Robert L. Wick

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.

Efficacy of ozone in killing Listeria monocytogenes on alfalfa seeds and sprouts and effects on sensory quality of sprouts.

A study was done to determine the efficacy of aqueous ozone treatment in killing Listeria monocytogenes on inoculated alfalfa seeds and sprouts. Reductions in populations of naturally occurring aerobic microorganisms on sprouts and changes in the sensory quality of sprouts were also determined. The treatment (10 or 20 min) of seeds in water (4 degrees C) containing an initial concentration of 21.8 +/- 0.1 microg/ml of ozone failed to cause a significant (P < or = 0.05) reduction in populations of L. monocytogenes. The continuous sparging of seeds with ozonated water (initial ozone concentration of 21.3 +/- 0.2 microg/ml) for 20 min significantly reduced the population by 1.48 log10 CFU/g. The treatment (2 min) of inoculated alfalfa sprouts with water containing 5.0 +/- 0.5, 9.0 +/- 0.5, or 23.2 +/- 1.6 microg/ml of ozone resulted in significant (P < or = 0.05) reductions of 0.78, 0.81, and 0.91 log10 CFU/g, respectively, compared to populations detected on sprouts treated with water. Treatments (2 min) with up to 23.3 +/- 1.6 microg/ml of ozone did not significantly (P > 0.05) reduce populations of aerobic naturally occurring microorganisms. The continuous sparging of sprouts with ozonated water for 5 to 20 min caused significant reductions in L. monocytogenes and natural microbiota compared to soaking in water (control) but did not enhance the lethality compared to the sprouts not treated with continuous sparging. The treatment of sprouts with ozonated water (20.0 microg/ml) for 5 or 10 min caused a significant deterioration in the sensory quality during subsequent storage at 4 degrees C for 7 to 11 days. Scanning electron microscopy of uninoculated alfalfa seeds and sprouts showed physical damage, fungal and bacterial growth, and biofilm formation that provide evidence of factors contributing to the difficulty of killing microorganisms by treatment with ozone and other sanitizers.

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.

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 km2. 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 (HE) value of 0.112 (SE = 0.006) and a Nei’s expected heterozygosity (HJ) value of 0.190 (SE = 0.009), indicating low genetic diversity within the population. Analysis of molecular variation (ΦPT = 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.

Recent Outbreak of Fusarium Wilt of Basil in Florida

Symptoms of wilt, leaf chlorosis, leaf drop, and shoot and plant death were observed in commercial fields of basil (Ocimmum basilicum L.). Disease incidence ranged from 10 to 80% among individual fields. Initial isolations from infected stem tissue were made on water agar amended with streptomycin sulfate. Single-spore isolates transferred onto corn leaf agar were identified as Fusarium oxysporum Schlechtend.:Fr. f. sp. basilicum Dzidzariya. Pathogenicity tests were performed on 10-cm-tall basil plants, cv. Siam Queen, for three Florida isolates and one Massachusetts isolate. An inoculum concentration of 1 × 106 conidia per ml was applied to soil around the roots. Symptoms of wilt, external stem discoloration, and death of basil occurred after 14 days, and F. oxysporum f. sp. basilicum was reisolated from plants inoculated with all four isolates. Controls were disease-free. Identification of the isolates as F. oxysporum f. sp. basilicum was done with a set of DNA primers developed by Pan and Wick (2) for a 0.7-kb DNA fragment unique to this pathogen. This report confirms the existence of F. oxysporum f. sp. basilicum in Florida (1), and identifies this disease as a potential threat to commercial basil production. References: (1) S. A. Alfieri et al. Diseases and Disorders of Plants in Florida. Bull. No. 14. Fla. Dept. Agric. Consumer Serv., 1993. (2) Z. Pan and R. L. Wick. Phytopathology 85:1559, 1995.

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), β-tubulin (β-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.

In Vitro Production of Pectolytic and Cellulolytic Enzymes by Fusarium Tricinctum

The ability of Fusarium tricinctum to produce polygalacturonase, pectin methylesterase, pectin trans-eliminase and carboxymethylcellulase in vitro was investigated. The optimum temperature and pH for polygalacturonase activity was 35-40 C and 4.0, respectively. The absorption maximum for pectin trans-eliminase reaction products was approximately 550 nm. The activity of all the pectic enzymes investigated increased with culture age. The degradation of carboxymethylcellulose was also demonstrated.

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 Tukeys 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.