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Research Article
Cryptic diversity found in Didymellaceae from Australian native legumes
expand article infoElizabeth C. Keirnan‡, Yu Pei Tan‡, Matthew H. Laurence§, Allison A. Mertin§, Edward C. Y. Liew§, Brett A. Summerell§, Roger G. Shivas|
‡ The University of Adelaide, Adelaide, Australia
§ Royal Botanic Gardens and Domain Trust, Sydney, Australia
| Department of Agriculture and Fisheries, Ecosciences Precinct, Dutton Park, Australia
¶ University of Southern Queensland, Toowoomba, Australia

Corresponding author: Elizabeth C. Keirnan ( elizabeth.keirnan@adelaide.edu.au )

Academic editor: Imke Schmitt
© 2021 Elizabeth C. Keirnan, Yu Pei Tan, Matthew H. Laurence, Allison A. Mertin, Edward C. Y. Liew, Brett A. Summerell, Roger G. Shivas.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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
Open Access

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 garlbiwalawarda sp. nov., Nothophoma naiawu sp. nov. and Nothophoma ngayawang sp. nov., were isolated from Senna artemisioides. The other species described here are Epicoccum djirangnandiri sp. nov. from Swainsona galegifolia and Neodidymelliopsis tinkyukuku sp. 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.

Keywords

Alternative host, multilocus phylogeny, pathogen reservoir

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.

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).

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.

Table 1.
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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

1 BRIP, 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. 2 NSW, New South Wales; SA, South Australia; VIC, Victoria; WA, Western Australia. 3 ITS, internal transcribed spacer region; rpb2, RNA polymerase II second subunit. T ex-type strain.

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.

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).

Figure 2. 

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

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.

MycoBank No: 833692
Fig. 3

Type

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

Figure 3. 

Neodidymelliopsis tinkyukuku: a leaf lesions on Hardenbergia violacea b 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 CLA e pycnidia f pycnidial wall g conidia. Scale bars: 300 µm (d, e); 10 µm (f); 7 µm (g).

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.

MycoBank No: 833693
Fig. 4

Type

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

Figure 4. 

Nothophoma garlbiwalawarda: a pin-prick leaf spots on Senna artemisioides from Wudinna SA b 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 CLA e pycnidia and pycnidial ooze on OA f pycnidia on PDA g conidia. Scale bars: 300 µm (d, e, f); 7 µm (g).

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.

MycoBank No: 833694
Fig. 5

Type

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

Figure 5. 

Nothophoma naiawu: a pin-prick leaf spots on Senna artemisioides b 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 CLA e pycnidia f conidia. Scale bars: 300 µm (d, e); 10 µm (f).

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.

MycoBank No: 833695
Fig. 6

Type

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

Figure 6. 

Nothophoma ngayawang: a leaf and pod lesions on Senna artemisioides b 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).

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.

Acknowledgements

This research formed part of a Master of Philosophy by the first author. The authors thank the University of Adelaide and the Royal Botanic Gardens and Domain Trust, Sydney, for financial and facilities support. We acknowledge and are grateful to Professor Eileen Scott (University of Adelaide) and Associate Professor Jenny Davidson (South Australian Research and Development Institute and University of Adelaide) for providing access to facilities and resources and for general guidance. Kaylene Bransgrove (Department of Agriculture and Fisheries) is thanked for assistance with specimen curation.

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