Characterization of Quinone Outside Inhibitor Fungicide Resistance in Cercospora sojina and Development of Diagnostic Tools for its Identification.

Frogeye leaf spot of soybean, caused by the fungus Cercospora sojina, reduces soybean yields in most of the top-producing countries around the world. Control strategies for frogeye leaf spot can rely heavily on quinone outside inhibitor (QoI) fungicides. In 2010, QoI fungicide-resistant C. sojina isolates were identified in Tennessee for the first time. As the target of QoI fungicides, the cytochrome b gene present in fungal mitochondria has played a key role in the development of resistance to this fungicide class. The cytochrome b genes from three QoI-sensitive and three QoI-resistant C. sojina isolates were cloned and sequenced. The complete coding sequence of the cytochrome b gene was identified and found to encode 396 amino acids. The QoI-resistant C. sojina isolates contained the G143A mutation in the cytochrome b gene, a guanidine to cytosine transversion at the second position in codon 143 that causes an amino acid substitution of alanine for glycine. C. sojina-specific polymerase chain reaction primer sets and TaqMan probes were developed to efficiently discriminate QoI-resistant and -sensitive isolates. The molecular basis of QoI fungicide resistance in field isolates of C. sojina was identified as the G143A mutation, and specific molecular approaches were developed to discriminate and to track QoI-resistant and -sensitive isolates of C. sojina.

Evaluation of QoI Fungicide Application Strategies for Managing Fungicide Resistance and Potato Early Blight Epidemics in Wisconsin.

Potato early blight (Alternaria solani) is a yield-limiting disease and control depends primarily on multiple fungicide applications. Azoxystrobin, registered in the United States in 1999, initially provided outstanding early blight control. Within 3 years, approximately 80% of the total potato acreage was being treated with azoxystrobin and other quinone outside inhibitor (QoI), fungicides registered subsequently. Alternaria solani isolates with decreased in vitro sensitivity to azoxystrobin were detected in Wisconsin during 2001. Field experiments were conducted in 2001 to 2003 to evaluate season-long fungicide programs and test fungicide resistance management strategies. The fungicide program recommended to growers at that time, which consisted of three applications of azoxystrobin for weeks 1, 3, and 5 alternated with applications of chlorothalonil at label recommended rates, was effective in controlling early blight when conditions were conducive to disease development. Mean sensitivity in vitro of A. solani isolates from fungicide efficacy field experiments in 2001 to 2003 was numerically highest for isolates from the untreated control plots, chlorothalonil-alone plots, or plots treated with three applications of azoxystrobin alternated with chlorothalonil compared with other treatments tested. Three single-nucleotide polymorphisms (SNPs) can cause the F129L substitution (TTC to TTA, CTC, or TTG) that results in decreased sensitivity to azoxystrobin of A. solani. The TTA mutant was the most frequently recovered mutant type in the field experiments. The frequency of recovery of wild-type isolates in experiments was 22% in 2001, 4% in 2002, and 22% in 2003.

Monitoring and Tracking Changes in Sensitivity to Azoxystrobin Fungicide in Alternaria solani in Wisconsin.

Azoxystrobin is a common fungicide used by farmers of Solanaceous crops against Alternaria solani, but there was growing concern about decreased sensitivity with repeated applications. In 2002 and 2003, monitoring of A. solani from commercial potato fields in Wisconsin indicated increased frequency and a statewide distribution of isolates with decreased in vitro sensitivity to azoxystrobin. Mean effective concentration in inhibiting spore germination by 50% values gathered in 2002 and 2003 were approximately 20-fold higher than baseline isolates of A. solani collected in 1998 from fields that had never been treated with azoxystrobin. This sensitivity decrease was correlated with site-specific mutations in the cytochrome b detected by quantitative real-time polymerase chain reaction. The F129L and the G143A substitution have been shown to cause a reduction in sensitivity or resistance, respectively, to quinone outside inhibitors. All of the recovered A. solani isolates collected in 2002 and 2003 were wild type at position 143. However, all three mutations responsible for the F129L substitution (TTA, CTC, and TTG) were detected in our samples. In addition, the frequency of this amino acid substitution in A. solani isolates was statistically different across sampling sites and years, indicating that sensitivity changes depended on specific disease management practices.

Seroepidemiología del virus herpes simplex 2 en una población indígena Yukpa: estado Zulia, Venezuela / Seroepidemiology of herpes simplex virus type 2 in the amerindian Yukpa population of Zulia state, Venezuela

Background: Yukpa aborigines start early in life their sexual activity and promiscuity with native and foreign individuals is common among women. This behavior allows the installation and propagation of Herpes simplex virus type 2 (HSV-2). Aim: To determine the presence of antibodies to HSV-2 in a Yukpa Amerindian community at Zulia State, Venezuela. Subjects and methods : We studied 96 natives, between 13 and 49 years old (62.3 percent of the whole population, 100 percent of individuals in fertile age). Control population consisted of 76 individuals, with similar sex and age range distribution, of the urban population from Maracaibo, Zulia State, Venezuela. HSV-2 serologic status was determined by an indirect fluorescent antibody test. Results: Antibodies to HSV-2 were detected in 53.1 percent of Yukpa population and 21.1percent of urban population. Aboriginal women aged 21 to 40 years old had a prevalence of antibodies against HSV-2 of 66.7 percent. Among pregnant native women, the prevalence of antibodies against HSV-2 was 54.6 percent. Conclusions: There is a high prevalence of antibodies against HSV-2 among Yukpa natives in Venezuela

Characterization of laboratory mutants of Venturia inaequalis resistant to the strobilurin-related fungicide kresoxim-methyl.

Several agricultural fungicides related to the antifungal strobilurins act as inhibitors of respiration by binding to mitochondrial cytochrome b. Two types of laboratory mutants resisting higher doses of the strobilurin-related inhibitor kresoxim-methyl were characterized for Venturia inaequalis, the causal agent of apple scab. Selection of mutagenized conidia by kresoxim-methyl yielded mutants altered in the expression of alternative respiration during the stage of conidia germination. Cytochrome b sequences were not affected in the respective mutants. Selection of conidia on media containing the alternative oxidase inhibitor salicylhydroxamic acid in addition to kresoxim-methyl yielded a highly resistant mutant distinguished by a G143A exchange in cytochrome b. The status of mitochondrial cytochrome b genes remained heteroplasmic, and mitochondria containing wild-type cytochrome b returned to high frequencies during cultivation on inhibitor-free medium. However, continuation of the selection process led to a more pronounced replacement of sensitive by mutated mitochondria. The G143A mutation of cytochrome b causing resistance of V. inaequalis to a strobilurin-related inhibitor has been reported previously for mouse mitochondria; and a permanent G143A exchange rendering naturally resistant mitochondria has been reported for the strobilurin-producing basidiomycete Mycena galopoda and for the sea urchin Paracentrotus lividus. At the corresponding position, alanine was also present in chloroplast cytochrome b6 exhibiting low binding of strobilurin-related inhibitors. The mutation of cytochrome b reported here for V. inaequalis describes the first example of a mutation in filamentous ascomycetes and is part of an assessment of resistance risks inherent to strobilurin fungicides.