Nicholas J. Brazee

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.

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.

Insights into the phylogeny of Northern Hemisphere Armillaria: Neighbor-net and Bayesian analyses of translation elongation factor 1-α gene sequences

ABSTRACT Armillaria possesses several intriguing characteristics that have inspired wide interest in understanding phylogenetic relationships within and among species of this genus. Nuclear ribosomal DNA sequence–based analyses of Armillaria provide only limited information for phylogenetic studies among widely divergent taxa. More recent studies have shown that translation elongation factor 1-α (tef1) sequences are highly informative for phylogenetic analysis of Armillaria species within diverse global regions. This study used Neighbor-net and coalescence-based Bayesian analyses to examine phylogenetic relationships of newly determined and existing tef1 sequences derived from diverse Armillaria species from across the Northern Hemisphere, with Southern Hemisphere Armillaria species included for reference. Based on the Bayesian analysis of tef1 sequences, Armillaria species from the Northern Hemisphere are generally contained within the following four superclades, which are named according to the specific epithet of the most frequently cited species within the superclade: (i) Socialis/Tabescens (exannulate) superclade including Eurasian A. ectypa, North American A. socialis (A. tabescens), and Eurasian A. socialis (A. tabescens) clades; (ii) Mellea superclade including undescribed annulate North American Armillaria sp. (Mexico) and four separate clades of A. mellea (Europe and Iran, eastern Asia, and two groups from North America); (iii) Gallica superclade including Armillaria Nag E (Japan), multiple clades of A. gallica (Asia and Europe), A. calvescens (eastern North America), A. cepistipes (North America), A. altimontana (western USA), A. nabsnona (North America and Japan), and at least two A. gallica clades (North America); and (iv) Solidipes/Ostoyae superclade including two A. solidipes/ostoyae clades (North America), A. gemina (eastern USA), A. solidipes/ostoyae (Eurasia), A. cepistipes (Europe and Japan), A. sinapina (North America and Japan), and A. borealis (Eurasia) clade 2. Of note is that A. borealis (Eurasia) clade 1 appears basal to the Solidipes/Ostoyae and Gallica superclades. The Neighbor-net analysis showed similar phylogenetic relationships. This study further demonstrates the utility of tef1 for global phylogenetic studies of Armillaria species and provides critical insights into multiple taxonomic issues that warrant further study.

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.

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.