Publications

The homothallic mating-type locus of the conifer needle endophyte Phialocephala scopiformis DAOMC 229536 (order Helotiales)

Brent M. Robicheau, Adèle L. Bunbury-Blanchette, Kurt LaButti, Igor V. Grigoriev, Allison K. Walker. 2017. Fungal Biology. https://doi.org/10.1016/j.funbio.2017.08.007

ABSTRACT We describe the complete mating-type (MAT) locus for Phialocephala scopiformis Canadian Collection of Fungal Cultures (DAOMC) 229536 – a basal lineage within Vibrisseaceae. This strain is of interest due to its ability to produce the important antiinsectan rugulosin. We also provide some of the first insights into the genome structure and gene inventory of nonclavicipitalean endophytes. Sequence was obtained through shotgun sequencing of the entire P. scopiformis genome, and the MAT locus was then determined by comparing this genomic sequence to known MAT loci within the Phialocephala fortinii s.l.–Acephala applanata species complex. We also tested the relative levels of sequence conservation for MAT genes within Vibrisseaceae (n = 10), as well as within the Helotiales (n = 27). Our results: (1) show a homothallic gene arrangement for P. scopiformis [MAT1-1-1, MAT1-2-1, and MAT1-1-3 genes are present], (2) increase the genomic survey of homothallism within Vibrisseaceae, (3) confirm that P. scopiformis contains a unique S-adenosyl-l-methionine-dependent methyltransferase (SAM-Mtase) gene proximal to its MAT locus, while also lacking a cytoskeleton assembly control protein (sla2) gene, and (4) indicate that MAT1-1-1 is the more useful molecular marker amongst the MAT genes for phylogenetic reconstructions aimed at tracking evolutionary shifts in reproductive strategy and/or MAT loci gene composition within the Helotiales.

The complete mitochondrial genome of the conifer needle endophyte, Phialocephala scopiformis DAOMC 229536 confirms evolutionary division within the fungal Phialocephala fortinii s.l. – Acephala appalanata species complex

Brent M. Robicheau, Alexander P. Young, Kurt LaButti, Igor V. Grigoriev,  Allison K. Walker. 2016. Fungal Biology http://dx.doi.org/10.1016/j.funbio.2016.11.007
ABSTRACT Despite the recent surge in mitochondrial (mt) genome sequencing, Kingdom Fungi remains underrepresented with respect to mtDNA. We describe the 36,919 bp mt genome of the conifer needle endophyte, Phialocephala scopiformis DAOMC 229536 (Helotiales, Ascomycota). This strain of P. scopiformis is of interest to the Canadian forestry industry as it produces the potent anti-insectan compound rugulosin. Mitochondrial sequence was obtained from whole genome shotgun sequencing of P. scopiformis DAOMC 229536. Evolutionary comparison to the only other published complete Phialocephala mt genome, Phialocephala subalpina, indicates that the suite of common mt genes – cox1-3 and cob, nad1-6 and 4L, atp6, 8 and 9, as well as rrnL and rrnS – has retained an identical gene order. Nad4L remains one of the most conserved mitochondrial genes within the genus. Members of the closely related Phialocephala fortinii s.l. – Acephala appalanata species complex (PAC) share too much sequence similarity to properly resolve evolutionary branches using ITS barcoding alone. Using P. scopiformis mt sequence as a reference outgroup we determined ancestral gene states that help confirm clades within Phialocephala. Our results show: (1) the complete mt genome of P. scopiformis, representing the 10th complete mt genome for the diverse fungal order Helotiales (containing >3,800 species), and (2) how large-scale genomic patterns, such as mitochondrial gene order, can be used to confirm evolutionary branches within fungal species complexes.

Fungal diversity of marine biofilms on artificial reefs in the north-central Gulf of Mexico

Amy L. Salamone, Brent M. Robicheau, Allison K. Walker. 2016. Botanica Marina 59(5): 291–305. https://doi.org/10.1515/bot-2016-0032
ABSTRACT: We present the first characterization of fungal community diversity of natural mixed-species biofilms on artificial marine reefs. Four artificial reefs in the Mississippi (MS) Sound, USA, representing low-profile (underwater) and high-profile (periodically air-exposed) conditions were sampled every 3 months over a 23-month period to investigate changes in fungal diversity within reef biofilms. Fungal presence was assessed via PCR amplification of the internal transcribed spacer (ITS) region of fungal ribosomal DNA, and by terminal restriction fragment length polymorphism (T-RFLP) analysis of fungal ITS regions ? the latter being used to track variation in fungal community structure with respect to season, location, and reef profile type. Fungal communities were also characterized taxonomically through both morphological identification and phylogenetic comparisons of ITS gene sequences, with 36 fungal genera cultured from reef biofilms. Using a multivariate statistical approach, significant temporal and spatial differences in fungal biofilm communities were detected. High-profile reefs differed significantly in biofilm fungal community composition across the 10 sampling periods. This assessment of marine fungal biofilm communities over time provides novel insights into the fungal diversity present on artificial reefs in an understudied region, the north-central Gulf of Mexico.

‘Marine fungi’ and ‘marine-derived fungi’ in natural product chemistry research: Toward a new consensual definition

Ka-Lai Pang · David P. Overy · E. B. Gareth Jones, Maria da Luz Calado, Gaëtan Burgaud, Allison K. Walker, John A. Johnson, Russell G. Kerr, Hyo-Jung Cha, Gerald F. Bills. 2016. Fungal Biology Reviews 30(4):163–175. http://www.sciencedirect.com/science/article/pii/S1749461316300380
ABSTRACT: The discovery of new natural products from fungi isolated from substrata in marine environment has increased dramatically over the last few decades, cumulating in over 1000 new metabolites. The term ‘marine-derived fungi’ is used extensively in these reports, and it refers to the environment from which the fungi are isolated, in contrast to the classical ecological definition of ‘marine fungi’ as obligate and facultative inhabitants of the marine environment. In a significant number of reports, the origins of substrata or habitat relationships of strains referred to as ‘marine-derived fungi’ are unknown or whether a seawater medium was used for their isolation. In August 2014, a workshop held at the University of Prince Edward Island, Canada was convened to discuss a series of topics related to marine fungal natural product research. A central discussion topic was “What constitutes a marine fungus?” There was a general agreement that a review of the definition of a marine fungus would be beneficial to the marine fungal natural product community, together with an evaluation of the suitability and relevance of the use of the term ‘marine-derived fungi’. We here propose a revised, broad definition of a marine fungus as ‘any fungus that is recovered repeatedly from marine habitats because: 1) it is able to grow and/or sporulate (on substrata) in marine environments; 2) it forms symbiotic relationships with other marine organisms; or 3) it is shown to adapt and evolve at the genetic level or be metabolically active in marine environments’.

Full Genome of Phialocephala scopiformis DAOMC 229536, a Fungal Endophyte of Spruce Producing the Potent Anti-Insectan Compound Rugulosin

Allison K Walker, Samantha L Frasz, Keith A Seifert, J. David MillerStephen J. Mondo, Kurt LaButti, Anna Lipzen, Rhyan B. Dockter, Megan C. Kennedy, Igor V. Grigoriev, Joseph W Spatafora. 2016. Genome Announcements 4(1) doi: 10.1128/genomeA.01768-15.
ABSTRACT: We present the full genome of Phialocephala scopiformis DAOMC 229536 (Helotiales, Ascomycota), a foliar endophyte of white spruce from eastern Quebec. DAOMC 229536 produces the anti-insectan compound rugulosin, which inhibits a devastating forestry pest, the spruce budworm. This genome will enable fungal genotyping and host-endophyte evolutionary genomics in inoculated trees.

Antimicrobial dihydrobenzofurans and xanthenes from a foliar endophyte of Pinus strobus

Susan N Richardson, Tienabe K Nsiama, Allison K Walker, David R McMullin, J David Miller. 2015. Phytochemistry 117: 436-443.

ABSTRACT Foliar fungal endophytes of Pinus strobus (eastern white pine) were collected from different sites across south-eastern New Brunswick, Canada and screened for the production of bioactive metabolites. From one site, two fungal isolates representing a formerly unknown genus and species within the family Massarinaceae (Pleosporales, Dothideomycetes, Ascomycota) were resolved by phylogenetic analysis. These isolates produced crude organic extracts that were active against Microbotryum violaceum and Saccharomyces cerevisiae. From these strains, DAOM 242779 and 242780, four dihydrobenzofurans (1–4) and two xanthenes (5–6) were characterized. Structures were elucidated by HRMS, interpretation of NMR spectra and other spectroscopic techniques. All isolated metabolites displayed antimicrobial activity against the biotrophic fungal pathogen M. violaceum and Bacillus subtilis. Phytochem 2015

Distribution of the foliar fungal endophyte Phialocephala scopiformis and its toxin in the crown of a mature white spruce tree as revealed by chemical and qPCR analyses.

Samantha L Frasz, Allison K Walker, Tienabe Nsiama, Gregory Adams, J David Miller. 2014. Canadian Journal of Forest Research 44(9): 1138-1143.  cjfr-2014-0171

ABSTRACT Phialocephala scopiformis is a foliar fungal endophyte of white spruce that produces the anti-insect compound rugulosin and other compounds in lower amounts. Seedlings inoculated with this and other toxigenic endophytes have increased tolerance to the spruce budworm, Choristoneura fumiferana. The presence of rugulosin in the diet and in needles infected by P. scopiformis reduces the growth rate of the insect. One of 300 white spruce trees inoculated as a seedling in 2001 was chosen to investigate the distribution of P. scopiformis and its principal toxin rugulosin throughout the crown. To facilitate the detection of the fungus in small samples, a qPCR assay was developed based on the ITS region of fungal ribosomal DNA targeting a genetic polymorphism unique to P. scopiformis. The assay was specific, with a method limit of detection 100 ng mycelium/g needle sample with high reproducibility and accuracy. We found that 11 years after inoculation, P. scopiformis DAOM 229536 and its toxin were detectable in needle samples distributed throughout the crown. Of the 109 samples tested, 100% of the rugulosin-positive samples also tested positive for P. scopiformis DAOM 229536 DNA in the qPCR assay. The mean and median needle rugulosin concentrations were > 2 times that required to reduce the growth of spruce budworm.

Griseofulvin-producing Xylaria endophytes of Pinus strobus and Vaccinium angustifolium: evidence for a conifer-understory species endophyte ecology.

Susan N Richardson, Allison K Walker, Tienabe K Nsiama, Jordan McFarlane, Mark W Sumarah, Ashraf Ibrahim, J David Miller. 2014. Fungal Ecology  11:107-113. http://authors.elsevier.com/a/1PcwR_dv8-dfa2

ABSTRACT During three decades of research on conifer endophytes of the Acadian forest, numerous insights have been gained in conifer-fungal ecology and secondary metabolite production. Recently, we have explored endophyte assemblages of understory plants commonly occurring with pine. Here we report for the first time the production of the potent antifungal compound griseofulvin by a fungal endophyte isolated from eastern white pine (Pinus strobus) needles and lowbush blueberry (Vaccinium angustifolium) stems from the Acadian forest of New Brunswick and Nova Scotia, Canada. Maximum likelihood phylogenetic analysis has placed the endophyte strains as an undescribed Xylaria sp. Our study highlights the complexity of endophyte-host lifecycles and points to the existence of a pine-blueberry ecotype. Aside from griseofulvin, piliformic acid was isolated from one of the pine endophytes. This compound has been reported from Xylaria and related species but not from Penicillium species known to produce griseofulvin.

Ophiognomonia acadiensis. Fungal Planet 274.

Allison K. Walker, Yuuri Hirooka, Donald M. Walker. 2014. Persoonia – Molecular Phylogeny and Evolution of Fungi 32:290-291.  Ophiognomonia acadiensis, species novo

 Marine fungal diversity: a comparison of natural and created salt marshes of the north-central Gulf of Mexico.

Allison K Walker, Jinx Campbell. 2010. Mycologia 102(3):513-21.  Marine fungi_GOM_saltmarshes

ABSTRACT Marine fungal communities of created salt marshes of differing ages were compared with those of two reference natural salt marshes. Marine fungi occurring on the lower 30 cm of salt marsh plants Spartina alterniflora and Juncus roemerianus were inventoried with morphological and molecular methods (ITS T-RFLP analysis) to determine fungal species richness, relative frequency of occurrence and ascomata density. The resulting profiles revealed similar fungal communities in natural salt marshes and created salt marshes 3 y old and older with a 1.5 y old created marsh showing less fungal colonization. A 26 y old created salt marsh consistently exhibited the highest fungal species richness. Ascomata density of the dominant fungal species on each host was significantly higher in natural marshes than in created marshes at all three sampling dates. This study indicates marine fungal saprotroph communities are present in these manmade coastal salt marshes as early as 1 y after marsh creation. The lower regions of both plant hosts were dominated by a small number of marine ascomycete species consistent with those species previously reported from salt marshes of the East Coast of the USA.

First records of the seagrass parasite Plasmodiophora diplantherae from the northcentral Gulf of Mexico

Allison K Walker, Jinx Campbell. 2009. Gulf and Caribbean Research 21:63-65. seagrass parasite

New Clavulina species from the Pakaraima Mountains of Guyana

Terry W. Henkel, Ryan Meszaros, M. Catherine Aime, Allison Kennedy. 2005. Mycological Progress 4(4):343-350.  New species of Clavulina from Guyana
 ABSTRACT Five new species of Clavulina (Clavulinaceae, Cantharellales, Basidiomycota) are described from the Pakaraima Mountains of Guyana, occurring in rain forestsdominated by the ectomycorrhizal tree Dicymbe corymbosa (Caesalpiniaceae). These clavarioid fungi have simple (i.e., unbranching) basidiomata, which is a relatively uncommon phenotypic feature for the genus Clavulina. Macromorphological, micromorphological, and habitat data are provided for each taxon, and nuclear ribosomal DNA sequences of the 28S subunit and internal transcribed spacer region were obtained for each holotype collection.

 

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