Evaluating In Vitro Fitness Parameters of G143A-Containing and Wild-Type Corynespora cassiicola Isolates from Mississippi Soybean.

Quinone outside inhibitor (QoI) fungicides have been widely used to manage diseases of soybean including target spot caused by Corynespora cassiicola. However, resistance to QoI fungicides has recently been reported within the C. cassiicola population from Alabama, Arkansas, Mississippi, and Tennessee as a result of isolates in the population containing the G143A amino acid substitution. Therefore, the relative fitness and stability of isolates containing the G143A substitution compared with wild-type C. cassiicola isolates from Mississippi soybean were investigated by analyzing several fitness parameters in vitro. In addition, in vivo virulence assays were conducted in the greenhouse on a target spot-susceptible cultivar. The evaluations of fitness considered the difference between isolates from the wild-type and G143A-containing genotypes by evaluating colony growth parameters following the first and the 10th subcultures on microbiological media. When considered as an average of all G143A-containing isolates, the G143A-containing isolates following the 10th subculture produced 6.2% greater colony diameter growth but produced 2.3% less conidia. Conversely, over the same period, wild-type isolates produced 6.7% less colony growth but produced 10.9% more conidia. Based on our results, the C. cassiicola isolates that contained the G143A substitution appear stable since successive subculturing did not significantly affect the measured fitness parameters. The lack of fitness cost accompanying the genotypic shift to the G143A amino acid substitution which confers fungicide resistance in C. cassiicola indicates that these isolates may have fitness advantages and may remain stable in the population as well as displace wild-type isolates with repeated fungicide applications of QoI-containing products.

Defining Fungicide Resistance Mechanisms in the Corynespora cassiicola Population from Mississippi Soybean.

Target spot, caused by Corynespora cassiicola, is a common lower canopy soybean disease in the southern United States. Recently, target spot has resurged in importance especially following the identification of resistance to the quinone outside inhibitor (QoI) fungicides. As a result, a survey of C. cassiicola from soybean throughout Mississippi began in 2018. A total of 819 C. cassiicola monoconidial isolates were obtained from 228 fields in 75 counties. The molecular mechanism of QoI resistance was determined, which resulted from an amino acid substitution from glycine (G) to alanine (A) at position 143 using a PCR-RFLP method and comparing nucleotide sequences of the cytochrome b gene. Five previously defined geographic regions were used to present the distribution of the G143A substitution and included the Capital, Coast, Delta, Hills, and Pines. The Capital had the greatest proportion of G143A-containing isolates (95.0%), followed by the Coast (92.9%), Delta (89.8%), Pines (78.8%), and Hills (69.4%). In all, 85.8% of the C. cassiicola isolates carried the G143A substitution. In addition, the effective fungicide concentration (EC50) of randomly selected C. cassiicola isolates to azoxystrobin was used to characterize isolates as resistant (n = 14) (based on the presence of the G143A substitution and EC50 values >52 µg/ml) or sensitive (n = 11) (based on the absence of the G143A substitution and EC50 values <46 µg/ml). The EC50 values varied among isolates (P < 0.0001), with QoI-sensitive isolates exhibiting lower EC50 values than QoI-resistant isolates. The current study revealed that a reduction in sensitivity to QoI fungicides has likely resulted based on the percentage of C. cassiicola isolates containing the G143A substitution identified in Mississippi.

Allele-Specific Detection Methods for QoI Fungicide-Resistant Erysiphe necator in Vineyards.

Grapevine powdery mildew (GPM), caused by the fungus Erysiphe necator, is a constant threat to worldwide production of grape berries, requiring repeated use of fungicides for management. The frequent fungicide applications have resulted in resistance to commonly used quinone outside inhibitor (QoI) fungicides and the resistance is associated with single-nucleotide polymorphisms (SNPs) in the mitochondrial cytochrome b gene (cytb). In this study, we attempted to detect the most common SNP causing a glycine to alanine substitution at amino acid position 143 (i.e., G143A) in the cytb protein, to track this resistance using allele-specific TaqMan probe and digital-droplet PCR-based assays. Specificity and sensitivity of these assays showed that these two assays could discriminate SNPs and were effective on mixed samples. These diagnostic assays were implemented to survey E. necator samples collected from leaf and air samples from California and Oregon grape-growing regions. Sequencing of PCR amplicons and phenotyping of isolates also revealed that these assays accurately detected each allele (100% agreement), and there was an absolute agreement between the presence or absence of the G143A mutation and resistance to QoIs in the E. necator sampled. These results indicate that the developed diagnostic tools will help growers make informed decisions about fungicide selections and applications which, in turn, will facilitate GPM disease management and improve grape production systems.

Discovery of new group I-D introns leads to creation of subtypes and link to an adaptive response of the mitochondrial genome in fungi.

Group I catalytic introns are widespread in bacterial, archaeal, viral, organellar, and some eukaryotic genomes, where they are reported to provide regulatory functions. The group I introns are currently divided into five types (A-E), which are themselves distributed into several subtypes, with the exception of group I type D intron (GI-D). GI-D introns belong to the rarest group with only 17 described to date, including only one with a putative role reported in fungi, where it would interfere with an adaptive response in the cytochrome b (COB) gene to quinone outside inhibitor (QoI) fungicide resistance. Using homology search methods taking into account both conserved sequences and RNA secondary structures, we analysed the mitochondrial genomes or COB genes of 169 fungal species, including some frequently under QoI selection pressure. These analyses have led to the identification of 216 novel GI-D introns, and the definition of three distinct subtypes, one of which being linked with a functional activity. We have further uncovered a homing site for this GI-D intron type, which helps refine the accepted model of quinone outside inhibitor resistance, whereby mobility of the intron across fungal mitochondrial genomes, would influence a fungus ability to develop resistance to QoIs.

Transmission of the G143A QoI-resistance point mutation through anastomosis in Magnaporthe grisea.

BACKGROUND: Soon after the introduction of Qo inhibitor fungicides in 1996, the point mutation leading to the amino acid exchange glycine to alanine at the 143 position of the mitochondrial cytochrome b gene was identified as the main cause of resistance. The present study describes the role of anastomosis in the transmission of the G143A mutation in Magnaporthe grisea. RESULTS: Two M. grisea mutants were co-cultivated on oatmeal agar and also co-inoculated on barley leaves. The mutants differed by the presence of the G143A mutation in one isolate and a disrupted AOX gene by insertion of a hygromycin gene in the other (M-145). Specific resistant (r) or sensitive (s) phenotypes of 409 monosporic cultures were determined on media amended with either hygromycin (H) or azoxystrobin (S) plus SHAM. The phenotypes identified reflected not only the phenotypes of mutants M-145 and G143A but also the wild-type parent phenotype HsSs and a new HrSr isolate. CONCLUSION: Identification of the M. grisea phenotypes HrSr and HsSs suggests that anastomosis occurred during co-cultivation and co-inoculation of the mutants M-145 and G143A, allowing the transfer of the G143A point mutation from the QoI-resistant isolate to the susceptible isolate.