Analysis of 12 polymorphic simple sequence repeats identified in the genome sequence of Phytophthora ramorum, causal agent of 'sudden oak death', revealed genotypic diversity to be significantly higher in nurseries (91% of total) than in forests (18% of total). Our analysis identified only two closely related genotypes in US forests, while the genetic structure of populations from European nurseries was of intermediate complexity, including multiple, closely related genotypes. Multilocus analysis determined populations in US forests reproduce clonally and are likely descendants of a single introduced individual. The 151 isolates analysed clustered in three clades. US forest and European nursery isolates clustered into two distinct clades, while one isolate from a US nursery belonged to a third novel clade. The combined microsatellite, sequencing and morphological analyses suggest the three clades represent distinct evolutionary lineages. All three clades were identified in some US nurseries, emphasizing the role of commercial plant trade in the movement of this pathogen.
Potato common scab, caused by Streptomyces spp., is an annual production problem for potato growers, and not effectively controlled by current methods. A field with naturally occurring common scab suppression has been identified in Michigan, and confirmed to have a biological basis for this disease suppression. This field and an adjacent scab nursery conducive to disease were studied using pyrosequencing to compare the two microbial communities. Total DNA was extracted from both the disease-conducive and -suppressive soils. A phylogenetically taxon-informative region of the 16S rRNA gene was used to establish operational taxonomic units (OTUs) to characterize bacterial community richness and diversity. In total, 1,124 OTUs were detected and 565 OTUs (10% dissimilarity) were identified in disease-conducive soil and 859 in disease-suppressive soil, including 300 shared both between sites. Common phyla based on relative sequence abundance were Acidobacteria, Proteobacteria, and Firmicutes. Sequences of Lysobacter were found in significantly higher numbers in the disease-suppressive soil, as were sequences of group 4 and group 6 Acidobacteria. The relative abundance of sequences identified as the genus Bacillus was significantly higher by an order of magnitude in the disease-conducive soil.
A field in East Lansing, MI, showed a decline of potato common scab compared with an adjacent potato field. To confirm that the decline was due to biological factors, the soil was assayed. In the greenhouse, putative common-scab-suppressive soil (SS) was either treated with various temperatures or mixed with autoclaved SS at various ratios. Pathogenic Streptomyces scabies was incorporated into the treated soil at 106 CFU/cm3 of soil, followed by planting of either potato or radish. Disease severity was negatively correlated with the percentage of SS in the mixture and positively correlated with temperature above 60°C. The soil was screened for four groups of potential antagonists (general bacteria, streptomycetes, fluorescent pseudomonads, and bacilli) pairing in culture with S. scabies. The frequency of antagonistic bacteria in SS was higher than common-scab-conducive soil (CS) in all four groups but only pseudomonads and streptomycetes were significantly higher. The population of pathogenic Streptomyces spp. in the rhizosphere of CS was significantly higher than SS. Dilution plating of CS and SS samples showed no clear trends or differences in populations of total fungi, total bacteria, streptomycetes, fluorescent pseudomonads, and bacilli but terminal restriction fragment polymorphism analysis revealed two distinct microbial communities were present in SS and CS.
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.
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.
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