Cryptic diversity found in Didymellaceae from Australian native legumes

Abstract

Ascochyta koolunga (Didymellaceae, Pleosporales) was first described in 2009 (as Phoma koolunga) and identified as the causal agent of Ascochyta blight of Pisum sativum (field pea) in South Australia. Since then A. koolunga has not been reported anywhere else in the world, and its origins and occurrence on other legume (Fabaceae) species remains unknown. Blight and leaf spot diseases of Australian native, pasture and naturalised legumes were studied to investigate a possible native origin of A. koolunga.

Ascochyta koolunga was not detected on native, naturalised or pasture legumes that had leaf spot symptoms, in any of the studied regions in southern Australia, and only one isolate was recovered from P. sativum. However, we isolated five novel species in the Didymellaceae from leaf spots of Australian native legumes from commercial field pea regions throughout southern Australia. The novel species were classified on the basis of morphology and phylogenetic analyses of the internal transcribed spacer region and part of the RNA polymerase II subunit B gene region. Three of these species, Nothophoma garlbiwalawardasp. nov., Nothophoma naiawusp. nov. and Nothophoma ngayawangsp. nov., were isolated from Senna artemisioides. The other species described here are Epicoccum djirangnandirisp. nov. from Swainsona galegifolia and Neodidymelliopsis tinkyukukusp. nov. from Hardenbergia violacea. In addition, we report three new host-pathogen associations in Australia, namely Didymella pinodes on S. artemisioides and Vicia cracca, and D. lethalis on Lathyrus tingitanus. This is also the first report of Didymella prosopidis in Australia.

Introduction

The Didymellaceae was established to accommodate Ascochyta, Didymella, and other allied Phoma-like genera (de Gruyter et al. 2009). To date, more than 5,400 species from 31 genera have been recorded, including recently established genera such as Dimorphoma and Macroascochyta (Hou et al. 2020). Species of Didymellaceae are cosmopolitan and occupy a broad range of environments. Many species are plant pathogens that cause leaf and stem lesions, often with a broad host range (Aveskamp et al. 2009; Aveskamp et al. 2010; Chen et al. 2015b). Multilocus phylogenetics and a polyphasic approach to classify species have helped to revise taxa and refine systematic relationships in the Didymellaceae (Aveskamp et al. 2009, de Gruyter et al. 2009; Aveskamp et al. 2010; Chen et al. 2015a, de Gruyter 2012; Hou et al. 2020).

In Australia, reports of taxa in the Didymellaceae mostly refer to plant pathogenic species, particularly on crop and pasture legumes (Fabaceae). In Australia, the disease Ascochyta blight of Pisum sativum (field pea) is typically caused by three fungal species, Ascochyta koolunga, Didymella pinodella, and D. pinodes. A fourth species, Ascochyta pisi, is very rarely isolated. One species in particular, A. koolunga, is an important part of the Ascochyta blight disease complex of field pea in South Australia (Davidson et al. 2009a). First described in 2009, A. koolunga (syn. Phoma koolunga) had spread across southern Australia and had been detected in Victoria and Western Australia by 2015 (Davidson et al. 2011; Tran et al. 2015a).

Molecular techniques are now routinely used to understand the genetic diversity and population structure of Didymellaceae (Aveskamp et al. 2010; Salam et al. 2011, de Gruyter 2012; Chen et al. 2015a, Hou et al. 2020). To date, there has not been a systematic inventory of leaf spot pathogens associated with Australian native legume species despite international reports from a diversity of countries on Ascochyta blight since 2009 (Le May et al. 2009; Mathew et al. 2010; Panicker and Ramraj 2010; Skoglund et al. 2011; Soylu and Dervis 2011; Gaurilcikiene and Viciene 2013; Liu et al. 2013; Ahmed et al. 2015; Liu et al. 2016). Ascochyta koolunga is only known to occur in Australia, which suggests an Australasian origin, with perhaps an association with native legume species. The aim of this study was to determine the species of Didymellaceae associated with leaf spot diseases, and to investigate possible native sources of A. koolunga. To this end we collected legume specimens from both cultivated and neighbouring natural ecosystems. In particular, we collected specimens from Australian native, pasture and naturalised legumes in the field pea growing regions of eastern and southern Australia.

Materials and methods

Sample collection and culturing

Samples of leaf tissue displaying leaf spot disease symptoms on legumes were obtained from 22 field pea trial sites, from the immediate surrounds of experimental and commercial crops and roadsides around crops in field pea growing regions of southern Australia. In total, 124 samples (stems with multiple leaves and more rarely seed pods and flowers) were collected during four separate 4–5 day (d) periods in August, September and October 2017. In addition to trial sites, local agronomists were contacted to obtain approval to allow access to growers’ properties in Eyre Peninsula (South Australia) and Horsham (Victoria).

The national parks, or conservation areas, nearest to the field pea sampling sites were identified prior to field trips and permits were obtained to enable collections of samples from native plants that exhibited leaf disease symptoms within these neighbouring natural ecosystems. Leaf disease samples were also collected from two botanic gardens, Adelaide Botanic Garden, Adelaide, South Australia and the Australian Botanic Garden, Mount Annan, New South Wales. Plants with leaf spots were photographed in the field with a Samsung galaxy S5 or S8 mobile phone camera and the GPS locations recorded. Representative leaf samples were placed in plastic bags, labelled and stored at 4 °C.

Within 5 d of collection, leaf specimens were surface disinfected by spraying with 70% v/v ethanol and blotted dry with fresh, non-sterilised tissue paper. Excised leaf pieces were placed on plates of potato dextrose agar (PDA) (Oxoid) acidified by supplementation with 1 ml of 85% v/v lactic acid per litre (APDA) to minimise bacterial contamination. Incubation was under a 12 hour (h) black and fluorescent light /12 h dark cycle at 22 °C for 7–10 d, when fungal colonies were examined microscopically for pycnidia and conidia. Representative isolates were subcultured onto PDA using hyphal tips and deposited in the culture collection of the Queensland Plant Pathology Herbarium (BRIP).

DNA extraction, PCR and sequencing

Genomic DNA was extracted from 7 d old mycelium grown on PDA from the subculture isolates using the FastDNA Kit (Q-biogene Inc. Irvine, California, USA) according to the manufacturer’s instructions. A section of DNA from the internal transcribed spacer (ITS) region was amplified with the primers ITS1 and ITS4 (White et al. 1990), and the partial region of the RNA polymerase II subunit B (rpb2) gene was amplified with the primers RPB2-5F2 (Sung et al. 2007) and RPB2-7cR (Liu et al. 1999). The PCR conditions were as described by White et al. (1990) for ITS and O’Donnell et al. (2007) for rpb2. All PCRs were undertaken in 25 μl reaction volumes containing the final concentrations; 1 unit of PCR 5X buffer (Promega Corporation, Madison, Wisconsin, USA), 1.6 mM of 25 mM MgCl₂ (Sigma-Aldrich Corporation, Louis, Missouri, USA), 0.025 U/μl of GoTaq™ (Promega), 0.6 mM of primer 1 and primer 2 and 1.6 mM of each dNTP (Promega). The PCR amplicons were purified using ExoSAP-IT (USB Corporation) following the manufacturer’s instructions. The purified amplicons were sent to the Ramaciotti Centre for Gene Function Analysis (University of New South Wales, Kensington, NSW), where DNA sequences were determined using an ABI PRISM 3700 DNA Analyser (Applied Biosystems Inc).

Phylogenetic analysis

Forward and reverse sequences were assembled using Geneious v. 11.1.5 (Biomatters Ltd) and deposited in GenBank (Table 1, in bold). The sequences were aligned with selected reference sequences of Didymellaceae (Table 1) using the multiple alignment MAFFT algorithm (Katoh et al. 2009) in Geneious. Neoascochyta desmazieri strain CBS 267.69 was included as the outgroup. The sequences of each locus were aligned separately and manually adjusted where necessary.

Table 1.

Didymellaceae isolates examined in this study. Novel taxa and newly generated sequences are indicated in bold.

Species	Strain 1	Host	Locality 2	GenBank accessions 3
				ITS	rpb2
Ascochyta astragalina	CBS 113797	Lathyrus vernus	Sweden	KT389482	MT018257
Ascochyta benningiorum	CBS 144957 T	Soil	The Netherlands	MN823581	MN824606
Ascochyta coronillae-emeri	MFLUCC 13-0820 T	Hippocrepis emerus	Italy	MH069661	MH069679
Ascochyta fabae	CBS 524.77	Phaseolus vulgaris	Belgium	GU237880	MT018241
Ascochyta herbicola	CBS 629.97	Water	USA, Montana, Missoula	GU237898	KP330421
Ascochyta koolunga	DAR 78535 T	Pisum sativum	Australia, SA, Minnipa	EU338416	EU874849
	BRIP 70265	Pisum sativum	Australia, SA, Riverton	MN567671	MN604922
	BRIP 69590	Pisum sativum	Australia, SA, Mundulla	MN567672	MN604923
Ascochyta lentis	CBS 370.84	Lens culinaris	Unknown	KT389474	MT018246
Ascochyta medicaginicola	CBS 112.53 T	Medicago sativa	USA	GU237749	MT018251
Ascochyta nigripycnidia	CBS 116.96 T	Vicia cracca	Russia	GU237756	MT018253
Ascochyta phacae	CBS 184.55 T	Phaca alpine	Switzerland	KT389475	MT018255
Ascochyta pilosella	CBS 583.97 T	Clintonia uniflora	Canada	MN973590	MT018258
Ascochyta pisi	CBS 122785	Pisum sativum	The Netherlands	GU237763	MT018244
Ascochyta rabiei	CBS 237.37 T	Cicer arietinum	Bulgaria	KT389479	MT018256
Ascochyta rosae	MFLUCC 15-0063 T	Rubus ulmifolius	Italy	KY496751	KY514409
Ascochyta syringae	CBS 545.72 T	Syringa vulgaris	The Netherlands	KT389483	MT018245
Ascochyta versabilis	CBS 876.97	Silene sp.	The Netherlands, Wageningen	GU237909	KT389561
Ascochyta viciae	CBS 451.68	Vicia sepium	The Netherlands, Baarn, Praamgracht	KT389484	KT389562
Ascochyta viciae-pannonicae	CBS 254.92	Vicia pannonica	Czechoslovakia	KT389485	MT018250
Ascochyta viciae-villosae	CBS 255.92	Vicia villosa	Czechoslovakia	MN973584	MT018249
Didymella americana	CBS 185.85	Zea mays	USA, Georgia	FJ426972	KT389594
Didymella anserina	CBS 253.80		Germany	KT389498	KT389595
Didymella arachidicola	CBS 333 .75 T	Arachis hypogaea	South Africa, Cape Province	GU237833	KT389598
Didymella aurea	CBS 269.93 T	Medicago polymorpha	New Zealand, Auckland	GU237818	KT389599
Didymella chlamydospora	YW23-14 T	Soil	South Korea	MK836111	LC480708
Didymella coffeae-arabicae	CBS 123380 T	Coffea Arabica	Ethiopia	FJ426993	KT389603
Didymella combreti	CBS 137982 T	Combretum mossambiciensis	Zambia	MN973525	MT018139
Didymella curtisii	CBS 251.92	Nerine sp.	The Netherlands	FJ427038	MT018131
Didymella degraaffiae	CBS 144956 T	Soil	The Netherlands	MN823444	MN824470
Didymella eucalyptica	CBS 377.91	Eucalyptus sp.	Australia, WA	GU237846	KT389605
Didymella gardeniae	CBS 626.68 T	Gardenia jasminoides	India	FJ427003	KT389606
Didymella glomerata	CBS 528.66	Chrysanthemum sp.	The Netherlands	FJ427013	GU371781
Didymella guttulata	CBS 127976 T	Soil	Zimbabwe	MN973524	MT018138
Didymella heteroderae	CBS 109.92 T	Undefined food material	The Netherlands	FJ426983	KT389601
Didymella keratinophila	UTHSC DI16-200 T	Homo sapiens	USA	LT592901	LT593039
Didymella lethalis	CBS 103.25			GU237729	KT389607
	BRIP 69584	Lathyrus tingitanus	Australia, SA, Brownhill Creek	MN567674	MN604925
Didymella magnoliae	MFLUCC 18-1560 T	Magnolia grandiflora	China	MK347814	MK434852
Didymella maydis	CBS 588.96 T	Zea mays	USA, Wisconsin, Hancock	FJ427086	GU371782
Didymella mitis	CBS 443.72 T	Soil	South Africa	MN973523	MT018137
Didymella musae	CBS 463.69	Mangifera indica	India	FJ427026	MT018148
Didymella nigricans	CBS 444.81	Acer palmatum	Japan	KY742075	KY742158
Didymella pinodella	CBS 318.90	Pisum sativum	The Netherlands	FJ427051	MN983533
	BRIP 69589	Pisum sativum	Australia, VIC, Rainbow	MN567675	MN604926
Didymella pinodes	CBS 525.77 T	Pisum sativum	Belgium	GU237883	KT389614
	BRIP 69581	Senna artemisioides	Australia, SA, Blanchetown	MN567676	MN604927
	BRIP 69593	Senna artemisioides	Australia, SA, Blyth	MN567677	MN604928
	BRIP 69596	Senna artemisioides	Australia, SA, Wudinna	MN567678	MN604929
	BRIP 69578	Vicia cracca	Australia, NSW, Cowra	MN567679	MN604930
Didymella pomorum	CBS 539.66	Polygonum tataricum	The Netherlands	FJ427056	KT389618
Didymella prolaticolla	CBS 126182 T	Soil	Namibia	MN973533	MT018157
Didymella prosopidis	CBS 136414 T	Prosopis sp.	South Africa	KF777180	MT018149
	BRIP 69579	Gastrolobium celsianum	Australia, SA, Adelaide	MN5676780	MN604931
Didymella protuberans	CBS 381.96 T	Lycium halifolium	The Netherlands	GU237853	KT389620
Didymella sancta	CBS 281.83 T	Ailanthus altissima	South Africa	FJ427063	KT389623
Didymella sinensis	CGMCC 3.18348 T	Cerasus pseudocerasus	China	KY742085	MT018127
Didymella subglobispora	CBS 364.91 T	Ananas sativus		MN973531	MT018153
Didymella subglomerata	CBS 110.92	Triticum sp.	USA, North Dakota	FJ427080	KT389626
Epicoccum brahmansense	CBS 990.95 T	Soil	Papua New Guinea	MN973513	MT018119
Epicoccum brasiliense	CBS 120105 T	Amaranthus sp.	Brazil	GU237760	KT389627
Epicoccum camelliae	CGMCC 3.18343 T	Camellia sinensis	China	KY742091	KY742170
Epicoccum catenisporum	CBS 181.80 T	Oryza sativa	Guinea-Bissau	FJ427069	LT623253
Epicoccum dendrobii	CGMCC 3.18359 T	Dendrobium fimbriatum	China	KY742093	MT018084
Epicoccum dickmanii	CBS 124671 T	Acropora Formosa	Australia	MN973509	MT018113
Epicoccum djirangnandiri sp. nov.	BRIP 69585 T	Swainsona galegifolia	Australia, NSW, Mount Annan	MN567673	MN604924
Epicoccum draconis	CBS 186.83	Dracaena sp.	Rwanda	GU237795	KT389628
Epicoccum duchesneae	CGMCC 3.18345 T	Duchesnea indica	China	KY742095	MT018115
Epicoccum henningsii	CBS 104.80	Acacia mearnsii	Kenya	GU237731	KT389629
Epicoccum hordei	CGMCC 3.18360 T	Hordeum vulgare	Australia	KY742097	MT018102
Epicoccum huancayense	CBS 105.80 T	Solanum sp.	Peru	GU237732	KT389630
Epicoccum italicum	CGMCC 3.18361 T	Acca sellowiana	Italy	KY742099	KY742172
Epicoccum keratinophilum	UTHSC DI16-271 T	Homo sapiens	USA	LT592930	LT593068
Epicoccum latusicollum	CGMCC 3.18346 T	Sorghum bicolor	China	KY742101	KY742174
Epicoccum longiostiolatum	CBS 886.95 T	Stellaria sp.	Papua New Guinea	FJ427074	MT018108
Epicoccum mackenziei	MFLUCC 16-0335 T	Ononis spinose	Italy	KX698039	KX698035
Epicoccum mezzettii	CBS 173.38 T	Populus pulp	Italy	MN973496	MT018095
Epicoccum nigrum	CBS 173.73 T	Dactylis glomerata	USA	FJ426996	KT389632
Epicoccum ovisporum	CBS 180.80 T	Zea mays	South Africa	FJ427068	LT623252
Epicoccum phragmospora	CGMCC 3.19339 T	Saccharum officinarum	China	MN215619	MN255460
Epicoccum pimprinum	CBS 246.60 T	Soil	India	FJ427049	MT018100
Epicoccum plurivorum	CBS 558.81 T	Setaria sp.	New Zealand	GU237888	KT389634
Epicoccum pneumoniae	UTHSC DI16-257 T	Homo sapiens	USA	LT592927	LT593065
Epicoccum poaceicola	MFLUCC 15-0448 T	Poaceae	Thailand	KX965727	KX898365
Epicoccum poae	CGMCC 3.18363 T	Poa annua	USA	KY742113	KY742182
Epicoccum polychromum	CBS 141502 T	Paspalum dilinateum	France	MN973506	MT018109
Epicoccum proteae	CBS 114179 T	Protea compacta x Protea neriifolia	South Africa, Somerset West	JQ044433	LT623251
Epicoccum pseudokeratinophilum	MFLUCC 18-1593 T	Prunus avium	China	MH827002	MH853659
Epicoccum purpurascens	CBS 128906	Soil	USA	MN973488	MT018083
Epicoccum sorghinum	CBS 179.80	Sorghum bicolor	Puerto Rico	FJ427067	KT389635
Epicoccum tobaicum	CBS 384.36 T	Soil	Indonesia	MN973493	MT018092
Epicoccum variabile	CBS 119733 T	Coffea Arabica	Brazil	MN973501	MT018103
Epicoccum viticis	CGMCC 3.18344 T	Vitex negundo	China	KY742118	KY742186
Neoascochyta desmazieri (outgroup)	CBS 297.69 T	Lolium perenne	Germany, Hohenlieth	KT389508	KT389644
Neodidymelliopsis achlydis	CBS 256.77 T	Achlys triphylla	Canada, British Columbia, Vancouver Island	KT389531	MT018293
Neodidymelliopsis cannabis	CBS 234.37	Cannabis sativa	Unknown	GU237804	KP330403
Neodidymelliopsis farokhinejadii	CBS 142853	Conocarpus erectus	Iran	KY449009	KY464922
Neodidymelliopsis longicolla	CBS 382.96 T	Soil	Israel, En Avdat, Negev desert	KT389532	MT018298
Neodidymelliopsis moricola	MFLUCC 17-1063	Morus alba	Russia	KY684939	KY684943
Neodidymelliopsis negundinis	JZB380011	Acer negundo	Russia	MG564165	MG564166
Neodidymelliopsis polemonii	CBS 109181 T	Polemonium caeruleum	The Netherlands	GU237746	KP330427
Neodidymelliopsis ranunculi	CBS 286.72	Citrus limonium	Italy	MN973612	MT018294
Neodidymelliopsis tillae	CBS 519.95 T	Tilia sp.	Italy	MN973610	MT018287
Neodidymelliopsis tinkyukuku sp. nov.	BRIP 69592 T	Hardenbergia violacea	Australia, SA, Clare	MN5676781	MN604932
Neodidymelliopsis xanthina	CBS 383.68 T	Delphinium sp.	The Netherlands, Baarn	GU237855	KP330431
Nothophoma acaciae	CBS 143404 T	Acacia melanoxylon	Australia	MG386056	MG386144
Nothophoma anigozanthi	CBS 381.91 T	Anigozanthus maugleisii	The Netherlands	GU237852	KT389655
Nothophoma arachidis-hypogaeae	CBS 125.93	Arachis hypogaea	India, Madras	GU237771	KT389656
Nothophoma brennandiae	CBS 145912 T	Soil	The Netherlands	MN823579	MN824604
Nothophoma garlbiwalawarda sp. nov.	BRIP 69580	Senna artemisioides	Australia, SA, Adelaide	MN5676782	MN604933
	BRIP 69586	Senna artemisioides	Australia, SA, Berri	MN5676783	MN604934
Nothophoma garlbiwalawarda sp. nov.	BRIP 69587	Senna artemisioides	Australia, SA, Berri	MN5676784	MN604935
	BRIP 69594	Senna artemisioides	Australia, SA, Kimba	MN5676785	MN604936
	BRIP 69595 T	Senna artemisioides	Australia, SA, Wudinna	MN5676786	MN604937
Nothophoma eucalyptigena	CBS 142535 T	Eucalyptus sp.	Australia	KY979771	KY979852
Nothophoma gossypiicola	CBS 377.67	Gossypium sp.	USA, Texas	GU237845	KT389658
Nothophoma infossa	CBS 123395 T	Fraxinus pennsylvanica	Argentina, Buenos Aires Province, La Plata	FJ427025	KT389659
Nothophoma infuscata	CBS 121931 T	Acacia longifolia	New Zealand	MN973559	MN973559
Nothophoma macrospora	UTHSC DI16-199 T	Homo sapiens	USA, Arizona	LN880536	LT593073
Nothophoma naiawu sp. nov.	BRIP 69583 T	Senna artemisioides	Australia, SA, Blanchetown	MN5676787	MN604938
	BRIP 69582 T	Senna artemisioides	Australia, SA, Blanchetown	MN5676788	MN604939
Nothophoma nullicana	CPC 32330 T	Acacia falciformis	Australia	NR_156665	MG386143
Nothophoma pruni	MFLUCC 18-1600	Prunus avium	China	MH827005	MH853662
Nothophoma quercina	CBS 633.92	Microsphaera alphitoides from Quercus sp.	Ukraine	GU237900	KT389657
Nothophoma variabilis	UTHSC DI16-285 T	Homo sapiens	USA	LT592939	LT593078

¹BRIP, Queensland Plant Pathology Herbarium, Brisbane, QLD, Australia; CBS, Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; CGMCC, China General Microbiological Culture Collection, Beijing, China; MFLUCC, Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; UTHSC, Fungus Testing Laboratory at the University of Texas Health Science Center, San Antonio, Texas, USA. ²NSW, New South Wales; SA, South Australia; VIC, Victoria; WA, Western Australia. ³ITS, internal transcribed spacer region; rpb2, RNA polymerase II second subunit. ^T ex-type strain.

Maximum likelihood (ML) analysis was run using the RAxML v. 7.2.8 (Stamatakis and Alachiotis 2010) plug-in in Geneious v. 11.1.5 starting from a random tree topology. The nucleotide substitution model used was general time-reversible (GTR) with a gamma-distributed rate variation. The Bayesian analysis was performed using the MrBayes v.3.2.1 (Ronquist and Huelsenbeck 2003) plug-in in Geneious v. 11.1.5. To remove the need for a priori model testing, the Markov chain Monte Carlo (MCMC) analysis was set to sample across the entire GTR model space with a gamma-distributed rate variation across the nucleotide sites. Ten million random trees were generated using the MCMC procedure with four chains. The sample frequency was set at 2000 and the temperature of the heated chain was 0.1. “Burn-in” was set at 25%, after which the log-likelihood values were stationary.

Morphology

Fungal isolates were cultured on four media types; PDA, oatmeal agar (OA), malt extract agar (MEA) (Boerema et al. 2004; Chen et al. 2015a), and carnation leaf agar (CLA). The colonies were measured at 7 d, and morphology examined after 12–14 d incubation in the same light and temperature conditions described above. Images of the colonies were captured by an Epson Perfection V700 scanner at a 300 dpi resolution. Colony colour was determined on surface and reverse using the colour charts of Rayner (1970). Isolates were characterised microscopically from the PDA plates. Lactic acid (100 % v/v) was used as the mounting fluid. Specimens were examined using a Leica DM5500B compound microscope with a Leica DFC 500 camera fitted to capture images under Nomarski differential interference contrast illumination. Micromorphological measurements and descriptions of pycnidia, pycnidial wall cells and conidia were taken from up to 20 samples, and septation and colour recorded. Images of pycnidia were taken from CLA plates using a Leica M165C stereo microscope and Lecia DFC 500 camera. The NaOH spot test on MEA culture plates helped distinguish taxa (Boerema et al. 2004).

Results

From 124 samples of legumes collected at 22 locations, 194 isolates were obtained of which 54 isolates were identified as Didymellaceae by ITS sequences. Of these, 36 isolates were further sequenced (rpb2 locus). Duplicate isolates were excluded where they were from the same host species, which left 18 isolates for multilocus sequence analysis and inclusion in the phylogenetic analysis.

Phylogeny

A multilocus sequence analysis based on the ITS region and partial region of the rpb2 gene was used to infer the relationship of the 18 isolates and recognised species in Didymellaceae (Table 1). The resulting concatenated aligned dataset comprised 124 ingroup isolates from 111 taxa, and consisted of 1,090 characters (493 for ITS, and 596 for rpb2, including alignment gaps). The ML tree based on the combined dataset is presented, with bootstrap support values (BS) greater than 70% and Bayesian posterior probabilities (PP) greater than 0.95 indicating four well-supported clades, and limited support for Nothophoma (Fig. 1). The ITS phylogeny, using either ML or Bayesian analysis, provided poor resolution at the genus and species level (data not shown). The phylogenetic tree based on the concatenated alignment of ITS and rpb2 indicates the placement of the 18 isolates (Fig. 1), five of which represent novel species (Figs 2–6).

Figure 1.

Phylogenetic tree based on maximum likelihood analysis of the combined multilocus (rpb2 and ITS) alignment. RAxML bootstrap values (bs) greater than 70 % and Bayesian posterior probabilities (pp) greater than 0.95 are given at the nodes (bs/pp). Genera are delimited in coloured boxes, with the genus name indicated to the right. Isolates identified in this study are in bold, and novel taxa are in red bold. Ex-type isolates are marked with ^T. The outgroup is Neoascochyta desmazieri (CBS 297.69).

Figure 2.

Epicoccum djirangnandiri: a leaf lesions on Swainsona galegifoliab 14-d old colonies on PDA, MEA, OA (left, top to bottom) and lower surface (right) c upper surface d pycnidia on CLAe conidia. Scale bars: 200 µm (d); 7 µm (e).

Figure 6.

Nothophoma ngayawang: a leaf and pod lesions on Senna artemisioidesb 14-d old colonies, top to bottom on PDA, MEA, OA (left, top to bottom) and lower surface (right) c upper surface d pycnidia e pycnidial wall f conidia. Scale bars: 250 µm (d); 8 µm (e); 3 µm (f).

We identified three new host-pathogen associations, and one new record for Australia Didymella pinodes (strains BRIP 69581, 69593, and 69596) was isolated from native S. artemisioides from three locations in South Australia separated by over 400 km. Didymella pinodes (strain BRIP 69578) was also isolated from naturalised Vicia cracca (tufted vetch) in New South Wales from an area which did not cultivate P. sativum. Didymella lethalis (strain BRIP 69584) was isolated from the naturalised Lathyrus tingitanus (tangier pea) from a recreational walking area within an urban environment. Didymella prosopidis (strain BRIP 69579) was isolated from Gastrolobium celsianum from the botanic gardens in the capital city of South Australia, Adelaide.

Taxonomy

Multilocus sequence analysis and morphological comparisons classified nine fungal isolates from legumes in southern Australia into five novel species from three Didymellaceae genera. The novel species are described and illustrated in Figs 2–6. Nomenclatural novelties are registered in MycoBank.

The species epithets were derived from Indigenous Australian Peoples’ language groups to provide a uniquely Australian theme. Permission to use words from the local language of the area in which the fungi were collected was granted by elders or community representatives.

Epicoccum djirangnandiri

E.C. Keirnan, M.H. Laurence, R.G. Shivas & Y.P. Tan sp. nov.

215FEA5D-B1DB-576B-85B6-6BF8A79E7087

833689 Fig. 2

Type.

Australia, New South Wales, Mount Annan, Swainsona galegifolia, 19 Jan. 2017, E.C. Keirnan (holotype BRIP 69585, includes culture ex-type).

Description.

Colonies on OA, 76–80 mm diam. after 7 d, covered in dense aerial mycelium, variable shades of grey, pale cinnamon towards centre; reverse dark vinaceous; on MEA, 70–72 mm after 7 d, margin entire, covered in low dense aerial mycelium, pale mouse grey with lighter patches; reverse olivaceous with radiating spokes; on PDA, 73–80 mm after 7 d, margin entire, mycelia felty, mouse grey becoming vinaceous buff towards centre; reverse fuscous black. NaOH spot test: negative. Conidiomata on CLA, pycnidial, globose 100–200 μm diam., pale brown becoming black, solitary, glabrous, non-papillate; pycnidial wall composed of textura globulosa, pale brown, cells 5–15 μm diam. Conidiogenous cells phialidic, cylindral, thin-walled, hyaline, rounded ends. Conidia aseptate, 5–7 × 2–3 μm.

Etymology.

From the language of the Indigenous Australian Dharawal people, meaning leaf spot. The Dharawal people are from the western Sydney region in New South Wales, which includes Mount Annan, where the holotype was collected.

Notes.

Epicoccum djirangnandiri is phylogenetically close to E. pneumoniae ex-type strain UTHSC DI16-257 (Fig. 1) and is distinguished in rpb2 sequences with 99% identity. Morphological comparisons could not be made as E. pneumoniae was sterile in culture (Valenzuela-Lopez et al. 2018). Epicoccum djirangnandiri is only known from one specimen on Swainsona galegifolia.

Neodidymelliopsis tinkyukuku

E.C. Keirnan, M.H. Laurence, R.G. Shivas & Y.P. Tan sp. nov.

99FBBB09-AB5C-588E-9815-F1D246805CCA

833692

Fig. 3

Figure 3.

Neodidymelliopsis tinkyukuku: a leaf lesions on Hardenbergia violaceab 12-d old colonies top to bottom on PDA, MEA, OA (left, top to bottom) and lower surface (right) c upper surface d pycnidia on CLAe pycnidia f pycnidial wall g conidia. Scale bars: 300 µm (d, e); 10 µm (f); 7 µm (g).

Type.

Australia, South Australia, Clare, Hardenbergia violacea, 17 Sep. 2017, E.C. Keirnan (holotype BRIP 69592, includes culture ex-type).

Description.

Colonies on OA, 26–28 mm diam. after 7 d, dense low aerial mycelium, buff with numerous grey patches, darker with abundant pycnidia at centre; reverse buff to rosy buff with darker concentric rings towards centre; on MEA, 28–30 mm after 7 d, margin entire, dense low aerial mycelium, vinaceous buff paler at margin; reverse rosy buff to buff at margin with abundant scattered pycnidia; on PDA, 35–38 mm after 7 d, margin entire, dense low aerial mycelium, pale mouse grey lighter at margin; reverse cinnamon with concentric dark rings, darker at centre. NaOH spot test: light yellow. Conidiomata on CLA pycnidial, globose to ampulliform, 250–350 μm diam., brown becoming black, solitary, abundant in centre of colony, zonate, glabrous, non-papillate; ostiole c. 25 μm diam.; pycnidial wall composed of textura angularus, pale brown, cells 5–8 μm diam. Conidiogenous cells phialidic, cylindrical, thin-walled, hyaline. Conidia occasionally septate, 6–9 × 2–3 μm, cylindrical, hyaline, thin-walled.

Etymology.

From the language of the Indigenous Australian Kaurna people, meaning leaf disease. The Kaurna people are from the Adelaide plains region, which includes Clare, the locality where the holotype was collected.

Notes.

Neodidymelliopsis tinkyukuku (strain BRIP 69592) is sister to a clade that includes N. farokhinejadii (strain CBS 142853), N. longicolla (ex-type strain CBS 382.96) and N. ranunculi (strain CBS 286.72) (Fig. 1). Neodidymelliopsis conidial dimensions are distinct from N. farokhinejadii (4.6–7.5 × 2.4–3.9 μm), N. longicolla (12–15 × 4–7 μm), and N. ranunculi (3–5 × 7.5–10 μm). Neodidymelliopsis tinkyukuku can be easily distinguished from these three species by DNA sequences of the rpb2 locus.

Nothophoma garlbiwalawarda

E.C. Keirnan, M.H. Laurence, R.G. Shivas & Y.P. Tan sp. nov.

1CA11AC0-D737-5DA2-9F63-DDFFEA13D2AA

833693

Fig. 4

Figure 4.

Nothophoma garlbiwalawarda: a pin-prick leaf spots on Senna artemisioides from Wudinna SAb 12-d old colonies top to bottom on PDA, MEA, OA (left, top to bottom) and lower surface (right) c upper surface d pycnidia on CLAe pycnidia and pycnidial ooze on OAf pycnidia on PDAg conidia. Scale bars: 300 µm (d, e, f); 7 µm (g).

Type.

Australia, South Australia, Wudinna, Senna artemisioides, 19 Aug. 2017, E.C. Keirnan (holotype BRIP 69595, includes culture ex-type).

Description.

Colonies on OA, 27–30 mm diam. after 7 d, flat with scant aerial mycelia with a few zonate rings, vinaceous to dark vinaceous; vinaceous to dark vinaceous; on MEA, 23–25 mm after 7 d, margin entire, flat, scant aerial mycelium towards centre, amber with abundant pycnidia; reverse amber darker towards centre; on PDA, 28–30 mm after 7 d, margin irregular, flat with aerial mycelia tufted in centre, dark with abundant pycnidia in concentric rings, buff at margin; reverse dark becoming buff at margin. NaOH spot test: reddish. Conidiomata pycnidial, globose to subglobose, 130–320 μm diam., pale brown, scattered, abundant, zonate, glabrous, non-papillate; ostiole c. 25 μm diam.; pycnidial wall composed of textura angularus, pale to medium brown, cells 5–12 μm diam. Conidiogenous cells phialidic, cylindrical, thin-walled, hyaline 5–12 × 2–4 μm long, narrower at the apex. Conidia aseptate, 5–7.0 × 2.0–3.0 μm, parallel to narrowly ellipsoidal, hyaline, wall c. 0.5 μm.

Etymology.

From the native language of the Indigenous Australian Barngarla people, meaning leaf-fun-guy. The Barngarla people are from the Eyre Peninsula region, which includes Wudinna, the locality where the holotype was collected.

Additional material examined.

Australia, South Australia, Adelaide, Senna artemisioides, 26 Oct. 2016, E.C. Keirnan (BRIP 69580); Berri, Senna artemisioides, 01 Jul. 2017, E.C. Keirnan (BRIP 69586); ibid, 01 Jul. 2017, E.C. Keirnan (BRIP 69587); Kimba, Senna artemisioides, 17 Sep. 2017, E.C. Keirnan (BRIP 69594).

Notes.

Nothophoma garlbiwalawarda is phylogenetically closest to No. anigozanthi and two novel species (see below for notes) (Fig. 2). Nothophoma garlbiwalawarda is distinguished from No. anigozanthi by its larger conidia (cf. 3.5–5 × 1.5–2.5 μm), rpb2 sequence (93% identity), and its reaction to NaOH spot test on MEA (dull green then black).

Nothophoma naiawu

E.C. Keirnan, M.H. Laurence, R.G. Shivas & Y.P. Tan sp. nov.

4045D173-B744-5746-9924-46212C012F80

833694

Fig. 5

Figure 5.

Nothophoma naiawu: a pin-prick leaf spots on Senna artemisioidesb 14-d old colonies top to bottom on PDA, MEA, OA (left, top to bottom) and lower surface (right) c upper surface d pycnidia on CLAe pycnidia f conidia. Scale bars: 300 µm (d, e); 10 µm (f).

Type.

Australia, South Australia, Blanchetown, from Senna artemisioides, 22 Oct. 2016, E.C. Keirnan, holotype BRIP 69583 (includes culture ex-type).

Description.

Colonies on OA, 21–25 mm diam. after 7 d, flat with scant aerial mycelia, rosy vinaceous, dark at centre; reverse rosy buff, dark at centre, with a few dark radiating fissures; on MEA, 27–30 mm after 7 d, margin entire, flat, with sparse aerial mycelium towards centre rosy vinaceous; reverse peach, darker at centre; on PDA, 27–30 mm after 7 d, margin entire, flat felty, rosy buff; reverse peach, dark at centre. NaOH spot test: slightly yellow. Conidiomata pycnidial, globose to subglobose, 200–300 μm diam., pale brown becoming black, semi-immersed, confluent on MEA, glabrous, non-papillate; ostiole c. 25 μm diam.; pycnidial wall composed of textura globulosa, pale brown, cells 5–8 μm diam.. Conidiogenous cells phialidic, cylindrical, very thin-walled, hyaline. Conidia aseptate or 1-septate, 8–12 × 4–6 μm, cylindrical to narrow ellipsoidal, pale yellow.

Etymology.

A variation of the Indigenous Australian Ngayawang people’s language group, who lived in the Murray River region of South Australia, which includes Blanchetown, the locality where this specimen was collected.

Notes.

Nothophoma naiawu is phylogenetically close to No. eucalyptigena and No. infuscata (Fig. 2). Nothophoma naiawu is easily distinguished from No. eucalyptigena and No. infuscata by the ITS region (98 % identity to both) and the rpb2 locus (95%, and 94% identity, respectively). Nothophoma infuscata produce a pale red discolouration in response to NaOH spot test on MEA media, which is distinct from the slightly yellow response by No. naiawu.

Nothophoma ngayawang

E.C. Keirnan, M.H. Laurence, R.G. Shivas & Y.P. Tan sp. nov.

A9352891-7546-5110-9BB1-05B9446D1859

833695

Fig. 6

Type.

Australia, South Australia, Blanchetown, Senna artemisioides, 22 Oct. 2016, E.C. Keirnan, holotype BRIP 69582 (includes culture ex-type).

Description.

Colonies on OA, 18–20 mm diam. after 7 d, covered by scant tufted aerial mycelia at centre becoming abundant and floccose towards margin, rosy buff becoming darker towards centre; reverse salmon with centre and margins pale isabelline; on MEA, 15–20 mm after 7 d, margin irregular, felty buff becoming white towards the margin; reverse pale rosy buff, darker at centre becoming paler near margin; on PDA, 18–21 mm after 7 d, margin regular, aerial mycelia tufted in centre becoming floccose toward the margin, white to pale rosy buff; reverse pale rosy buff with few scattered vinaceous spots. NaOH spot test: slightly yellow. Conidiomata pycnidial, globose to subglobose, 200–300 μm diam., pale brown becoming black, solitary, abundant in centre of colony, glabrous, non-papillate; ostiole c. 25 μm diam.; pycnidial wall composed of textura globulosa, pale brown, cells 5–8 μm diam. Conidiogenous cells phialidic, cylindrical, thin-walled, hyaline. Conidia aseptate, 2.5–4.0 × 1.0–2.0 μm, cylindrical to narrow ellipsoidal, hyaline, thin-walled.

Etymology.

Named after the Indigenous Australian Ngayawang people’s language group, who existed in the Murray River region of South Australia, which includes Blanchetown, the locality where this specimen was collected.

Notes.

Nothophoma ngayawang is phylogenetically close to No. anigozanthi ex-type strain CBS 381.91 (Fig. 2). Nothophoma ngayawang is distinguished from No. variabilis by the ITS region (98 % identity) and the rpb2 locus (93% identity). The NaOH spot test of No. variabilis was negative on MEA, which is distinguished from the slightly yellow reaction of No. ngayawang.

Discussion

Our investigations did not identify A. koolunga from native Australian legumes. In fact, the incidence was low in that only one isolate (BRIP 69590) was collected from P. sativum in South Australia. It is difficult to make an association between the low incidence of A. koolunga on P. sativum and the absence of A. koolunga on other legumes. While the current evidence suggests that A. koolunga is unlikely to have originated from Australian native legumes, additional field surveys may be required to investigate the possible source of A. koolunga.

Our investigations instead uncovered five novel Didymellaceae species not yet known to science. Epicoccum djirangnandiri on S. galegifolia was collected from the botanic garden in New South Wales, where the host is endemic. Neodidymelliopsis tinkyukuku on H. violacea was collected from a public garden in South Australia. Growing in the same garden is V. sativa from which D. pinodes (strain BRIP 69578), a known Ascochyta blight pathogen, was isolated. Hardenbergia violacea has a wide distribution in southern and eastern Australia. These three native Australian legume species were found in a cultivated environment rather than in a natural environment. Further studies are warranted to understand how widespread these fungal species may be in cultivated or natural environments, and if they are host specific.

Leaf spots were commonly seen on the native legume S. artemisioides throughout the regions sampled in South Australia. Three novel Nothophoma species were isolated from S. artemisioides. Nothophoma garlbiwalawarda was collected from five locations across South Australia, separated by over 400 km, in field pea and non-field pea growing regions. Nothophoma naiawu and No. ngayawang were collected from the South Australian Murray River region on the roadside of a main highway. The leaf spot symptoms for the three Nothophoma species were similar (small pin-prick lesions), with some larger spots on the seed pods caused by No. ngayawang.

Our investigations also identified new host-pathogen associations, namely D. pinodes on S. artemisioides and V. cracca, and D. lethalis on L. tingitanus. These hosts could be a reservoir of Ascochyta blight inoculum if found growing adjacent to field pea crops. The discovery of an alternative host has implications for disease epidemiology and management. The symptoms of D. pinodes on S. artemisioides are indistinguishable from the pin-prick leaf spot symptoms caused by the three Nothophoma species described in this study. Didymella pinodes was isolated from five locations. Four of these locations also yielded a novel Nothophoma species. Didymella prosopidis was isolated from the Australian native G. celsianum, a species first described as associated with stem disease of Prosopis sp. (also a member of the Fabaceae family) in South Africa (Crous et al. 2013). This is the first report of D. prosopidis outside of South Africa.

At the outset, our study sought to identify if any A. koolunga could be isolated from Australian native legumes causing leaf spot disease. This study uncovered five novel isolates in the Didymellaceae from Australian native legumes, and identified three new legume host-pathogen associations for Australia. Ascochyta koolunga was not isolated from hosts other than field pea, which might be an artefact of the low incidence of the fungus during the collection period. Further investigations using a longitudinal systematic survey are needed to identify any native hosts of A. koolunga and to further investigate the diversity and prevalence of Didymellaceae species on Australian native, pasture and naturalised legumes, to classify novel isolates and to identify new Australian hosts for known species.

Citation

Keirnan EC, Tan YP, Laurence MH, Mertin AA, Liew ECY, Summerell BA, Shivas RG (2021) Cryptic diversity found in Didymellaceae from Australian native legumes. MycoKeys 78: 1–20. https://doi.org/10.3897/mycokeys.78.60063