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 ). 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. 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. 2010;Chen et al. 2015a, de Gruyter 2012Hou 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 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 2012Chen 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.

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 MgCl 2 (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.
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). 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.

Epicoccum djirangnandiri
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.
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.
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.

Nothophoma ngayawang
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 extype 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.