Wongia gen. nov. (Papulosaceae, Sordariomycetes), a new generic name for two root-infecting fungi from Australia.

The classification of two root-infecting fungi, Magnaporthe garrettii and M. griffinii, was examined by phylogenetic analysis of multiple gene sequences. This analysis demonstrated that M. garrettii and M. griffinii were sister species that formed a well-supported separate clade in Papulosaceae (Diaporthomycetidae, Sordariomycetes), which clusters outside of the Magnaporthales. Wongia gen. nov, is established to accommodate these two species which are not closely related to other species classified in Magnaporthe nor to other genera, including Nakataea, Magnaporthiopsis and Pyricularia, which all now contain other species once classified in Magnaporthe.

INTRODUCTION

The taxonomic and nomenclatural problems that surround generic names in the Magnaporthales (Sordariomycetes, Ascomycota), together with recommendations for the suppression and protection of some of these names, were explained by the Pyricularia/Magnaporthe Working Group established under the auspices of the International Commission on the Taxonomy of Fungi (ICTF; Zhang ). One of these generic names, Magnaporthe, was proposed for suppression by Zhang because Magnaporthe is congeneric with Nakataea (Hara 1939) as the types of both genera, Magnaporthe salvinii (syn. Leptosphaeria salvinii) and Nakataea sigmoidea (syn. Helminthosporium sigmoideum) are conspecific(Krause & Webster 1972, Luo & Zhang 2013).

Magnaporthe was morphologically characterised by having dark perithecia with long necks immersed in host tissue, unitunicate asci, and 4-celled fusiform hyaline to pale brown ascospores (Krause & Webster 1972). Subsequently, seven species were assigned to Magnaporthe based on morphology, namely, M. salvinii (Krause & Webster 1972), M. grisea (Barr 1977), M. rhizophila (Scott & Deacon 1983), M. poae (Landschoot & Jackson 1989), M. oryzae (Couch & Kohn 2002), and M. garrettii and M. griffinii (Wong et al. 2012). Most of these species belong to other genera, specifically Magnaporthiopsis, Nakataea, and Pyricularia (Luo & Zhang 2013). The two exceptions are the Australian ectotrophic species, M. garrettii and M. griffinii, which infect roots of some turf grasses (Wong ). One of these species, M. griffinii, was found by Klaubauf to be distant from Sordariomycetes based on ITS sequences (GenBank JQ390311, JQ390312).

This study aims to resolve the classification of M. garrettii and M. griffinii using molecular sequence data from the type specimens. Four loci from the nuclear genome namely, ITS) and the large subunit (LSU) of rDNA, translation elongation factor 1-alpha (TEF1), and the largest subunit of RNA polymerase II (RPB1) were selected for analysis.

MATERIALS AND METHODS

Fungal cultures and DNA extraction

Dried specimens of the holotypes of Magnaporthe garrettii (DAR 76937) and M. griffinii (DAR 80512) were borrowed from the Plant Pathology Herbarium, New South Wales Agriculture (DAR). Dried perithecia were excised with a needle and soaked in extraction buffer overnight at 65 ºC before extraction of DNA with an UltraClean® Microbial DNA Isolation Kit (MoBIO Laboratories) as per the manufacturer’s instructions. An additional culture of M. griffinii (BRIP 60377) was grown on PDA for 6 wk before enough mycelium was produced for DNA extraction.

PCR amplification

The primer pairs ITS1/ITS4 (White ), RPB-Ac/RPB-Cr (Castlebury , Matheny ), LR5/LROR and EF1983F/2218R (Schoch ) were used to amplify ITS, RPB1, LSU, and TEF1 sequences, respectively. PCR amplifications were conducted in a 20 μl reaction volume containing 1 μl of 5-10 ng DNA, 10 μl of high fidelity Phusion DNA Polymerase (New England Biolabs), 1 μl of primers (10 μM) and 7 μl of sterile water with the thermal cycling program as follows: 98 ºC for 30s, 30 cycles of 98 ºC for 10 s, 58–62 ºC for 30 s and 72 ºC for 1 min, and a final extension of 72 ºC for 10 min. PCR products were sent to Macrogen (Korea) for direct sequencing using the amplification primers.

Phylogenetic analysis

All sequences were assembled with Sequencher v. 5.1 (Gene Codes, Ann Arbor, MI). Alignments were generated for individual loci using MAFFT v. 6.611 (Katoh & Toh 2008), and then the alignments concatenated for the phylogenetic analyses. DNA sequences were deposited in GenBank with the accession numbers listed in Table 1 and the final curated alignment deposited in TreeBASE under accession no. ID 19968. Phylogenetic trees were reconstructed with two phylogenetic criteria, Maximum likelihood (ML) and Bayesian Inference (BI). ML was carried out with RAxML v. 7.2.6 using GTRGAMMA as the model of evolution (Stamatakis 2006), choosing the rapid bootstrap analysis (command –f a) with a random starting tree and 1000 maximum likelihood bootstrap replications. BI was done with MrBayes v. 3.1.2 (Ronquist ), utilizing four parallel MCMC chains, which were allowed to run for 10 million generations, with sampling every 1000 generations and saving trees every 5 000 generations. The cold chain was heated at a temperature of 0.25. All phylogenetic trees were visualized using FigTree (Morariu ).

Table 1.

Collection details and GenBank accession numbers of isolates included in this study.

Species	Voucher1	Substrate	Locality	GenBank accession no.2
				ITS	LSU	RPB1	TEF1
Annulusmagnus triseptatus	CBS 128831	Decayed wood	France		GQ996540
Bambusicularia brunnea	CBS 133599T	Sasa sp.	Japan	KM484830	KM484948	KM485043
Barretomyces calatheae	CBMAI 1060T	Calathea longifolia	Brazil	GU294490
Brunneosporella aquatica	HKUCC 3708	Submerged wood	Hong Kong		AF132326
Budhanggurabania cynodonticola	BRIP 59305T	Cynodon dactylon	Australia	KP162134	KP162140	KP162143	KP162138
Buergenerula spartinae	ATCC 22848T	Spartina alterniflora	-	JX134666	DQ341492	JX134720	JX134692
Calosphaeria pulchella	CBS 115999	Prunus avium	France		AY761075
Camarops ustulinoides	AFTOL-ID 72	-	-		DQ470941	DQ471121	DQ471050
Coniochaeta ligniaria	NRRL 30616	Soil	-		AY198388
Cordana pauciseptata	CBS 121804	-	Spain		HE672160
Cryphonectria havanensis	CBS 505.63	Eucalyptus saligna	Russia		AF408339
C. parasitica	ATCC 38755	Castanea dentata	USA	Genome3	Genomea	Genome3	Genome3
Diaporthe eres	CBS 109767	Acer campestre	Austria		AF408350
Diaporthe phaseolorum	ATCC 64802	-	-		AY346279
Fluminicola coronata	HKUCC 3717	-	Hong Kong		AF132332
Gaeumannomyces oryzinus	CBS 235.32	Oryza sativa	USA	JX134669	JX134681	JX134723	JX134695
Harknessia eucalypti	CBS 342.97	Eucalyptus regnans	Australia		AF408363
Lecythophora luteoviridis	CBS 206.38	-	Switzerland		FR691987
Magnaporthiopsis agrostidis	BRIP 59300T	Agrostis stolonifera	Australia	KT364753	KT364754	KT364755	KT689623
M. poae	ATCC 64411	Triticum aestivum	USA	JF414836	JF414885	JF710433	JF710415
Nakataea oryzae	ATCC 44754	Oryza sativa	Japan	JF414838	JF414887	JF710441	JF701406
Neurospora crassa	MUCL 19026	-	-		AF286411
Ophioceras leptosporum	CBS 894.70	Dead stem	UK	JX134678	JX134690	JX134732	JX134704
O. dolichostomum	CBS 114926	Rotten wood	China	JX134677	JX134689	JX134731	JX134703
O. commune	YMF1.00980	Rotten wood	China	JX134675	JX134687	JX134729	JX134701
Ophiostoma floccosum	AU55-6 in G	Pinus sp.	Canada		AF234836
O. stenoceras	AFTOL-ID 1038	-	-		DQ836904
Papulosa amerospora	AFTOL-ID 748	-	-		DQ470950	DQ471143	DQ471069
Pseudophialophora eragrostis	RUTTP-CM12m9T	Eragrostis sp.	USA	KF689648	KF689638	KF689618	KF689628
Pseudopyricularia kyllingae	CBS 133597T	Kyllinga brevifolia	Japan	KM484876	KM484992	KM485096
Pyricularia grisea	M 83	Digitaria sp.	USA	JX134671	JX134683	JX134725	JX134697
P. oryzae	70-15	-	USA	Genome4	Genome4	Genome4	Genome4
Togniniella acerosa	CBS 113648	Decayed wood	New Zealand		AY761076
Wongia garrettii	DAR 76937T	Cynodon dactylon	Australia	KU850474		KU850469	KU850467
W. griffinii	DAR 80512T	Cynodon dactylon × transvaalensis	Australia	KU850473	KU850471
W. griffinii	BRIP 60377	Cynodon dactylon × transvaalensis	Australia	KU850472	KU850470	KU850468	KU850466

1AFTOL: Assembling the Fungal Tree of Life; ATCC: American Type Culture Collection, Manassas, VA; BRIP: Plant Pathology Herbarium, Department of Agriculture and Forestry, Queensland, Australia; CBMAI: Coleção Brasileira de Microrganismos para Ambiente e Indústria, Paulinia, Brazil ; CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; DAR: Plant Pathology Herbarium, Orange Agriculture Institute, NSW, Australia; F: Field Museum Mycology Herbarium, Chicago, IL; G: Culture Collection of the Wood Science Department, University of British Columbia, Vancouver, BC, Canada; HKUCC: Hong Kong University Culture Collection; MUCL: Mycothèque de l′Université Catholique de Louvain, Louvain-la-Neuve, Belgium; NRRL: American Research Service (ARS) culture collection, Beltsville, MD; RUTPP = Rutgers Mycological Herbarium, New Brunswick, NJ; YMF: Yunnan Microbiological Fermentation Culture Collection Center, Kunming, Yunnan, China.

2 GenBank accession numbers of sequences newly generated in this study are in bold.

3 Joint Genome Institute, Walnut Creek, CA.

4 Broad Institute, Cambridge, MAA.

T Type specimen or ex-type culture.

RESULTS

Molecular phylogeny

The phylogenetic trees recovered from the ML and BI analyses had identical topologies and were well-supported by bootstrap and posterior probabilities (Fig. 1). The analyses comprised 36 taxa belonging to eight orders and two families in the subclass Diaporthomycetidae (Sordariomycetes). Camarops ustulinoides (Boniliales, Sordariomycetes) was used as the outgroup (Table 1). The phylogenetic analysis revealed Magnaporthe garrettii (DAR 76937) and M. griffinii (DAR 80512) as sister species that formed a distinct well-supported (100/1.0) monophyletic clade in Papulosaceae that sat outside Magnaporthales. The analysis provided moderate support (67/0.93) for placement of M. garrettii and M. griffinii in Papulosaceae, which has not yet been assigned to any order of Diaporthomycetidae. Based on this analysis, a new generic name is established here to accommodate M. garrettii and M. griffinii.

Fig 1.

Phylogenetic tree obtained from a maximum likelihood analysis of the combined ITS/LSU/RPB1/TEF1 alignment. The bootstrap support values from 1 000 replicates and posterior probabilities obtained in Bayesian analysis are indicated at the nodes. The scale bar indicates the expected changes per site. Ex-type cultures of species are indicated in bold.

TAXONOMY

Wongia Khemmuk, Geering & R.G. Shivas, gen. nov. MycoBank MB817529

Etymology: Named after the eminent Australian mycologist and plant pathologist, Percy T.W. Wong (University of Sydney), who first studied and classified these fungi.

Diagnosis: Differs from all other genera in the subclass Diaporthomycetidae in having non-amyloid apical rings in the asci with 3-septate ascospores that have dark brown middle cells and pale brown to subhyaline shorter distal cells.

Type species: Wongia garrettii (P. Wong & M.L. Dickinson) Khemmuk et al. 2016

Classification: Ascomycota, Sordariomycetes, Diaportho-mycetidae.

Description: Mycelium comprised of brown, straight or flexuous hyphae, with simple hyphopodia. Ascomata perithecial, superficial and immersed, mostly solitary or sometimes aggregated in small groups, globose, black, ostiolate, with a long or short neck, perithecial wall composed of textura epidermoidea, external cell much darker. Paraphyses thin-walled, hyaline, filiform, septate.

Asci unitunicate in structure, cylindrical, mostly straight, short stalked, tapered towards a rounded apex, with a light refractive, non-amyloid apical ring, 8-spored. Ascospores uniseriate, cylindrical to fusiform, straight or slightly curved with rounded ends, 3-septate, middle cells dark brown and distal cells pale brown to subhyaline and shorter.

Wongia garrettii (P. Wong & M.L. Dickinson) Khemmuk, Geering & R.G. Shivas, comb. nov.

(Fig. 2A–B)

Fig 2.

Morphological features of Wongia species. A–B. W. garrettii (DAR 76937 – holotype). A. Perithecium. B. Ascospores. C–D. W. griffinii (BRIP 60378). C. Perithecium. D. Ascospores. Bars: A, D = 100 μm; B, D = 10 μm.

MycoBank MB817530

Basionym: Magnaporthe garrettii P. Wong & M.L. Dickinson, Australasian Plant Pathology 41: 326 (2012).

Type: Australia: South Australia: Adelaide, Colonel Light Gardens Bowling Club, on Cynodon dactylon, 30 Oct. 2004, M.L. Dickinson (DAR 76937 – holotype).

Description and illustration: Wong .

Wongia griffinii (P.Wong & A.M. Stirling) Khemmuk, Geering & R.G. Shivas, comb. nov.

(Fig. 2C–D)

MycoBank MB817531

Basionym: Magnaporthe griffinii P. Wong & A.M. Stirling, Australasian Plant Pathology 41: 327 (2012).

Type: Australia: Queensland: Coolum, Hyatt Coolum Golf Club, on Cynodon dactylon × transvaalensis, 13 Mar. 2008, M. Whatman (DAR 80512 – holotype).

Description and illustration: Wong

Other specimens examined: Australia: New South Wales: Cobbitty, on Cynodon dactylon, 19 Apr. 2013, G. Beehag, (BRIP 60378). Queensland: Brisbane, on on Cynodon dactylon × transvaalensis, Jan. 2000, A.M. Stirling (BRIP 60377).

DISCUSSION

Magnaporthe is a synonym of Nakataea as their respective type species, Magnaporthe salvinii and Nakataea sigmoidea, refer to the same species (Krause & Webster 1972, Luo & Zhang 2013, Klaubauf , Zhang ). This led us to re-examine two Australian species, M. garrettii and M. griffinii, pathogenic on roots of couch (Cynodon dactylon) and hybrid couch (C. dactylon × transvaalensis) (Wong ). We establish Wongia here to accommodate these two species, based on molecular and morphological analysis.

Multigene analyses placed W. garrettii and W. griffinii in Papulosaceae (Diaporthomycetidae, Sordariomycetes; Maharachchikumbura ) with moderate bootstrap support (Fig. 1). The Papulosaceae has not yet been placed in an order within Sordariomycetes (Winka & Erikson 2000). Wongia is the fourth genus to be placed in Papulosaceae, along with Brunneosporella (Ranghoo & Hyde 2001), Fluminicola (Wong ). and Papulosa (Kohlmeyer & Volkmann-Kohlmeyer 1993). Most members in this family are found on submerged wood in freshwater habitats and grow slowly in culture on potato dextrose agar (Ranghoo & Hyde 2001). Wongia garrettii and W. griffinii are morphologically different from other genera of Papulosaceae in having non-amyloid apical rings in the asci using Melzer’s reagent, while others have amyloid apical rings (Winka & Eriksson (2000). The long perithecial necks of W. garrettii differentiate it from W. griffinii (Wong ), which also has larger ascospores (24–35 x 6–9 μm) than W. garrettii (19–25 x 5–7 μm) (Wong ). Asexual morphs have not been found in either W. garrettii or W. griffinii in nature or in cultures grown on artificial media under laboratory conditions (Wong ).