A method was developed for enriching bacterial cells from soybean stems which was recalcitrant for a culture-independent analysis of bacterial community due to the interference with plant DNA. Stem homogenates were fractionated by a series of differential centrifugations followed by a Nycodenz density gradient centrifugation. The efficiency of bacterial cell enrichment was assessed by ribosomal intergenic spacer analysis (RISA). The intensity and the number of bacterial amplicons of RISA were markedly increased in the DNA extracted from the enriched bacterial cells compared to that in the DNA directly extracted from soybean stems. The phylogenetic diversity of the enriched bacterial cells was evaluated by analyzing a clone library of 16S rRNA gene in comparison with those of the culturable fractions of the enriched and non-enriched stem-associated bacteria, endophytic bacteria, and epiphytic bacteria. The results indicated that the method was able to enrich both endophytic and epiphytic bacteria from soybean stems, and was useful to assess the bacterial diversity based on a 16S rRNA gene clone library. When the sequence data from all clones (1,332 sequences) were combined, 72 operational taxonomic units were affiliated with Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteroidetes, which also provided the most comprehensive set of data on the bacterial diversity in the aerial parts of soybeans.

Demethylation inhibitors (DMIs) have been an important tool in the management of grapevine powdery mildew caused by Erysiphe necator. Long-term, intensive use of DMIs has resulted in reduced sensitivity in field populations. To further characterize DMI resistance and understand resistance mechanisms in this pathogen, we investigated the cyp51 sequence of 24 single-spored isolates from Virginia and surrounding states and analyzed gene expression in isolates representing a wide range of sensitivity. Two cyp51 alleles were found with respect to the 136th codon of the predicted EnCYP51 sequence: the wild-type (TAT) and the mutant (TTT), which results in the known Y136F amino acid change. Some isolates possessed both alleles, demonstrating gene duplication or increased gene copy number and possibly a requirement for at least one mutant copy of CYP51 for resistance. Cyp51 was over-expressed 1.4- to 19-fold in Y136F-mutant isolates. However, the Y136F mutation was absent in one isolate with moderate to high resistance factor. Two additional synonymous mutations were detected as well, one of which, A1119C was present only in isolates with high cyp51 expression. Overall, our results indicate that at least two mechanisms, cyp51 over-expression and the known target-site mutation in CYP51, contribute to resistance in E. necator, and may be working in conjunction with each other.

Microorganisms associated with the stems and roots of nonnodulated (Nod؊ soybeans, whereas the abundance of basidiomycetes was just the opposite. The phylogenetic analyses suggested that these basidiomycetous fungi might represent a root-associated group in the Auriculariales. Principal-component analysis of the ARISA results showed that there was no clear relationship between nodulation phenotype and bacterial community structure in the stem. In contrast, both the bacterial and fungal community structures in the roots were related to nodulation phenotype. The principal-component analysis further suggested that bacterial community structure in roots could be classified into three groups according to the nodulation phenotype (Nod ؊ , Nod ؉

Colcol, J. F., Rallos, L. E., and Baudoin, A. B. 2012. Sensitivity of Erysiphe neeator to demethylation inhibitor fungicides in Virginia. Plant Dis. 96:111-116.Grape powdery mildew {Erysiphe necator) isolates were collected from 2(K)5 to 2007 from vineyards mostly in Virginia but also some in Maryland, North Carolina, and Pennsylvania. Using a leaf disc assay, the isolates were tested against five demethylation inhibitor (DMI) fungicides. Most isolates exhibited reduced sensitivity to the live DMIs when compared with a sensitive group (H = 12) and compared with unexposed populations reported from other areas. The median resistance factor (RF) was highest for tebucona/ole (RF = 399) and myclobutanil (RF = 378), followed by tritlumizole (RF = 70), triadimefon (RF = 62), and fenarimol (RF = 44). Tbe sensitive group used as the basis for comparison appears to have been more sensitive than unexposed isolates in New York and California. Our finding that the greatest resistance shift occurred with tebucona/ole and myclobutanil contrasts with earlier reports from New York and California, whore the greatest resistance shift was observed with triadinielbn or triadimenol. Sensitivities to all five DMI fungicides were strongly correlated (pairwise r values of 0.70 to 0.87) but our data suggest that some may retain greater utility than others.

Rallos, L. E. E., Johnson, N. G., Schmale, D. G., Ill, Prussin, A. J., II, and Baudoin, A. B. 2014. Fitness of Erysiphe necator with G143A-based resistance to quinone outside inhibitors. Plant Dis. 98:1494-1502.Management of grape powdery mildew (Erysiphe necator) using qui none outside inhibitors (Qols) has eroded in an increasing number of regions due to resistance development. To determine persistence of re sistance when Qols are withdrawn, competition assays were conducted on unsprayed grape plants (Vitis vinifera 'Chardonnay') by cycling mixtures of resistant and sensitive isolates characterized as genetically diverse based on microsatellite analyses. Under laboratory conditions, %G143A, quantified by quantitative polymerase chain reaction (qPCR), increased significantly, indicating competitiveness of the resistant fraction. To confirm competitiveness in the field, trials using potted plants were conducted. Percent G143A tended to decrease in one growing season, probably due to spore migration and mixing of populations with natural background inoculum. In a second season, Qol resistance persisted at high frequency for 4 weeks. Resistant popu lations were also found to persist in one vineyard without Qol applica tion for four consecutive years. The frequency was still about 25% in the fourth year, with higher frequency (36%) in a hotspot section. Qolresistant populations with >5% G143A also harbored Y136F in the cyp51 gene that confers some resistance to sterol demethylation inhibi tors, another fungicide class for powdery mildew control. Double re sistance could have been partly responsible for persistence of Qol resistance at this location.