A De Novo-Assembly Based Data Analysis Pipeline for Plant Obligate Parasite Metatranscriptomic Studies.

Current and emerging plant diseases caused by obligate parasitic microbes such as rusts, downy mildews, and powdery mildews threaten worldwide crop production and food safety. These obligate parasites are typically unculturable in the laboratory, posing technical challenges to characterize them at the genetic and genomic level. Here we have developed a data analysis pipeline integrating several bioinformatic software programs. This pipeline facilitates rapid gene discovery and expression analysis of a plant host and its obligate parasite simultaneously by next generation sequencing of mixed host and pathogen RNA (i.e., metatranscriptomics). We applied this pipeline to metatranscriptomic sequencing data of sweet basil (Ocimum basilicum) and its obligate downy mildew parasite Peronospora belbahrii, both lacking a sequenced genome. Even with a single data point, we were able to identify both candidate host defense genes and pathogen virulence genes that are highly expressed during infection. This demonstrates the power of this pipeline for identifying genes important in host-pathogen interactions without prior genomic information for either the plant host or the obligate biotrophic pathogen. The simplicity of this pipeline makes it accessible to researchers with limited computational skills and applicable to metatranscriptomic data analysis in a wide range of plant-obligate-parasite systems.

Phytophthora species recovered from the Connecticut River Valley in Massachusetts, USA.

Little is currently known about the assemblage of Phytophthora species in northeastern North America, representing a gap in our understanding of species incidence. Therefore, Phytophthora species were surveyed at 20 sites in Massachusetts, with 16 occurring in the Connecticut River Valley. Many of the sampled waterways were adjacent to active agricultural lands, yet were buffered by mature floodplain forests composed of Acer, Platanus, Populus and Ulmus. Isolates were recovered with three types of baits (rhododendron leaves, pear, green pepper) in 2013 and water filtration in 2014. Overall, 457 isolates of Phytophthora were recovered and based on morphological characters and rDNA internal transcribed spacer (ITS), ß-tubulin (ß-tub) and cytochrome oxidase c subunit I (cox1) sequences, 18 taxa were identified, including three new species: P. taxon intercalaris, P. taxon caryae and P. taxon pocumtuck. In addition, 49 isolates representing five species of Phytopythium also were identified. Water filtration captured a greater number of taxa (18) compared to leaf and fruit baits (12). Of the three bait types rhododendron leaves yielded the greatest number of isolates and taxa, followed by pear and green pepper, respectively. Despite the proximity to agricultural lands, none of the Phytophthora species baited are considered serious pathogens of vegetable crops in the region. However, many of the recovered species are known woody plant pathogens, including four species in the P. citricola s.l. complex that were identified: P. plurivora, P. citricola III, P. pini and a putative novel species, referred to here as P. taxon caryae. An additional novel species, P. taxon pocumtuck, is a close relative of P. borealis based on cox1 sequences. The results illustrate a high level of Phytophthora species richness in the Connecticut River Valley and that major rivers can serve as a source of inoculum for pathogenic Phytophthora species in the northeast.

Basil Downy Mildew (Peronospora belbahrii): Discoveries and Challenges Relative to Its Control.

Basil (Ocimum spp.) is one of the most economically important and widely grown herbs in the world. Basil downy mildew, caused by Peronospora belbahrii, has become an important disease in sweet basil (O. basilicum) production worldwide in the past decade. Global sweet basil production is at significant risk to basil downy mildew because of the lack of genetic resistance and the ability of the pathogen to be distributed on infested seed. Controlling the disease is challenging and consequently many crops have been lost. In the past few years, plant breeding efforts have been made to identify germplasm that can be used to introduce downy mildew resistance genes into commercial sweet basils while ensuring that resistant plants have the correct phenotype, aroma, and tastes needed for market acceptability. Fungicide efficacy studies have been conducted to evaluate current and newly developed conventional and organic fungicides for its management with limited success. This review explores the current efforts and progress being made in understanding basil downy mildew and its control.

Genotypic diversity of Armillaria gallica from mixed oak forests in Massachusetts.

The population structure of Armillaria gallica, an important pathogen of Quercus spp., was investigated from mixed oak forests in central Massachusetts, encompassing a sampling area over 500 km(2). From 16 plots at four sites a total of 153 isolates (34-40 isolates per site) was analyzed with amplified fragment length polymorphisms (AFLPs). Analyses of 204 polymorphic loci detected 38 AFLP genotypes from a sample area of 4.51 hectares (ha). Genets ranged in distribution from five to 33 genets per hectare (GPH), with a mean of eight GPH and the average A. gallica genet occupying 0.13 ha. Allele frequencies produced an unbiased expected heterozygosity (H(E)) value of 0.112 (SE = 0.006) and a Nei's expected heterozygosity (H(J)) value of 0.190 (SE = 0.009), indicating low genetic diversity within the population. Analysis of molecular variation (Φ(PT) = 0.301; P < 0.001) indicates high genetic differentiation, with 70% of the molecular variation explained at the site-level within A. gallica subpopulations. However, results of the Mantel test, used to assess the isolation-by-distance hypothesis, were inconclusive in determining whether the subpopulations were truly isolated by distance. A neighbor-joining tree constructed from a genetic distance matrix grouped genotypes from the same site (subpopulation) together, but from three of four sites genotypes were randomly clustered at the plot level. The results suggest that basidiospore dispersal is an important means of new genet formation at linear distances up to 2000 m.

Evaluation of partial tef1, rpb2, and nLSU sequences for identification of isolates representing Armillaria calvescens and Armillaria gallica from northeastern North America.

Armillaria calvescens and Armillaria gallica are two of the most closely-related species of Armillaria in North America and have been difficult to distinguish from one another using morphological and molecular techniques. In an attempt to better distinguish these two species, partial sequences of the elongation factor-1 alpha (tef1), RNA polymerase II (rpb2), and nuclear large subunit (nLSU) genes were generated for 32 total isolates; 12 isolates each for A. calvescens and A. gallica, along with two isolates each of Armillaria gemina, Armillaria mellea, Armillaria sinapina, and Armillaria solidipes. Within the tef1 amplicon, five single nucleotide polymorphisms (SNPs) were present between A. calvescens and A. gallica. Phylogenetic reconstruction using the maximum likelihood (ML) and maximum parsimony (MP) methods showed that tef1 was the only gene capable of distinguishing A. calvescens from A. gallica, and additionally, all isolates representing the six northeastern North American species. Partial tef1 sequences grouped A. calvescens into a strongly-supported, monophyletic clade with bootstrap support (BS) values of 98/98% (ML/MP), while A. gallica was grouped into a monophyletic clade with lower BS support (76/59%). The results illustrate the utility of partial tef1 sequences for the identification of field isolates of Armillaria from northeastern North America.

Effects of Hydrolyzable Tannins on In Vitro Growth of Armillaria calvescens and A. gallica.

We hypothesized that Armillaria gallica, which is abundant in oak-dominated forests, is more successful at oxidizing and metabolizing polyphenols than A. calvescens, which is mostly restricted to maple-dominated forests. Isolates were challenged with up to seven concentrations of tannic acid (TA), gallic acid (GA), and black oak root bark extracts (RBE). Six concentrations of glucose and ethanol were also tested to determine the influence of available carbon on growth. Colony area and biomass values were analyzed using a GLM and Tukey's HSD test. When challenged with 0.12% concentrations of TA, GA, and RBE, A. gallica produced a significantly larger biomass in all treatments and larger colony areas in four of the five treatments compared to control values. A. gallica also produced a significantly larger number of rhizomorphs than A. calvescens on RBE medium. In contrast, A. calvescens generated significantly larger biomass over control treatments only when RBE was added, and values were substantially less compared to A. gallica. Growth of both species was significantly greater when ethanol was added, especially on GA medium, while glucose had little effect. Results from this study suggest that A. gallica is better at oxidizing and metabolizing polyphenols than A. calvescens.

Toxicity of nanoparticulate and bulk ZnO, Al2O3 and TiO2 to the nematode Caenorhabditis elegans.

Limited information is available on the environmental behavior and associated potential risk of manufactured oxide nanoparticles (NPs). In this research, toxicity of nanoparticulate and bulk ZnO, Al(2)O(3) and TiO(2) were examined to the nematode Caenorhabditis elegans with Escherichia coli as a food source. Parallel experiments with dissolved metal ions from NPs were also conducted. The 24-h median lethal concentration (LC(50)) and sublethal endpoints were assessed. Both NPs and their bulk counterparts were toxic, inhibiting growth and especially the reproductive capability of the nematode. The 24-h LC(50) for ZnO NPs (2.3 mg L(-1)) and bulk ZnO was not significantly different, but significantly different between Al(2)O(3) NPs (82 mg L(-1)) and bulk Al(2)O(3) (153 mg L(-1)), and between TiO(2) NPs (80 mg L(-1)) and bulk TiO(2) (136 mg L(-1)). Oxide solubility influenced the toxicity of ZnO and Al(2)O(3) NPs, but nanoparticle-dependent toxicity was indeed observed for the investigated NPs.