Morphological and phylogenetic analyses reveal three new species of Diaporthe from Yunnan, China

Shengting Huang; Jiwen Xia; Xiuguo Zhang; WenXiu Sun

doi:10.3897/mycokeys.78.60878

Research Article

Morphological and phylogenetic analyses reveal three new species of Diaporthe from Yunnan, China

Shengting Huang^‡, Jiwen Xia^§, Xiuguo Zhang^§, WenXiu Sun^‡

‡ Yangtze University, Jingzhou, China

§ Shandong Agricultural University, Taian, China

Corresponding author: WenXiu Sun ( wenxiusun@163.com )

Academic editor: Nattawut Boonyuen

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: Huang S, Xia J, Zhang X, Sun W (2021) Morphological and phylogenetic analyses reveal three new species of Diaporthe from Yunnan, China. MycoKeys 78: 49-77. https://doi.org/10.3897/mycokeys.78.60878

Abstract

Species of Diaporthe have often been reported as plant pathogens, endophytes or saprobes, commonly isolated from a wide range of plant hosts. Sixteen strains isolated from species of ten host genera in Yunnan Province, China, represented three new species of Diaporthe, D. chrysalidocarpi, D. machili and D. pometiae as well as five known species D. arecae, D. hongkongensis, D. middletonii, D. osmanthi and D. pandanicola. Morphological comparisons with known species and DNA-based phylogenies based on the analysis of a multigene (ITS, TUB, TEF, CAL and HIS) dataset support the establishment of the new species. This study reveals that a high species diversity of Diaporthe with wide host ranges occur in tropical rainforest in Yunnan Province, China.

Keywords

Diaporthaceae, Diaporthales, phylogeny, taxonomy, three taxa new to science

Introduction

The genus Diaporthe (Diaporthaceae Diaporthales) with asexual morphs previously known as Phomopsis spp. is based on the type species Diaporthe eres Nitschke (1870) from Ulmus sp. in Germany. Rossman et al. (2015) proposed to use the name Diaporthe over Phomopsis in the context of the one fungus – one name initiative, because it was described first, is encountered commonly in literature and includes the majority of known species. The sexual morph of Diaporthe is characterised by immersed ascomata and an erumpent pseudostroma with elongated perithecial necks; asci are unitunicate, clavate to cylindrical; and ascospores are fusoid, ellipsoid to cylindrical, hyaline, biseriate to uniseriate in the ascus, sometimes with appendages (Udayanga et al. 2011; Senanayake et al. 2017, 2018). The asexual morph is characterised by ostiolate pycnidia with cylindrical phialides often producing three types of hyaline, aseptate conidia called α-conidia, β-conidia and γ-conidia (Udayanga et al. 2011; Gomes et al. 2013). The α-conidia and β-conidia are produced frequently, but the γ-conidia are rarely observed (Gomes et al. 2013; Guarnaccia and Crous 2017; Guo et al. 2020).

Currently, more than 1100 epithets of Diaporthe are listed in Index Fungorum (http://www.indexfungorum.org/; accessed 1 Nov. 2020), but only one-fifth of these taxa have been well-studied with ex-type cultures and supplementary DNA barcodes (Guo et al. 2020; Yang et al. 2020; Zapata et al. 2020). Species of Diaporthe are widely distributed and have a broad range of hosts including economically significant agricultural crops and ornamental plants such as species of Camellia, Castanea, Citrus, Glycine, Helianthus, Juglans, Persea, Pyrus, Vaccinium, Vitis and many more (van Rensburg et al. 2006; Santos and Phillips 2009; Crous et al. 2011a, b, 2016; Santos et al. 2011; Thompson et al. 2011; Grasso et al. 2012; Huang et al. 2013; Lombard et al. 2014; Gao et al. 2015, 2016, 2017; Udayanga et al. 2012, 2015; Guarnaccia et al. 2016; Dissanayake et al. 2017; Guarnaccia and Crous 2017; Fan et al. 2018; Senanayake et al. 2018; Guo et al. 2020). Diaporthe species have been reported as destructive plant pathogens, harmless endophytes or saprobes (Murali et al. 2006; Udayanga et al. 2012; Gomes et al. 2013; Ménard et al. 2014; Guarnaccia et al. 2016; Torres et al. 2016; Senanayake et al. 2018). However, the biology and lifestyle of some of these fungi remain unclear (Vilka and Volkova 2015).

In the past, methods of species identification of Diaporthe had previously been based only on host as well as morphological characters such as the size and shape of ascomata and conidiomata. Nowadays, molecular phylogenetic studies demonstrate that determining species boundaries only by morphological characters is not possible due to lack of host specificity and their variability under changing environmental conditions (Gomes et al. 2013). Phylogenetic analysis using a five-locus dataset (ITS-TUB-TEF-CAL-HIS) has been determined to be the optimal combination to identify species of Diaporthe species, as revealed by Santos et al. (2017). Many Diaporthe species are described based on a polyphasic approach together with morphological characterisation (Rehner and Uecker 1994; Udayanga et al. 2011; Gao et al. 2017; Guarnaccia and Crous 2017; Yang et al. 2018a, 2020; Crous et al. 2020; Dayarathne et al. 2020; Guo et al. 2020; Hyde et al. 2020; Li et al. 2020; Zapata et al. 2020).

The aim of this study was to explore the diversity of Diaporthe species from symptomatic leaves of plants in Yunnan Province. We present three novel species and five known species of Diaporthe, collected from species belonging to ten host genera, based on morphological characters and phylogenetic analysis.

Materials and methods

Isolation and morphological studies

Leaves of samples were collected in Yunnan Province, China. Isolations from surface sterilized leaf tissues were conducted following the protocol of Gao et al. (2014). Tissue fragments (5 × 5 mm) were taken from the margin of leaf lesions and surface-sterilized by immersing them in 75% ethanol solution for 1 min, 5% sodium hypochlorite solution for 30 s, and then rinsing in sterile distilled water for 1 min. The pieces were dried with sterilized paper towels and placed on potato dextrose agar (PDA) (Cai et al. 2009). PDA plates (90 mm) were incubated in an incubator at 25 °C for 2–4 days, and hyphae were picked out of the periphery of the colonies and inoculated onto new PDA plates.

Following 2–3 weeks of incubation, photographs of colonies were taken at 7 days and 15 days using a Powershot G7X mark II digital camera. Colour notations was done using the colour charts of Rayner (1970). Micromorphological characters were observed using an Olympus SZX10 stereomicroscope and Olympus BX53 microscope, both fitted with Olympus DP80 high definition colour digital cameras to document fungal structures. All fungal strains were stored in 10% sterilized glycerin at 4 °C for further studies. Voucher and type specimens were deposited in the Herbarium of Plant Pathology, Shandong Agricultural University (HSAUP). Living cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org).

DNA extraction and amplification

Genomic DNA was extracted from fungal mycelium on PDA, using a modified cetyltrimethylammonium bromide (CTAB) protocol as described in Guo et al. (2000). The internal transcribed spacer regions with intervening 5.8S nrRNA gene (ITS), part of the beta-tubulin gene region (TUB), partial translation elongation factor 1-alpha (TEF), histone H3 (HIS) and calmodulin (CAL) genes were amplified and sequenced by using primers pairs ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), EF1-728F/EF1-986R (Carbone and Kohn 1999), CAL-228F/CAL-737R (Carbone and Kohn 1999) and CYLH3F/H3-1b (Glass and Donaldson 1995; Crous et al. 2004), respectively.

PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions were performed in a 25 μL reaction volume, which contained 12.5 μL Green Taq Mix (Vazyme, Nanjing, China), 1 μL of each forward and reverse primer (10 μM) (Biosune, Shanghai, China), and 1 μL template genomic DNA in amplifier, and were adjusted with distilled deionized water to a total volume of 25 μL.

PCR parameters were as follows: 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at a suitable temperature for 30 s, extension at 72 °C for 1 min and a final elongation step at 72 °C for 10 min. Annealing temperature for each gene were 55 °C for ITS, 60 °C for TUB, 52 °C for TEF, 54 °C for CAL and 57 °C for HIS. The PCR products were visualised on 1% agarose electrophoresis gel. Sequencing was done bi-directionally, conducted by the Biosune Company Limited (Shanghai, China). Consensus sequences were obtained using MEGA 7.0 (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank (Table 1).

Table 1.

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Species and Genbank accession numbers of DNA sequences used in this study. New sequences in bold.

Species	Voucher	Host/Substrare	GeneBank accession number					Reference
Species	Voucher	Host/Substrare	ITS	TUB	TEF	CAL	HIS	Reference
Diaporthe acuta	PSCG 046	Pyrus pyrifolia	MK626958	MK691224	MK654803	MK691124	MK726162	Guo et al. 2020
Diaporthe acuta	PSCG 047*	Pyrus pyrifolia	MK626957	MK691225	MK654802	MK691125	MK726161	Guo et al. 2020
D. acutispora	LC6160	Camellia sasanqua	KX986763	KX999194	KX999154	KX999273	KX999234	Gao et al. 2017
D. acutispora	LC6161	Coffea sp.	KX986764	KX999195	KX999155	KX999274	KX999235	Gao et al. 2017
D. amaranthophila	MAFF 246900	Amaranthus tricolor	LC459575	LC459579	LC459577	LC459583	LC459581	Rossman et al. 2015
D. amaranthophila	MAFF 246901	Amaranthus tricolor	LC459576	LC459580	LC459578	LC459584	LC459582	Rossman et al. 2015
D. angelicae	CBS 111592*	Heracleum sphondylium	KC343027	KC343995	KC343753	KC343269	KC343511	Gomes et al. 2013
D. anhuiensis	CNUCC 201901*	Cunninghamia lanceolata	MN219718	MN227008	MN224668	MN224549	MN224556	Zhou and Hou 2019
D. anhuiensis	CNUCC 201902	Cunninghamia lanceolata	MN219727	MN227009	MN224669	MN224550	MN224557	Zhou and Hou 2019
D. arctii	DP0482	Arctium sp.	KJ590736	KJ610891	KJ590776	KJ612133	KJ659218	Udayanga et al. 2015
D. arecae	CBS 161.64*	Areca catechu	KC343032	KC344000	KC343758	KC343274	KC343516	Gomes et al. 2013
	CBS 535.75	Citrus sp.	KC343033	KC344001	KC343759	KC343275	KC343517	Gomes et al. 2013
	SAUCC194.18	*Persea americana*	MT822546	MT855743	MT855860	MT855631	MT855515	This study
D. arengae	CBS 114979*	Arenga engleri	KC343034	KC344002	KC343760	KC343276	KC343518	Gomes et al. 2013
D. aseana	MFLUCC 12-0299a*	On dead leaves	KT459414	KT459432	KT459448	KT459464	–	Dissanayake et al. 2017
D. beilharziae	BRIP 54792*	Indigofera australis	JX862529	KF170921	JX862535	–	–	Tan et al. 2013
D. biconispora	ZJUD 60	Citrus sinensis	KJ490595	KJ490416	KJ490474	–	KJ490537	Huang et al. 2017
	ZJUD 61	Fortunella margarita	KJ490596	KJ490417	KJ490475	–	KJ490538	Huang et al. 2017
	ZJUD 62	Citrus grandis	KJ490597	KJ490418	KJ490476	–	KJ490539	Huang et al. 2017
D. brasiliensis	CBS 133183*	Aspidosperma tomentosus	KC343042	KC344010	KC343768	KC343284	KC343526	Gomes et al. 2013
D. caatingaensis	URM 7486*	Tacinga inamoena	KY085926	KY115600	KY115603	KY115597	KY115605	Crous et al. 2017
D. camporesii	JZB320143	Urtica dioidca	MN535309	MN561316	MN984254	–	–	Hyde et al. 2020
D. caricae-papayae	NIBM-ABIJP	Carica papaya	MN335224	–	–	–	–	Rossman et al. 2015
D. caryae	CFCC 52563	Carya illinoensis	MH121498	MH121580	MH121540	MH121422	MH121458	Yang et al. 2018
D. caryae	CFCC 52564	Carya illinoensis	MH121499	MH121581	MH121541	MH121423	MH121459	Yang et al. 2018
D. cercidis	CFCC 52565	Cercis chinensis	MH121500	MH121582	MH121542	MH121424	MH121460	Yang et al. 2018
*D. chrysalidocarpi*	SAUCC194.33	*Chrysalidocarpus lutescens*	MT822561	MT855758	MT855874	MT855645	MT855530	This study
*D. chrysalidocarpi*	SAUCC194.35*	*Chrysalidocarpus lutescens*	MT822563	MT855760	MT855876	MT855646	MT855532	This study
D. cichorii	MFLUCC 17-1023*	Cichorium intybus	KY964220	KY964104	KY964176	KY964133	–	Dissanayake et al. 2017
D. compacta	LC3083*	Camellia sinensis	KP267854	KP293434	KP267928	–	KP293508	Gao et al. 2016
D. cucurbitae	CBS 136.25	Cucumis sativus	KC343031	KC343999	KC343757	KC343273	KC343515	Udayanga et al. 2014
D. cuppatea	CBS 117499	Aspalathus linearis	KC343057	KC344025	KC343783	KC343299	KC343541	Udayanga et al. 2012
D. decedens	CBS 109772	Corylus avellana	KC343059	KC344027	KC343785	KC343301	KC343543	Gomes et al. 2013
D. eugeniae	CBS 444.82	Eugenia aromatica	KC343098	KC344066	KC343824	KC343340	KC343582	Gomes et al. 2013
D. fraxini-angustifoliae	BRIP 54781*	Fraxinus angustifolius	JX862528	KF170920	JX862534	–	–	Tan et al. 2013
D. fulvicolor	PSCG 051*	Pyrus pyrifolia	MK626859	MK691236	MK654806	MK691132	MK726163	Guo et al. 2020
D. fulvicolor	PSCG 057	Pyrus pyrifolia	MK626858	MK691233	MK654810	MK691131	MK726164	Guo et al. 2020
D. ganjae	CBS 180.91*	Cannabis sativa	KC343112	KC344080	KC343838	KC343354	KC343596	Gomes et al. 2013
D. guangxiensis	JZBH 320094*	Vitis vinifera	MK335772	MK500168	MK523566	MK736727	–	Manawasinghe et al. 2019
D. gulyae	MF-Ha 17-042*	Helianthus annuus	MK024252	MK033488	MK039420	–	–	Thompson et al. 2011
D. hongkongensis	CBS 115448*	Dichroa febrifuga	KC343119	KC344087	KC343845	KC343361	KC343603	Gomes et al. 2013
	CGMCC 3.17102	Lithocarpus glaber	KF576275	KF576299	KF576250	KF576227	–	Gao et al. 2015
	LC 3478	Camellia sinensis	KP267904	KP293484	KP267978	–	KP293553	Gao et al. 2017
	SAUCC194.81	*Millettia reticulata*	MT822609	MT855806	MT855921	MT855688	MT855577	This study
	SAUCC194.87	*Camellia sinensis*	MT822615	MT855812	MT855927	MT855694	MT855583	This study
D. huangshanensis	CNUCC 201903	Camellia oleifera	MN219729	MN227010	MN224670	–	MN224558	Zhou and Hou 2019
D. huangshanensis	CNUCC 201904	Camellia oleifera	MN219730	MN227011	MN224671	–	MN224559	Zhou and Hou 2019
D. infecunda	CBS 133812*	Schinus terebinthifolius	KC343126	KC344094	KC343852	KC343368	KC343610	Gomes et al. 2013
D. krabiensis	MFLUCC 17-2481*	Bruguiera sp.	MN047101	MN431495	MN433215	–	–	Dayarathne et al. 2020
D. litchiicola	BRIP 54900*	Litchi chinensis	JX862533	KF170925	JX862539	–	–	Tan et al. 2013
D. limonicola	CPC 28200*	Citrus limon	MF418422	MF418582	MF418501	MF418256	MF418342	Guarnaccia and Crous 2017
D. lusitanicae	CBS 123212*	Foeniculum vulgare	KC343136	KC344104	KC343862	KC343378	KC343620	Phillips and Santos 2009
*D. machili*	SAUCC194.69	*Pometia pinnata*	MT822597	MT855794	MT855909	MT855677	MT855565	This study
*D. machili*	SAUCC194.111*	*Machilus pingii*	MT822639	MT855836	MT855951	MT855718	MT855606	This study
D. malorum	CAA752*	Malus domestica	KY435643	KY435671	KY435630	KY435661	KY435651	Santos et al. 2017
D. malorum	CAA740	Malus domestica	KY435642	KY435670	KY435629	KY435660	KY435650	Santos et al. 2017
D. manihotia	CBS 505.76	Manihot utilissima	KC343138	KC344106	KC343864	KC343380	KC343622	Gomes et al. 2013
D. mayteni	CBS 133185*	Maytenus ilicicolia	KC343139	KC344107	KC343865	KC343381	KC343623	Gomes et al. 2013
D. melitensis	CPC 27873*	Citrus limon	MF418424	MF418584	MF418503	MF418258	MF418344	Guarnaccia and Crous 2017
D. middletonii	BRIP 54884e*	Rapistrum rugostrum	KJ197286	KJ197266	KJ197248	–	–	Thompson et al. 2015
	SAUCC194.27	*Litchi chinensis*	MT822555	MT855752	MT855868	MT855639	MT855524	This study
	SAUCC194.45	*Lithocarpus glaber*	MT822573	MT855770	MT855886	MT855654	MT855542	This study
	SAUCC194.46	*Lithocarpus glaber*	MT822574	MT855771	MT855887	MT855655	MT855543	This study
	SAUCC194.48	*Lithocarpus craibianus*	MT822576	MT855773	MT855889	MT855657	MT855545	This study
D. millettiae	GUCC9167*	Millettia reticulata	MK398674	MK502089	MK480609	MK502086	–	Long et al. 2019
D. multigutullata	ZJUD 98*	Citrus grandis	KJ490633	KJ490454	KJ490512	–	KJ490575	Huang et al. 2015
D. musigena	CBS 129519*	Musa sp.	KC343143	KC344111	KC343869	KC343385	KC343627	Crous et al. 2011
D. myracrodruonis	URM7972	Myracrodruon urundeuva	MK205289	MK205291	MK213408	MK205290	–	Silva et al. 2019
D. neoarctii	CBS 109490*	Ambrosia trifida	KC343145	KC344113	KC343871	KC343387	KC343629	Gomes et al. 2013
D. novem	CBS 127270*	Glycine max	KC343156	KC344124	KC343882	KC343398	KC343640	Santos et al. 2011
D. osmanthi	GUCC9165*	Osmanthus fragrans	MK398675	MK502091	MK480610	MK502087	–	Long et al. 2019
D. osmanthi	SAUCC194.21	*Litchi chinensis*	MT822549	MT855746	MT855862	MT855634	MT855518	This study
D. oxe	CBS 133186*	Maytenus ilicifolia	KC343164	KC344132	KC343890	KC343406	KC343648	Gomes et al. 2013
D. oxe	CBS 133187	Maytenus ilicifolia	KC343165	KC344133	KC343891	KC343407	KC343649	Gomes et al. 2013
D. pandanicola	MFLUCC 17-0607	Pandanus sp.	MG646974	MG646930	–	–	–	Tibpromma et al. 2018
D. pandanicola	SAUCC194.82	*Millettia reticulata*	MT822610	MT855807	MT855922	MT855689	MT855578	This study
D. paranensis	CBS 133184*	Maytenus ilicifolia	KC343171	KC344139	KC343897	KC343413	KC343655	Gomes et al. 2013
D. pascoei	BRIP 54847*	Persea americana	JX862532	KF170924	JX862538	–	–	Tan et al. 2013
D. perseae	CBS 151.73	Persea gratissima	KC343173	KC344141	KC343899	KC343415	KC343657	Gomes et al. 2013
D. pescicola	MFLU 16-0105*	Prunus persica	KU557555	KU557579	KU557623	KU557603	–	Dissanayake et al. 2017
D. podocarpi-macrophylli	LC6155*	Podocarpus macrophyllus	KX986774	KX999207	KX999167	KX999278	KX999246	Gao et al. 2017
D. podocarpi-macrophylli	LC6200	Podocarpus macrophyllus	KX986769	KX999201	KX999161	KX999276	KX999240	Gao et al. 2017
*D. pometiae*	SAUCC194.19	*Persea americana*	MT822547	MT855744	MT855861	MT855632	MT855516	This study
	SAUCC194.72*	*Pometia pinnata*	MT822600	MT855797	MT855912	MT855679	MT855568	This study
	SAUCC194.73	*Heliconia metallica*	MT822601	MT855798	MT855913	MT855680	MT855569	This study
D. pseudomangiferae	CBS 101339*	Mangifera indica	KC343181	KC344149	KC343907	KC343423	KC343665	Gomes et al. 2013
D. pseudophoenicicola	CBS 462.69*	Phoenix dactylifera	KC343184	KC344152	KC343910	KC343426	KC343668	Gomes et al. 2013
D. pterocarpicola	MFLUCC 10-0580a*	Pterocarpus indicus	JQ619887	JX275441	JX275403	JX197433	–	Udayanga et al. 2012
D. pterocarpicola	MFLUCC 10-0580b	Pterocarpus indicus	JQ619888	JX275442	JX275404	JX197434	–	Udayanga et al. 2012
D. pyracanthae	CAA487*	Pyracantha coccinea	KY435636	KY435667	KY435626	KY435657	KY435647	Santos et al. 2017
D. racemosae	CPC 26646*	Euclea racemosa	MG600223	MG600227	MG600225	MG600219	MG600221	Marin-Felix et al. 2018
D. raonikayaporum	CBS 133182*	Spondias mombin	KC343188	KC344156	KC343914	KC343430	KC343672	Gomes et al. 2013
D. rossmaniae	CAA 762*	Vaccinium corymbosum	MK792290	MK837914	MK828063	MK883822	MK871432	Hilario et al. 2020
D. sackstonii	BRIP 54669b*	Helianthus annuus	KJ197287	KJ197267	KJ197249	–	–	Thompson et al. 2015
D. salinicola	MFLU 18-0553*	Xylocarpus sp.	MN047098	–	MN077073	–	–	Dayarathne et al. 2020
D. salinicola	MFLU 17-2592	Xylocarpus sp.	MN047099	–	MN077074	–	–	Dayarathne et al. 2020
D. schini	CBS 133181*	Schinus terebinthifolius	KC343191	KC344159	KC343917	KC343433	KC343675	Gomes et al. 2013
D. schoeni	MFLU 15-2609	Schoenus nigricans	KY964229	KY964112	KY964185	KY964141	–	Dissanayake et al. 2017
D. sennae	CFCC 51636*	Senna bicapsularis	KY203724	KY228891	KY228885	KY228875	–	Yang et al. 2017
D. serafiniae	BRIP 55665a*	Helianthus annuus	KJ197274	KJ197254	KJ197236	–	–	Thompson et al. 2015
D. spinosa	PSCG 383*	Pyrus pyrifolia	MK626849	MK691234	MK654811	MK691129	MK726156	Guo et al. 2020
D. stewartii	CBS 193.36*	Cosmos bipinnatus	FJ889448	JX275421	GQ250324	JX197415	–	Santos et al. 2010; Udayanga et al. 2012
D. subordinaria	CBS 101711	Plantago lanceolata	KC343213	KC344181	KC343939	KC343455	KC343697	Gomes et al. 2013
D. subordinaria	CBS 464.90	Plantago lanceolata	KC343214	KC344182	KC343940	KC343456	KC343698	Gomes et al. 2013
D. taoicola	PSGG485	Prunus persica	MK626869	MK691227	MK654812	MK691120	MK726173	Dissanayake et al. 2017
D. tarchonanthi	CPC 37479	Tarchonanthus littoralis	MT223794	–	–	–	–	Crous et al. 2020
D. tectonigena	MFLUCC 12-0767*	Tectona grandis	KU712429	KU743976	KU749371	KU749358	–	Doilom et al. 2016
D. terebinthifolii	CBS 133180*	Schinus terebinthifolius	KC343216	KC344184	KC343942	KC343458	KC343700	Gomes et al. 2013
D. undulate	LC6624*	Unknown host	KX986798	KX999230	KX999190	–	KX999269	Gao et al. 2017
D. undulate	LC8110	Unknown host	KY491545	KY491565	KY491555	–	–	Gao et al. 2017
D. vawdreyi	BRIP 57887a*	Psidium guajava	KR936126	KR936128	KR936129	–	–	Crous et al. 2015
D. viniferae	JZBH 320071	Vitis vinifera	MK341550	MK500112	MK500107	MK500119	–	Manawasinghe et al. 2019
D. viniferae	JZBH 320072	Vitis vinifera	MK341551	MK500113	MK500108	MK500120	–	Manawasinghe et al. 2019
D. xishuangbanica	LC6707*	Camellia sinensis	KX986783	KX999216	KX999175	–	KX999255	Gao et al. 2017
Diaporthella corylina	CBS 121124	Corylus sp.	KC343004	KC343972	KC343730	KC343246	KC343488	Gomes et al. 2013

Phylogenetic analyses

Novel sequences generated from the sixteen strains in this study, and all reference sequences of Diaporthe species downloaded from GenBank, were used for phylogenetic analyses. Alignments of the individual locus were determined using MAFFT v. 7.110 by default settings (Katoh et al. 2017) and manually corrected where necessary. To establish the identity of the isolates at species level, phylogenetic analyses were conducted first individually for each locus and then as combined analyses of five loci (ITS, TUB, TEF, CAL and HIS regions). Phylogenetic analyses were based on maximum likelihood (ML) and Bayesian inference (BI) for the multi-locus analyses. For BI, the best evolutionary model for each partition was determined using MrModeltest v. 2.3 (Nylander 2004) and incorporated into the analyses. ML and BI were run on the CIPRES Science Gateway portal (https://www.phylo.org/) (Miller et al. 2012) using RaxML-HPC2 on XSEDE (8.2.12) (Stamatakis 2014) and MrBayes on XSEDE (3.2.7a) (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003; Ronquist et al. 2012), respectively. For ML analyses the default parameters were used and BI was carried out using the rapid bootstrapping algorithm with the automatic halt option. Bayesian analyses included five parallel runs of 5,000,000 generations, with the stop rule option and a sampling frequency of 500 generations. The burn-in fraction was set to 0.25 and posterior probabilities (PP) were determined from the remaining trees. The resulting trees were plotted using FigTree v. 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree) and edited with Adobe Illustrator CS5.1. New sequences generated in this study were deposited at GenBank (https://www.ncbi.nlm.nih.gov; Table 1) and the alignments and trees were deposited in TreeBASE: S27479 (http://treebase.org/treebase-web/home.html).

Results

Phylogenetic analyses

Sixteen strains of Diaporthe isolated from plant hosts from Yunnan, China, were grown in culture and used for analyses of molecular sequence data. Diaporthe spp. were analysed by using multilocus data (ITS, TUB, TEF, CAL and HIS) from 115 isolates of Diaporthe spp. and Diaporthella corylina (CBS 121124) as the outgroup taxon. A total of 3005 characters including gaps were obtained in the phylogenetic analysis, viz. ITS: 1–656, TUB: 657–1329, TEF: 1330–1860, CAL: 1861–2444, HIS: 2445–3005. Of these characters, 1349 were constant, 453 were variable and parsimony-uninformative, and 1203 were parsimony-informative. For the BI and ML analyses, the substitution model GTR+I+G for ITS, TUB, TEF and HIS, HKY+I+G for and CAL were selected and incorporated into the analyses. The ML tree topology confirmed the tree topologies obtained from the BI analyses, and therefore, only the ML tree is presented (Fig. 1).

Figure 1.

Phylogram of Diaporthe spp. based on combined sequence data of ITS, TUB, TEF, CAL and HIS genes. The ML and BI bootstrap support values above 50% and 0.90 BYPP are shown at the first and second position, respectively. Strains marked with “*” are ex-type or ex-epitype. Codes referring to strains from the current study are written in red. Some branches were shortened to fit them to the page as indicated by two diagonal lines with the number of times a branch was shortened indicated.

Figure 1.

Continued.

Figure 1.

Continued.

ML bootstrap support values (≥ 50%) and Bayesian posterior probability (≥ 0.90) are shown as first and second position above nodes, respectively. Based on the five-locus phylogeny and morphology, nine isolates were assigned to five species, including Diaporthe arecae (1), D. hongkongensis (2), D. middletonii (4), D. osmanthi (1) and D. pandanicola (1), whereas seven isolates formed distinct well supported clades, which refer to novel species named D. chrysalidocarpi (2), D. machili (2) and D. pometiae (3), respectively.

Taxonomy

Diaporthe arecae (H.C. Srivast., Zakia & Govindar.) R.R. Gomes, Glienke & Crous, Persoonia 31: 16. (2013)

Figure 2

Subramanella arecae H.C. Srivast., Zakia & Govindar., in Srivastava, Banu and Govindarajan (1962). Basionym.

Description

Asexual morph: Conidiomata pycnidial, several pycnidia grouped together, globose, black, erumpent, exuding creamy to yellowish conidial droplets from ostioles. Conidiophores hyaline, septate, branched, cylindrical, straight to sinuous, 25.0–32.0 × 1.4–2.5 μm. Conidiogenous cells 10.5–20.7 × 1.4–2.0 μm, phialidic, cylindrical, swollen at base, tapering towards apex, slightly curved. Alpha conidia hyaline, smooth, aseptate, ellipsoidal, guttulate, apex subobtuse, base subtruncate, 7.5–10.0 × 1.8–3.0 µm (mean = 8.2 × 2.4 μm, n = 20). Beta conidia hyaline, aseptate, filiform, slightly curved, tapering towards base, 18.5–26.5 × 1.0–1.8 µm (mean = 24.3 × 1.4 μm, n = 20). Gamma conidia not observed. Sexual morph not observed.

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 11.2–13.3 mm diam/day. Aerial mycelium white, cottony, feathery, abundant in center, sparse in margin, white on surface, reverse yellowish to tan.

Specimen examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Persea americana (Lauraceae). 19 April 2019, S.T. Huang, HSAUP194.18, living culture SAUCC194.18.

Figure 2.

Diaporthe arecae (SAUCC194.18) a infected leaf of Persea americana b, c surface and reverse of a colony after 15 days on PDA d conidiomata e–g conidiophores and conidiogenous cells h beta conidia i alpha conidia j alpha conidia and beta conidia. Scale bars: 10 μm (e–j).

Notes

Diaporthe arecae (CBS 161.64) was originally described as Subramanella arecae on fruit of Areca catechu in India (Srivastava et al. 1962) and placed in Diaporthe by Gomes et al. (2013). The Diaporthe isolate from fruits of Citrus sp. (CBS 535.75) in Suriname was also placed in D. arecae by Gomes et al. (2013). In the present study, strain (SAUCC194.18) from symptomatic leaves of Persea americana was congruent with D. arecae based on morphology and DNA sequences data (Fig. 1). We therefore consider the isolated strain as D. arecae.

Diaporthe chrysalidocarpi S.T. Huang, J.W. Xia, W.X. Sun, & X.G. Zhang, sp. nov.

MycoBank No: 837812

Figure 3

Etymology

Named after the host genus on which it was collected, Chrysalidocarpus lutescens.

Diagnosis

Diaporthe chrysalidocarpi can be distinguished from the phylogenetically most closely related species D. spinosa by longer beta conidia (28.0–32.5 × 1.2–1.6 vs. 18.5–30.5 × 1.0–1.5 μm), and from other species D. fulvicolor by the types of conidia (D. chrysalidocarpi produces only beta conidia, while D. fulvicolor produces only alpha conidia) and several loci (25/491 in the ITS region, 18/471 TUB, 4/298 TEF, 28/458 CAL and 13/441 HIS).

Type

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Chrysalidocarpus lutescens (Palmae). 19 April 2019, S.T. Huang, HSAUP194.35 holotype, ex-type living culture SAUCC194.35.

Description

Asexual morph: Leaf spots irregular, pale brown in center, brown to tan at margin. Conidiomata pycnidial, scattered or aggregated, black, erumpent, raising above surface of culture medium, subglobose, exuding white or yellowish creamy conidial droplets from central ostioles after 30 days in light at 25 °C; pycnidial wall consists of black to dark brown, thin-walled cells. Conidiophores 27.5–35.0 × 1.4–2.0 μm, hyaline, slightly branched, swelling at base, subcylindrical, septate, smooth, straight or curved. Conidiogenous cells 10.5–23.0 × 1.4–1.8 μm, phialidic, cylindrical, terminal, straight to sinuous, tapering towards apex. Beta conidia 28.0–32.5 × 1.2–1.6 μm (mean = 30.3 × 1.3 μm, n = 20), filiform, hyaline, straight or slightly curved, aseptate, base subtruncate, tapering towards the base. Alpha conidia and gamma conidia not observed. Sexual morph not observed.

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 13.3–15.2 mm diam/day, initially white, becoming greyish, reverse pale brown, with concentric rings of dense, sparse hyphae, irregular margin, fluffy aerial mycelium at center, pycnidia forming after 15 days.

Additional specimen examined

Figure 3.

Diaporthe chrysalidocarpi (SAUCC194.35) a diseased leaf of Chrysalidocarpus lutescens b, c surface and reverse of a colony after 15 days on PDA d, e conidiomata f, g conidiophores and conidiogenous cells h, i beta conidia. Scale bars: 10 μm (f–i).

Notes

Phylogenetic analysis of a combined five gene showed that D. chrysalidocarpi formed an independent clade (Fig. 1) and is phylogenetically distinct from D. spinosa and D. fulvicolor. This species can be distinguished from D. spinosa by 61 different nucleotides in the concatenated alignment (13/492 in the ITS region, 17/471 TUB, 4/298 TEF, 17/458 CAL and 10/441 HIS), and D. fulvicolor by 88 nucleotides (25/491 in the ITS region, 18/471 TUB, 4/298 TEF, 28/458 CAL and 13/441 HIS). Morphologically, D. chrysalidocarpi differs from D. spinosa in having longer beta conidia (28.0–32.5 × 1.2–1.6 vs. 18.5–30.5 × 1.0–1.5 μm) (Guo et al. 2020). Furthermore, Diaporthe chrysalidocarpi produces only beta conidia, while D. spinosa produces alpha conidia and beta conidia and D. fulvicolor produces only alpha conidia (Guo et al. 2020). Therefore, we establish this fungus as a novel species.

Diaporthe hongkongensis R.R. Gomes, Glienke, Crous, Persoonia 31: 23. (2013)

Figure 4

Description

Asexual morph: Conidiomata pycnidial, subglobose or globose, solitary, black, erumpent, coated with white hyphae, thick-walled, exuding creamy conidial droplets from central ostioles. Conidiophores hyaline, smooth, septate, unbranched, densely aggregated, cylindrical or clavate, straight to sinuous, swollen at base, tapering towards apex, 32.0–42.0 × 2.0–2.9 μm. Conidiogenous cells 20.0–24.2 × 1.3–2.3 μm, phialidic, cylindrical, terminal, slightly tapering towards apex. Alpha conidia, hyaline, smooth, aseptate, ellipsoidal or oval, 0–2 guttulate, apex subobtuse, base subtruncate, 5.5–7.0 × 2.0–2.5 µm (mean = 6.2 × 2.2 μm, n = 20). Beta conidia hyaline, aseptate, filiform, hamate, tapering towards both ends, mostly J-shaped, 21.5–27.0 × 1.4–1.8 µm (mean = 25.6 × 1.3 μm, n = 20). Gamma conidia not observed. Sexual morph not observed.

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 19.0–21.5 mm diam/day, cottony, radial with abundant aerial mycelium, sparse at margin, with an obvious pale brown concentric ring of dense hyphae, white to grayish on surface with age, white to pale brown on the reverse side.

Figure 4.

Diaporthe hongkongensis (SAUCC194.87) a diseased leaf of Camellia sinensis b, c surface and reverse of colony after 15 days on PDA d conidiomata e–g conidiophores and conidiogenous cells h beta conidia i alpha conidia. Scale bars: 10 μm (e–i).

Specimens examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, 19 April 2019, S.T. Huang. On diseased leaves of Millettia reticulata (Fabaceae) HSAUP194.81, living culture SAUCC194.81; on diseased leaves of Camellia sinensis (Theaceae) HSAUP194.87, living culture SAUCC194.87.

Notes

In the present study, two strains (SAUCC194.81 and SAUCC194.87) from symptomatic leaves of Millettia reticulata and Camellia sinensis were similar to Diaporthe hongkongensis (CGMCC 3.17102) (Gomes et al. 2013) and D. salinicola (MFLU 18-0553) (Dayarathne et al. 2020) based on DNA sequences data (Fig. 1). Morphologically, our strains were similar to Diaporthe hongkongensis, which was originally described with an asexual morph on fruits of Dichroa febrifuga in China, but the asexual morph of D. salinicola was undetermined. We therefore identify our strains as D. hongkongensis.

Diaporthe machili S.T. Huang, J.W. Xia, W.X. Sun, & X.G. Zhang, sp. nov.

MycoBank No: 837814

Figure 5

Etymology

Named after the host genus on which it was collected, Machilus pingii.

Diagnosis

Diaporthe machili differs from D. caryae and D. sackstonii in the types of conidia (D. machili only produces beta conidia, while D. caryae produces alpha conidia and beta conidia, and D. sackstonii only produces alpha conidia), and from D. caryae in longer beta conidia (29.0–39.0 × 1.3–1.5 vs. 15.5–34.0 × 1.1–1.4 μm).

Type

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Machilus pingii (Lauraceae). 19 April 2019, S.T. Huang, HSAUP194.111 holotype, ex-holotype living culture SAUCC194.111.

Figure 5.

Diaporthe machili (SAUCC194.111) a infected leaf of Machilus pingii b, c surface and reverse of colony after 15 days on PDA d, e conidiomata f–h conidiophores and conidiogenous cells i–k beta conidia. Scale bars: 10 μm (f–k).

Description

Asexual morph: Conidiomata pycnidial, aggregated, black, erumpent, subglobose to globose, exuding creamy conidial droplets from central ostioles after 30 days in light at 25 °C. Conidiophores 7.0–11.4 × 1.8–2.8 μm, hyaline, unbranched, densely aggregated, mostly ampulliform, cylindrical, guttulate, septate, straight or slightly curved, swelling at base, tapering towards apex. Beta conidia 29.0–39.0 × 1.3–1.5 μm (mean = 32.5 × 1.4 μm, n = 20), filiform, hyaline, aseptate, mostly curved, J-shaped, swelling in middle, tapering towards both ends. Alpha and gamma conidia not observed. Sexual morph not observed.

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 16.3–17.5 mm diam/day, aerial mycelium abundant, white on surface, reverse white to pale yellow, with an obvious concentric zonation, pycnidia forming after 15 days.

Additional specimen examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Pometia pinnata (Sapindaceae). 19 April 2019, S.T. Huang, HSAUP194. 69 paratype; living culture SAUCC194. 69.

Notes

In the phylogenetic tree, Diaporthe machili forms an independent clade and is phylogenetically distinct from D. caryae and D. sackstonii (Fig. 1). Diaporthe machili can be distinguished from D. caryae in ITS, TUB, TEF, CAL and HIS loci by 67 nucleotide differences in concatenated alignment (5/459 in ITS, 10/416 in TUB, 15/334 in TEF, 7/454 in CAL and 30/455 in HIS), and from D. sackstonii in ITS, TUB and TEF loci by 58 nucleotide differences (12/559 in ITS, 23/486 in TUB and 23/348 in TEF). Moreover, Diaporthe machili differs from D. caryae in having longer beta conidia (29.0–39.0 × 1.3–1.5 vs. 15.5–34.0 × 1.1–1.4 μm). Diaporthe machili only produces beta conidia, while D. caryae produces alpha conidia and beta conidia, and D. sackstonii only produces alpha conidia (Thompson et al. 2015; Yang et al. 2018b).

Diaporthe middletonii R.G. Shivas, L. Morin, S.M. Thomps. & Y.P. Tan, Persoonia 35: 45. (2015)

Figure 6

Description

Asexual morph: Leaf spots discoid to irregular. Conidiomata pycnidial, scattered or aggregated in groups of 3–5 pycnidia, globose, black, erumpent, coated with white to greyish hyphae, thick-walled, exuding creamy translucent conidial droplets from central ostioles. Conidiophores hyaline, smooth, septate, unbranched, densely aggregated, cylindrical, straight to sinuous, tapering towards apex, 10.0–14.0 × 1.3–2.3 μm. Conidiogenous cells 5.0–9.5 × 1.3–1.7 μm, phialidic, cylindrical, terminal, slightly tapering towards apex. Alpha conidia hyaline, smooth, aseptate, biguttulate, ellipsoidal, oval, apex subobtuse, base subtruncate, 5.5–7.0 × 2.5–3.2 µm (mean = 6.3 × 2.8 μm, n = 20). Beta conidia hyaline, aseptate, filiform, mostly curved by 90–180°, tapering towards both ends, 26.0–36.5 × 1.0–1.6 µm (mean = 21.5 × 1.2 μm, n = 20). Gamma conidia not observed. Sexual morph not observed.

Figure 6.

Diaporthe middletonii (SAUCC194.46) a infected leaf of Lithocarpus glaber b, c surface and reverse of colony after 15 days on PDA d, e conidiomata f–i conidiophores and conidiogenous cells j beta conidia k, l alpha conidia and beta conidia. Scale bars: 10 μm (f–l).

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 22.5–24.0 mm diam/day, fluffy with abundant aerial mycelium, margin fimbriate, white on surface, white to pale yellow on reverse.

Specimens examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, 19 April 2019, S.T. Huang. On diseased leaves of Litchi chinensis (Sapindaceae), HSAUP194.27, living culture SAUCC194.27; on diseased leaves of Lithocarpus glaber (Fagaceae), HSAUP194.45, living culture SAUCC194.45; on diseased leaves of Lithocarpus glaber (Fagaceae), 19 April 2019, S.T. Huang, HSAUP194.46, living culture SAUCC194.46; on diseased leaves of Lithocarpus craibianus (Fagaceae), HSAUP194.48, living culture SAUCC194.48.

Notes

Diaporthe middletonii was originally described from the stem of Rapistrum rugosum (BRIP 54884e) (Brassicaceae) and Chrysanthemoides monilifera subsp. rotundata (BRIP 57329) (Asteraceae) in Australia (Thompson et al. 2015). In the present study, four strains (SAUCC194.27, SAUCC194.45, SAUCC194.46 and SAUCC194.48) are closely related to D. middletonii in the combined phylogenetic tree (Fig. 1). The differences between nucleotides in the concatenated alignment (17/565 in ITS, 9/494 in TUB and 10/340 in TEF) were minor. Morphologically, our strains were similar to D. middletonii by slightly shorter and wider alpha conidia (5.0–7.0 × 2.5–3.2 vs. 6.0–7.5 × 2.0–2.5 μm), and longer beta conidia (26.0–36.5 × 1.0–1.6 vs. 20.0–35.0 × 1.0–1.5 μm) (Thompson et al. 2015). We therefore identify our strains as Diaporthe middletonii.

Diaporthe osmanthi H. Long, K.D. Hyde, & Yong Wang bis, MycoKeys 57: 120. (2019)

Figure 7

Description

Conidiomata pycnidial, globose, 5–10 pycnidia grouped together, dark brown to black, exuding creamy to yellowish conidial droplets from central ostioles. Conidiophores hyaline, smooth, densely aggregated, branched, cylindric-clavate, 20.5–32.0 × 1.8–2.4 μm. Conidiogenous cells phialidic, hyaline, terminal, cylindrical, straight, 14.0–20.5 × 1.5–2.0 μm, tapered towards apex. Alpha conidia hyaline, aseptate, fusiform, tapering towards both ends, guttulate, 7.3–9.3 × 1.8–2.3 μm (mean = 8.5 × 2.0 μm, n = 20). Beta conidia hyaline, aseptate, filiform, curved, 22.0–28.5 × 1.0–2.0 μm (mean = 27.2 × 1.3 μm, n = 20). Gamma conidia not observed. Sexual morph not observed.

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 12.0–13.5 mm diam/day, cottony with abundant aerial mycelium, sparse at margin. With several concentric rings of dense hyphae, white on surface, white to pale brown on reverse.

Specimen examined

Figure 7.

Diaporthe osmanthi (SAUCC194.21) a infected leaf of Litchi chinensis b, c surface and reverse of colony after 15 days on PDA d conidiomata e–g conidiophores and conidiogenous cells h, i beta conidia j, k alpha conidia. Scale bars: 10 μm (e–k).

Notes

Diaporthe osmanthi was originally described from the leaves of Osmanthus fragrans (Oleaceae) in Guangxi province, China (Long et al. 2019). In the present study, phylogenetic analyses (Fig. 1) indicated that the strain SAUCC194.21 is closely related to Diaporthe osmanthi and D. podocarpi-macrophylli (Gao et al. 2017). Morphological comparison indicated that this strain was most similar to D. osmanthi by the size of alpha conidia and beta conidia. We therefore identify this strain as belonging to D. osmanthi.

Diaporthe pandanicola Tibpromma & K.D. Hyde, MycoKeys 33: 44 (2018)

Figure 8

Description

Asexual morph: Conidiomata pycnidial, 3–5 pycnidia grouped together, superficial to embedded on PDA, erumpent, thin-walled, dark brown to black, globose or subglobose, exuding white creamy conidial mass from ostioles. Conidiophores hyaline, aseptate, cylindrical, smooth, straight to sinuous, unbranched, aggregated, 17.0–26.5 × 2.0–3.0 µm. Conidiogenous cells phialidic, cylindrical, terminal, 10.0–20.0 × 1.5–1.8 µm. Alpha conidia hyaline, smooth, aseptate, ellipsoidal, eguttulate, apex subobtuse, base subtruncate, 6.5–9.0 × 1.8–2.5 µm (mean = 7.5 × 2.0 μm, n = 20). Beta conidia hyaline, aseptate, filiform, curved, tapering towards apex, base truncate, 26.0–32.8 × 1.0–1.6 µm (mean = 29.0 × 1.3 μm, n = 20). Gamma conidia infrequent, aseptate, smooth, straight, hyaline, 12.5–14.5 × 1.3–1.8 µm (mean = 13.5 × 1.6 μm, n = 6). Sexual morph not observed.

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 12.8–15.0 mm diam/day, flat, cottony in centre, with aerial mycelium sparse toward margin, white on surface, white to pale yellow on reverse.

Figure 8.

Diaporthe pandanicola (SAUCC194.82) a infected leaf of Millettia reticulata b, c surface and reverse of colony after 15 days on PDA d conidiomata e–g conidiophores and conidiogenous cells h beta conidia i alpha conidia and gamma conidia j alpha conidia, beta conidia and gamma conidia. Scale bars: 10 μm (e–j).

Specimen examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Millettia reticulata (Fabaceae). 19 April 2019, S.T. Huang, HSAUP194.82, living culture SAUCC194.82.

Notes

Diaporthe pandanicola was originally described by Tibpromma et al. (2018) on healthy leaves of Pandanus sp. (Pandanaceae) as an endophytic fungus. Our strain (SAUCC194.82) is closely related to Diaporthe pandanicola based on phylogenetic analyses (Fig. 1). The differences of nucleotides in the concatenated alignment (19/533 in the ITS region and 11/351 in the TUB region) are less than 3%. Morphologically, our strain produces alpha conidia, beta conidia and gamma conidia, while Diaporthe pandanicola did not sporulate. We therefore identify our strains as Diaporthe pandanicola.

Diaporthe pometiae S.T. Huang, J.W. Xia, W.X. Sun, & X.G. Zhang, sp. nov.

MycoBank No: 837815

Figure 9

Etymology

Named after the host genus on which it was collected, Pometia pinnata.

Diagnosis

Diaporthe pometiae is similar to D. biconispora but differs in having smaller alpha conidia (5.7–8.3 × 2.2–3.0 vs. 6.0–10.5 × 2–3.5 μm) and types of conidia (D. pometiae produces beta conidia unlike D. biconispora).

Type

Description

Asexual morph: Leaf spots subcircular, fawn to dark brown. Conidiomata pycnidial, subglobose to globose, aggregated in groups, black, coated with white hyphae, thick-walled, exuding creamy droplets from ostioles. Conidiophores hyaline, smooth, slightly septate, branched, densely aggregated, cylindric-clavate, straight to slightly sinuous, 22.5–32.5 × 1.0–2.0 μm. Conidiogenous cells 15.0–22.5 × 1.0–1.5 μm, phialidic, cylindrical, multi-guttulate, terminal, tapering towards apex. Alpha conidia abundant in culture, 2–4 guttulate, hyaline, smooth, aseptate, ellipsoidal to oblong ellipsoidal, with both ends obtuse, 5.7–8.3 × 2.2–3.0 µm (mean = 6.7 × 3.1 μm, n = 20). Beta conidia, hyaline, aseptate, filiform, multi-guttulate, slightly curved, tapering towards to apex, 27.8–34.5 × 1.0–1.7 µm (mean = 21.7 × 1.4 μm, n = 20). Gamma conidia not observed. Sexual morph not observed.

Figure 9.

Diaporthe pometiae (SAUCC194.72) a infected leaf of Pometia pinnata b, c surface and reverse of colony after 15 days on PDA d conidiomata e, f conidiophores and conidiogenous cells g beta conidia h alpha conidia and beta conidia. Scale bars: 10 μm (e–h).

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 11.5–13.0 mm diam/day, cottony with abundant aerial mycelium, with a concentric zonation, white on surface, white to grayish on reverse.

Additional specimens examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, 19 April 2019, S.T. Huang. On diseased leaves of Persea americana (Lauraceae), HSAUP194.19 paratype, ex-paratype culture SAUCC194.19; on diseased leaves of Heliconia metallica (Musaceae), HSAUP194.73 paratype, ex-paratype culture SAUCC194.73.

Notes

Diaporthe pometiae is introduced based on the multi-locus phylogenetic analysis, with three isolates clustering separately in a well-supported clade (ML/BI = 100/1). Diaporthe pometiae is most closely related to D. biconispora, but distinguished based on ITS, TUB, TEF and HIS loci by 74 nucleotide differences in the concatenated alignment, in which 2/492 are distinct in the ITS region, 8/353 in the TUB region, 49/370 in the TEF region and 15/471 in the HIS region. Morphologically, Diaporthe pometiae differs from D. biconispora in its smaller alpha conidia (5.7–8.3 × 2.2–3.0 vs. 6.0–10.5 × 2–3.5 μm). Furthermore, Diaporthe pometiae produces beta conidia unlike D. biconispora (Huang et al. 2015).

Discussion

The Yunnan Province in southeastern China has a unique geography where three climatic regions meet: the eastern Asia monsoon region, the Tibetan plateau region, and the tropical monsoon region of southern Asia and Indo-China. The environment is conducive to growth of unusual microbial species. Species diversity in Yunnan Province is high compared to other parts of China.

Previously, species identification of Diaporthe relied on the assumption of host-specificity, leading to the proliferation of names. The morphological characters of Diaporthe could be changeable, as most taxa in culture do not produce all spore states of the asexual (alpha, beta and gamma conidia) or the sexual morph (Gomes et al. 2013). Based on a polyphasic approach and morphology, more than one species of Diaporthe can colonize a single host, while one species can be associated with several hosts (Gomes et al. 2013; Gao et al. 2017; Guarnaccia and Crous 2017; Guarnaccia et al. 2018; Guo et al. 2020). These studies revealed a high diversity of Diaporthe species from different hosts. Our study supports this phenomenon. For example, Diaporthe arecae (SAUCC194.18) and D. pometiae (SAUCC194.19) were collected from Persea americana; In addition, isolates of D. middletonii were obtained from three hosts (Litchi chinensis, Lithocarpus craibianus, L. glaber). As for host specificity, in our study, four species of Diaporthe, D. machili (SAUCC194.69), D. middletonii (SAUCC194.27), D. osmanthi (SAUCC194.21), and D. pometiae (SAUCC194.72) were isolated from Litchi chinensis and Pometia pinnata belong to the Sapindaceae, and D. litchiicola also was reported from Litchi chinensis in Queensland (Tan et al. 2013); however, D. machili (SAUCC194.111) also was isolated from Machilus pingii (Lauraceae), D. middletonii (SAUCC194.45) from Lithocarpus glaber (Fagaceae), D. osmanthi (GUCC 9165) from leaves of Osmanthus fragrans (Oleaceae) (Long et al. 2019), and D. pometiae (SAUCC194.19 and SAUCC194.73) from Persea americana (Lauraceae) and Heliconia metallica (Musaceae). These results provide evidence that many species are able to colonise diverse hosts and several different species could co-occur on the same host. It seems obvious that specificity does not occur at the family level.

For the current study, sixteen strains isolated from ten host genera represented three new species and five known species, based on morphological characters and phylogenetic analyses of the five combined loci (ITS, TUB, TEF, CAL and HIS). The descriptions and molecular data for species of Diaporthe represent an important resource for plant pathologists, plant quarantine officials and taxonomists.

Acknowledgements

This work was jointly supported by the National Natural Science Foundation of China (no. 31900014, 31770016, and 31750001) and the China Postdoctoral Science Foundation (no. 2018M632699).

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