrobin. In the absence of salicylhydroxamic acid (SHAM), the mean EC50 values were 10.49 ± 13.12 and 0.36 ± 0.48 mg l−1 for inhibiting mycelial growth and conidium germination, respectively. In the presence of SHAM, the mean EC50 values were 2.24 ± 1.29 and 0.22 ± 0.11 mg l−1. In 2010, five azoxystrobin-resistant isolates were detected with the resistance frequency of 2.25% in greenhouse tomatoes after 4 years of continuous exposure. These resistant isolates showed cross-resistance to other QOIs but not to boscalid. In addition, these resistant isolates had comparable growth, sporulation and pathogenicity ability as sensitive isolates and maintained resistance in plants and the presence of SHAM. The G143A point mutation predicted to cause a change from glycine to alanine at codon 143 of cyt b gene was found in all resistant isolates.

A molecular mechanism of azoxystrobin resistance in Penicillium digitatum UV mutants and a PCR-based assay for detection of azoxystrobin-resistant strains in packing- or store-house isolates

Sixty-five isolates of Pencillium digitatum (Pers.:Fr) Sacc., a causative agent of green mold of postharvest citrus, were collected from various locations in Zhejiang province in 2000, 2005 and 2006, and assayed for their sensitivity to the quinone outside inhibitor (QoI) fungicide azoxystrobin. The results showed that azoxystrobin is highly effective against P. digitatum, in vitro, and that the effective concentrations resulting in reduction of conidial germination and mycelial growth by 50% (EC 50) averaged 0.0426 µg/ml and 0.0250 µg/ml, respectively. Twenty-eight azoxystrobin-resistant mutants were obtained by UV mutagenesis and subsequent selection on medium amended with azoxystrobin (12 µg/ml) and salicylhydroxamic acid. All obtained mutants were highly resistant to azoxystrobin and their resistance was genetically stable. Analysis of the cytochrome b gene structure of P. digitatum ( Pdcyt b) showed the absence of type I intron in the first hot spot region of mutation. These results indicate that P. digitatum is likely to evolve high levels of resistance to azoxystrobin after its application. Analysis of partial sequences of Pdcyt b from both the azoxystrobin-sensitive parental isolate and the 28 azoxystrobin-resistant mutants revealed that a point mutation, which leads to the substitution at code 143 of alanine for glycine (G143A), is responsible for the observed azoxystrobin resistance in the laboratory mutants. Based on this point mutation, two allele-specific PCR primers were designed and optimized for allele-specific PCR detection of azoxystrobin-resistant isolates of P. digitatum.

Occurrence and molecular characterization of azoxystrobin resistance in cucumber downy mildew in Shandong province of China

Cucumber downy mildew caused byPseudoperonospora cubensis (Berk. and Curt.) Rostov. limits crop production in Shandong Province of China. Since management of downy mildew is strongly dependent on fungicides, a rational design of control programs requires a good understanding of the fungicide resistance phenomenon in field populations of the pathogen. A total of 106 and 97 isolates ofP. cubensis were obtained in 2006 and 2007, respectively. The EC50 values for the growth of all the 106 isolates collected in 2006 were 0.0063–0.0688µg ml−1 (average: 0.0196±0.0048µg ml−1) azoxystrobin and these were therefore considered sensitive isolates. However, 57 field isolates ofP. cubensis of the 97 collected in 2007 with EC50 values that ranged from 0.609 to >51.2µg ml−1 were considered resistant to azoxystrobin. Fragments of the fungicide-targeted mitochondrial cytochromeb gene from total pathogen DNA were amplified using polymerase chain reaction and their sequences analyzed to elucidate the molecular mechanism of resistance. A single point mutation (GGT to GCT) in the cytochromeb gene, resulting in substitution of glycine by alanine at position 143, was found in the three selected azoxystrobin-resistant isolates of downy mildew. This substitution in cytochromeb exhibited different resistance levels, with the resistance factor from 21.15 to greater than 2618.9. In addition, the different resistance levels seemed to appear within 1 year (between 2006 and 2007). Therefore, growers of Shandong Province in China now are faced with a challenge in managing the azoxystrobin resistance in cucumber downy mildew.

Characterization of iprodione-resistant Alternaria isolates from pistachio in California

Alternaria late blight caused by Alternaria spp. in the alternata, tenuissima, and arborescens species-groups is one of the most common fungal diseases of pistachio in California. In this study, a field iprodione-resistant (FIR) isolate of the arborescens species-group and a laboratory-induced iprodione-resistant (LIIR) isolate of the alternata species-group were characterized by fungicide and osmotic sensitivity, virulence on detached pistachio leaflets, and sequence of the coiled-coil region (six repeats of approximately 90-amino-acid domain) of the two-component histidine kinase ( HK) gene. Both FIR and LIIR isolates were sensitive to azoxystrobin and tebuconazole, and azoxystrobin-resistant isolates were sensitive to iprodione and tebuconazole. The LIIR isolate showed more sensitivity to osmotic stress than its wild-type parent. However, the FIR isolate did not show higher osmotic sensitivity compared to field iprodione-sensitive (FIS) isolates. Laboratory inoculation tests showed that both FIR and LIIR isolates remained highly virulent on pistachio. Analysis of DNA sequences of the HK coiled-coil region showed that there were no differences in deduced amino acid sequence of this region from the LIIR, FIR, and FIS Alternaria isolates from pistachio in California.

Resistance to Azoxystrobin in the Gummy Stem Blight Pathogen Documented in Georgia

Gummy stem blight, caused by the fungus Didymella bryoniae, is the most destructive disease of watermelon in Georgia and in many other watermelon producing areas of the U.S. The QoI fungicide azoxystrobin has been used for gummy stem blight control in Georgia since 1998. As early as 1999, reduced control of gummy stem blight with azoxystrobin was noted in several research sites and commercial fields of cucumber and watermelon in Georgia. Isolates from several of these fields were later confirmed to be resistant to azoxystrobin. To determine how widespread the resistance problem was in Georgia, extensive surveys of watermelon and muskmelon fields and transplant houses were conducted in 2001 and 2002 to determine the frequency of azoxystrobin-resistant isolates in populations of D. bryoniae. Of the 272 isolates collected in 2001, 247 (91%) were resistant to azoxystrobin. In 2002, 82% of the 170 isolates collected were resistant to azoxystrobin, and of the 40 isolates collected from watermelon transplants, all but one were resistant to azoxystrobin, suggesting that resistant isolates in the field may have originated from seed or transplants. Georgia melon growers are now advised to use alternative fungicides that are chemically unrelated to the QoIs for gummy stem blight control. Accepted for publication 2 November 2004. Published 7 December 2004.

A non-Mendelian inheritance of resistance to strobilurin fungicides in Ustilago maydis.

Mutants of Ustilago maydis (DC) Corda with high resistance to azoxystrobin (RF 164 to 4714, based on EC50 values), an inhibitor of mitochondrial electron transport at the cytochrome bc1 complex, were isolated in a mutation frequency of 2.3 x 10(-7) after nitrosoguanidine mutagenesis and selection on media containing 1 microgram ml-1 azoxystrobin in addition to 0.5 mM salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strains by azoxystrobin (1.5 micrograms ml-1) in addition to SHAM (1 mM), but not in the mutant isolates. Genetic analysis with nine such mutant isolates resulted in progeny phenotypes which did not follow Mendelian segregation, but satisfied the criteria of non-Mendelian (cytoplasmic) heredity. In crosses between three mutant isolates with the compatible wild-type strains, the sensitivity was inherited by progeny maternally from the wild-type parent strain (criterion of uniparental inheritance). In crosses between wild-type strains and remaining mutant isolates, a continuous distribution of sensitivity in the progeny was found (criterion of vegetative segregation). The third criterion of cytoplasmic resistance (criterion of intracellular selection) was fulfilled by experiments on the stability of resistance phenotypes. With two exceptions, a reduction of resistance was observed in the mutant strains when they were grown on inhibitor-free medium. Recovery of the high resistance level was observed after they were returned to the selection medium. Cross-resistance studies with other fungicides, which also inhibit electron transport through complex III of respiratory chain, showed that mutations for resistance to azoxystrobin were also responsible for reduced sensitivity to kresoxim-methyl (RF 18 to 1199) and to antimycin-A (RF 20 to 305), which act at the Qo and Qi sites of the cytochrome bc1 complex, respectively. Studies of the fitness of azoxystrobin-resistant isolates showed that these mutations appeared to be pleiotropic, having significant adverse effects on growth in liquid culture and pathogenicity on young corn plants.