Mariusz Tadych

Nomenclatural realignment of Neotyphodium species with genus Epichloë

Nomenclatural rule changes in the International Code of Nomenclature for algae, fungi and plants, adopted at the 18th International Botanical Congress in Melbourne, Australia, in 2011, provide for a single name to be used for each fungal species. The anamorphs of Epichloë species have been classified in genus Neotyphodium, the form genus that also includes most asexual Epichloë descendants. A nomenclatural realignment of this monophyletic group into one genus would enhance a broader understanding of the relationships and common features of these grass endophytes. Based on the principle of priority of publication we propose to classify all members of this clade in the genus Epichloë. We have reexamined classification of several described Epichloë and Neotyphodium species and varieties and propose new combinations and states. In this treatment we have accepted 43 unique taxa in Epichloë, including distinct species, subspecies, and varieties. We exclude from Epichloë the two taxa Neotyphodium starrii, as nomen dubium, and Neotyphodium chilense, as an unrelated taxon.

Improving ITS sequence data for identification of plant pathogenic fungi

SummaryPlant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.

Endophytic and pathogenic fungi of developing cranberry ovaries from flower to mature fruit: diversity and succession

Culturable fungal population diversity and succession was investigated in developing cranberry ovaries of fruit rot-resistant and rot-susceptible cranberry selections, from flower through mature fruit. Fungi were recovered in culture from 1185 of 1338 ovary tissues collected from June to September, yielding 2255 isolates that represented 42 morphotaxa. During the season, species richness varied from 2 to 17 and 2 to 18 in rot-resistant and rot-susceptible selections, respectively, increasing from wk1 to wk10 and then gradually declining to wk14. Shannon-Wiener diversity index varied from 0.27 to 2.32 in rot-resistant and 0.18 to 2.38 in rot-susceptible, and Pielou’s evenness index varied from 0.11 to 0.63 and 0.06 to 0.64 in rot-resistant and rot-susceptible selections, respectively, confirming that diversity of fungi in developing ovaries was similar among rot-resistant and rot-susceptible selections, but varied among sampling time points. Principal component analysis grouped samples collected at the same sampling time point together regardless of rot susceptibility of the selections, and detected the predominant fungal species associated with each stage of development. Successional changes were observed in populations of endophytic, pathogenic and saprophytic fungi throughout the season as ovaries matured.

Occurrence of Bacillus amyloliquefaciens as a systemic endophyte of vanilla orchids

We report the occurrence of Bacillus amyloliquefaciens in vanilla orchids (Vanilla phaeantha) and cultivated hybrid vanilla (V. planifolia × V. pompona) as a systemic bacterial endophyte. We determined with light microscopy and isolations that tissues of V. phaeantha and the cultivated hybrid were infected by a bacterial endophyte and that shoot meristems and stomatal areas of stems and leaves were densely colonized. We identified the endophyte as B. amyloliquefaciens using DNA sequence data. Since additional endophyte‐free plants and seed of this orchid were not available, additional studies were performed on surrogate hosts Amaranthus caudatus, Ipomoea tricolor, and I. purpurea. Plants of A. caudatus inoculated with B. amyloliquefaciens demonstrated intracellular colonization of guard cells and other epidermal cells, confirming the pattern observed in the orchids. Isolations and histological studies suggest that the bacterium may penetrate deeply into developing plant tissues in shoot meristems, forming endospores in maturing tissues. B. amyloliquefaciens produced fungal inhibitors in culture. In controlled experiments using morning glory seedlings we showed that the bacterium promoted seedling growth and reduced seedling necrosis due to pathogens. We detected the gene for phosphopantetheinyl transferase (sfp), an enzyme in the pathway for production of antifungal lipopeptides, and purified the lipopeptide “surfactin” from cultures of the bacterium. We hypothesize that B. amyloliquefaciens is a robust endophyte and defensive mutualist of vanilla orchids. Whether the symbiosis between this bacterium and its hosts can be managed to protect vanilla crops from diseases is a question that should be evaluated in future research. Microsc. Res. Tech. 77:874–885, 2014.

Hydrogen peroxide staining to visualize intracellular bacterial infections of seedling root cells

Visualization of bacteria in living plant cells and tissues is often problematic due to lack of stains that pass through living plant cell membranes and selectively stain bacterial cells. In this article, we report the use of 3,3′‐diaminobenzidine tetrachloride (DAB) to stain hydrogen peroxide associated with bacterial invasion of eukaryotic cells. Tissues were counterstained with aniline blue/lactophenol to stain protein in bacterial cells. Using this staining method to visualize intracellular bacterial (Burkholderia gladioli) colonization of seedling roots of switch grass (Panicum virgatum), we compared bacterial free seedling roots and those inoculated with the bacterium. To further assess application of the technique in multiple species of vascular plants, we examined vascular plants for seedling root colonization by naturally occurring seed‐transmitted bacteria. Colonization by bacteria was only observed to occur within epidermal (including root hairs) and cortical cells of root tissues, suggesting that bacteria may not be penetrating deeply into root tissues. DAB/peroxidase with counter stain aniline blue/lactophenol was effective in penetration of root cells to selectively stain bacteria. Furthermore, this stain combination permitted the visualization of the bacterial lysis process. Before any evidence of H2O2 staining, intracellular bacteria were seen to stain blue for protein content with aniline blue/lactophenol. After H2O2 staining became evident, bacteria were often swollen, without internal staining by aniline blue/lactophenol; this suggests loss of protein content. This staining method was effective for seedling root tissues; however, it was not effective at staining bacteria in shoot tissues due to poor penetration. Microsc. Res. Tech. 77:566–573, 2014.

Collaboration between grass seedlings and rhizobacteria to scavenge organic nitrogen in soils

This article describes a process (termed ‘oxidative nitrogen scavenging’) where grasses scavenge organic nitrogen from microbes on and around roots. The authors propose a diurnal process where during the day roots produce and release hydrogen peroxide that oxidizes microbial exoenzymes around roots; at night hydrogen peroxide production ceases, then roots and symbiotic rhizobacteria secrete proteases that degrade the oxidized proteins to form peptides that are absorbed by roots. The existence of a mechanism for organic nitrogen scavenging in grasses emphasizes the nutritional importance of non-pathogenic microbes that associate with roots. Future applications of this process could result in new methods for the cultivation of crop plants.

Epichloë spp. associated with grasses: new insights on life cycles, dissemination and evolution

Epichloë species with their asexual states are specialized fungi associated with cool-season grasses. They grow endophytically in tissues of aerial parts of host plants to form systemic and mostly asymptomatic associations. Their life cycles may involve vertical transmission through host seeds and/or horizontal transmission from one plant to other plants of the same species through fungal propagules. Vertical transmission has been well studied, but comparatively little research has been done on horizontal dissemination. The goal of this review is to provide new insights on modes of dissemination of systemic grass endophytes. The review addresses recent progress in research on (i) the process of growth of Epichloë endophytes in the host plant tissues, (ii) the types and development of reproductive structures of the endophyte, (iii) the role of the reproductive structures in endophyte dissemination and host plant infection processes and (iv) some ecological and evolutionary implications of their modes of dissemination. Research in the Epichloë grass endophytes has accelerated in the past 25 y and has demonstrated the enormous complexity in endophyte-grass symbioses. There still remain large gaps in our understanding of the role and functions of these fungi in agricultural systems and understanding the functions, ecology and evolution of these endophytes in natural grass populations.

Moelleriella zhongdongii: stroma development and identification of hirsutella-like and Aschersonia synanamorphs.

Collections of Moelleriella zhongdongii were made at the La Selva Biological Station in Costa Rica. Fresh collections were examined to evaluate developmental stages. Isolations were made from single part-ascospores and Aschersonia conidia. Moelleriella zhongdongii produces perithecia with evanescent asci and part-ascospores, and both hirsutella-like and Aschersonia synanamorphs. Both anamorphs were produced in pure cultures under cultural conditions optimal to induce the respective anamorphs. Low-nutrient conditions favoured production of the hirsutella-like anamorph while high-nutrient conditions favoured development of the Aschersonia anamorph. The teleomorph developed on leaves of host plants but were not produced in vitro.

Endogone maritima , a new species in the Endogonales from Poland

Endogone maritima sp. nov. is described and illustrated. This fungus was recovered from around the roots of endomycorrhizal Ammophila arenaria, Corynephorus canescens, Juncus balticus, J. articulatus , and ectomycorrhizal Pinus sylvestris colonizing a deflation hollow of the Slowinski National Park in Poland. Endogone maritima produces sunflower to brownish yellow zygosporangia enveloped by a separable hyphal mantle consisting of interwoven hyphae appearing in section as a dense net. The zygosporangia occur both singly in the soil and in small, non-peridial sporocatps typically containing 2-7 zygosporangia.

Glomus multiforum and G. verruculosum, two new species from Poland

Two new vesicular-arbuscular mycorrhizal fungal species, Glomus multiforum and G. verruculos- um (Glomales, Zygomycetes) coming from north- western Poland are described and illustrated. Glomus multiforum produces spores singly in the soil. The deep yellow to brown spores are globose to subglo- bose, (170-)215(-230) pim diam or ovoid, 150-220 X 180-310 pxm and have a single wall consisting of three layers. Outermost layer 1 is mucilaginous, hya- line, and stains reddish in Melzers reagent. Layer 2 of young spores is hyaline and ornamented with in- growths filling the pits of layer 3; it sloughs with spore age. Layer 3 is laminated, colored and ornamented with evenly distributed pits. Glomus verruculosum forms spores singly in the soil. Spores are yellow to orange, (150-)189(-265) Jim diam. Their single wall consists of two layers: a hyaline, semiflexible outer layer and a yellow to orange, laminated inner layer ornamented with warts projecting inward from the innermost lamina. None of these layers stain in Mel- zers reagent. Both G. multiforum and G. verruculos- um formed spores and vesicular-arbuscular mycorrhi- zae in single-species pot cultures with Plantago lan- ceolata and Sorghum sudanense.