Identification and pathogenicity of Aurifilum species (Cryphonectriaceae, Diaporthales) on Terminalia species in Southern China

Abstract

The family of Cryphonectriaceae (Diaporthales) contains many important tree pathogens and the hosts are wide-ranging. Tree species of Terminalia were widely planted as ornamental trees alongside city roads and villages in southern China. Recently, stem canker and cracked bark were observed on 2–6 year old Terminalianeotaliala and T.mantaly in several nurseries in Zhanjiang City, Guangdong Province, China. Typical conidiomata of Cryphonectriaceae fungi were observed on the surface of the diseased tissue. In this study, we used DNA sequence data (ITS, BT2/BT1, TEF-1α, rpb2) and morphological characteristics to identify the strains from Terminalia trees. Our results showed that isolates obtained in this study represent two species of Aurifilum, one previously described species, A.terminali, and an unknown species, which we described as A.cerciana sp. nov. Pathogenicity tests demonstrated that both A.terminali and A.cerciana were able to infect T.neotaliala and two tested Eucalyptus clones, suggesting the potential for Aurifilum fungi to become new pathogens of Eucalyptus.

Introduction

Cryphonectriaceae is a fungal family within the order Diaporthales. This family is well-known for containing several species that are serious pathogens of trees, causing a wide range of diseases such as blight, die-back, and cankers (Gryzenhout et al. 2004, 2005, 2009; Begoude et al. 2010; Chen et al. 2010, 2011, 2013a, b, 2016, 2018; Wang et al. 2018, 2020; Roux et al. 2020). Most members of this family are easily recognizable based on the disease symptoms, as well as their distinctive yellow to orange or brown stromata and which can turn purple in 3% potassium hydroxide (KOH) and yellow in lactic acid (Gryzenhout et al. 2006, 2009; Jiang et al. 2020).

Twenty-four genera have been described in the Cryphonectriaceae (Gryzenhout et al. 2009, 2010; Begoude et al. 2010; Vermeulen et al. 2011, 2013; Crous et al. 2012; Chen et al. 2013a, b, 2016, 2018; Crane and Burgess 2013; Beier et al. 2015; Ali et al. 2018; Jiang et al. 2018, 2019, 2020; Ferreira et al. 2019; Wang et al. 2020; Huang et al. 2022). Some of the more well-known genera in this family include Cryphonectriaparasitica, which caused chestnut blight, and is one of the best-known tree-killing pathogen (Fairchild 1913; Shear and Stevens 1913; Anagnostakis 1987; Heiniger and Rigling 1994; Gryzenhout et al. 2009); Chrysoportheaustroafricana causes a canker disease of Eucalyptus, Syzygium and Tibouchina species in Southern and Eastern Africa (Wingfield et al. 1989; Gryzenhout et al. 2004; Roux et al. 2005; Nakabonge et al. 2006; Gryzenhout et al. 2009); Chrysoporthecubensis causes a canker disease of Eucalyptus species in West Africa and South America, and also causes diseases in Melastomataceae and Myrtaceae trees (Alfenas et al. 1983; Gryzenhout et al. 2004, 2009; Roux 2010); Chrysoporthedeuterocubensis, causes a canker disease of Eucalyptus species in Africa, Australia, China and Hawaii, and is also reported on native or non-native Melastomataceae and Myrtaceae trees (Davison and Coates 1991; Roux et al. 2005; Nakabonge et al. 2006; Zhou et al. 2008; Gryzenhout et al. 2009; Chen et al. 2010; Van der Merwe et al. 2010; Wang et al. 2020).

In China, various species of Cryphonectriaceae have been found to cause diseases in plants belonging to the Myrtales order. Some of the affected hosts include Eucalyptus hybrid (Chen et al. 2010, 2011; Wang et al. 2018, 2020), Lagerstroemiaspeciosa (Lythraceae, Myrtales) (Chen et al. 2018), Melastomacandidum, M.sanguineum (Melastomataceae, Myrtales), Psidiumguajava (Myrtaceae) (Chen et al. 2016; Wang et 2018, 2020), Syzygiumcumini, S.hancei, S.jambos, S.samarangense (Myrtaceae, Myrtales) (Chen et al. 2010, 2011; Van der Merwe et al. 2010; Wang et al. 2018, 2020), Terminalianeotaliala (Combretaceae) (Wang et al. 2020), Rhodomyrtustomentosa (Myrtaceae, Myrtales) (Chen et al. 2016). Inoculation tests have confirmed that all the Cryphonectriaceae species from Combretaceae, Lythraceae, Melastomataceae, and Myrtaceae in China are pathogenic to their original hosts and Eucalyptus (Chen et al. 2010, 2011, 2016, 2018; Wang et al. 2018, 2020).

Seven of the nine families of Myrtales are commonly found in southern China, and Cryphonectriaceae has been identified as an important pathogen to Myrtales trees in previous studies (Chen et al. 2010, 2011, 2016, 2018; Wang et al. 2018, 2020). Given the diverse climate and host range in southern China, there is potential for the discovery of various Cryphonectriaceae species and potential pathogens on Myrtales trees.

Terminalia species are economically and ecologically important trees in southern China and are widely used for timber, medicine, and ornamental purposes (Editorial Committee of Flora of China 1988; Batawila et al. 2005; Kamtchouing et al. 2006; Angiosperm Phylogeny Group 2009). In 2019, cankers were observed on the stems of Terminalia trees during disease surveys on Myrtales trees in southern China, and fruiting structures of the fungi on the cankered stems exhibited typical Cryphonectriaceae morphological characteristics. The aims of this study were to identify the fungi isolated from these cankers based on DNA sequencing and morphological characteristics and to test their pathogenicity on Terminalia species and two widely planted E.grandis hybrid genotypes.

Materials and methods

Disease symptoms, samples and isolations

In May 2019, disease surveys on Terminalia trees were conducted in Zhanjiang City, Guangdong Province in southern China. Sporocarps with typical characteristics of Cryphonectriaceae were observed on the surfaces of cankers on the branches, stems, and roots of Terminalia trees. In order to identify the pathogens, five experimental sites were set every 30 to 50 kilometers. Diseased bark pieces, branches, twigs, and roots bearing fruiting structures were collected and transported to the laboratory. The fruiting structures were incised using a sterile scalpel blade under a stereoscopic microscope. The spore masses were then transferred to 2% (v/v) malt extract agar (MEA) and incubated at room temperature for three to five days until colonies developed. The pure cultures were obtained by transferring single hyphal tips from the colonies to 2% MEA plates and incubated at room temperature for 7–10 days. The pure cultures are stored in the culture collection (CSF) at the Research Institute of Fast-Growing Trees (RIFT) (previous institution: China Eucalypt Research Centre, CERC), Chinese Academy of Forestry (CAF) in Zhanjiang, Guangdong Province, China.

DNA extraction, polymerase chain reaction (PCR) amplification and sequencing

Representative isolates were selected for DNA sequence analyses, and actively growing mycelium on MEA cultures grown for one week at room temperature was scraped using a sterilized scalpel and transferred into 2.0 mL Eppendorf tubes. Total genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method described by Van Burik et al. (1998). The extracted DNA was dissolved in 30 μL TE buffer, and the concentration was measured using a Nano-Drop 2000 spectrometer (Thermo Fisher Scientific, Waltham, Massachusetts).

Based on previous research four gene regions, including internal transcribed spacer regions (ITS), two segments of β-tubulin (BT2/BT1), a partial segment of the translation elongation factor 1-α (TEF-1α) and RNA polymerase II (rpb2), were amplified and sequenced as described by Chen et al. (2010, 2016), Liu et al. (1999) and Jiang et al. (2022).

All amplified products were sequenced in both directions using the same primers that were used for the PCR amplification. Sequence reactions were performed by the Beijing Genomics Institute of Guangzhou, China. The nucleotide sequences were edited using Geneious 7.1.8 software. The sequences obtained in this study were submitted to GenBank (http://www.ncbi.nlm.nih.gov).

Phylogenetic analysis

The preliminary identities of the isolates sequenced in this study were obtained by conducting a standard nucleotide BLAST search using the ITS, BT2, and BT1 sequences. The BLAST results showed that the isolates collected in this study were mainly grouped in the genus Aurifilum. Phylogenetic analyses for strains identification in the current study were conducted for both genetic and species identification.

To determine the placement of Aurifilum species, two represent strains in this study were first determined by conducting phylogenetic analyses within Cryphonectriaceae species (Table 1) on combined datasets for the ITS and BT2/BT1 regions. Then, the strains in the Aurifilum genus were further analyzed and identified using separate and combined datasets for the ITS, BT2/BT1, TEF-1α, and rpb2 regions. Sequences of the Aurifilum isolates collected in this study and those from NCBI were aligned using MAFFT 7 (http://mafft.cbrc.jp/alignment/server) with the interactive refinement method (FFT-NS-i) setting (Katoh and Standley 2013). Then they were manually edited in MEGA X.

Table 1.

Isolates from previous studies used in the phylogenetic analyses in the current study.

Identity	Isolate No.a,b	Host	Location	GenBank accession no.
				ITS	BT2	BT1	TEF	rpb2
Amphilogiagyrosa	CMW10469T	Elaeocarpusdentatus	New Zealand	AF452111	AF525714	AF525707	MN271818	MN271782
	CMW10470	Ela.dentatus	New Zealand	AF452112	AF525715	AF525708	MN271819	MN271783
Aurantioporthecorni	MES1001	N/A	USA	KF495039	N/A	KF495069	N/A	N/A
	CTS1001	N/A	USA	KF495033	N/A	KF495063	N/A	N/A
	CMW10526	N/A	USA	DQ120762	AH015163	AH015163	N/A	N/A
Aurantiosacculusacutatus	CBS 132181T	Eucalyptusviminalis	Australia	JQ685514	N/A	N/A	MN271823	NA
Aurantiosacculuscastaneae	CFCC 52456	Castanea mollissima	China	MH514025	MH539688	MH539678	NA	MN271786
Aurantiosacculuseucalyptorum	CBS 130826T	Euc.globulus	Australia	JQ685515	N/A	N/A	MN271824	MN271785
Aurapexpenicillata	CMW10030T	Miconiatheaezans	Colombia	AY214311	AY214275	AY214239	N/A	N/A
	CMW10035	Mic.theaezans	Colombia	AY214313	AY214277	AY214241	N/A	N/A
Aurifilummarmelostoma	CBS124928T	Terminaliamantaly	Cameroon	FJ882855	FJ900590	FJ900585	MN271827	MN271788
	CBS124929	Ter.ivorensis	Cameroon	FJ882856	FJ900591	FJ900586	MN271828	MN271789
Aurifilumterminali	CSF10748	Ter.neotaliala	China	MN199834	MN258767	MN258772	MN258777	OQ942878
	CSF10757T	Ter.neotaliala	China	MN199837	MN258770	MN258775	MN258780	OQ942879
Capillaureumcaryovora	CBL02T	Caryocarbrasiliense	Brazil	MG192094	MG211808	MG211827	N/A	N/A
	CBL06	Car.brasiliense	Brazil	MG192096	MG211810	MG211829	N/A	N/A
Celoportheborbonica	CMW44128T	Tibouchinagrandiflora	La Réunion	MG585741	N/A	MG585725	N/A	N/A
	CMW44139	Tib.grandiflora	La Réunion	MG585742	N/A	MG585726	N/A	N/A
Celoporthecerciana	CERC 9128T	Eucalyptus hybrid tree 4	China, GuangDong	MH084352	MH084412	MH084382	MH084442	N/A
	CERC 9125	Eucalyptus hybrid tree 1	China, GuangDong	MH084349	MH084409	MH084379	MH084439	N/A
Celoporthedispersa	CMW 9976T	Syzygiumcordatum	South Africa	DQ267130	DQ267142	DQ267136	HQ730840	N/A
	CMW 9978	S.cordatum	South Africa	AY214316	DQ267141	DQ267135	HQ730841	N/A
Celoportheeucalypti	CMW 26900	Eucalyptus clone EC48	China	HQ730836	HQ730826	HQ730816	HQ730849	N/A
	CMW 26908T	Eucalyptus clone EC48	China	HQ730837	HQ730827	HQ730817	HQ730850	N/A
Celoporthefontana	CMW 29375	S.guineense	Zambia	GU726940	GU726952	GU726952	JQ824073	N/A
	CMW 29376T	S.guineense	Zambia	GU726941	GU726953	GU726953	JQ824074	N/A
Celoportheguangdongensis	CMW 12750T	Eucalyptus sp.	China	HQ730830	HQ730820	HQ730810	HQ730843	N/A
Celoportheindonesiensis	CMW 10781T	S.aromaticum	Indonesia	AY084009	AY084021	AY084033	HQ730842	N/A
Celoporthesyzygii	CMW 34023T	S.cumini	China	HQ730831	HQ730821	HQ730811	HQ730844	N/A
	CMW24912	S.cumini	China	HQ730833	HQ730823	HQ730813	HQ730846	N/A
Celoporthetibouchineae	CMW44126T	Tib.grandiflora	La Réunion	MG585747	N/A	MG585731	N/A	N/A
	CMW44127	Tib.grandiflora	La Réunion	MG585748	N/A	MG585732	N/A	N/A
Celoporthewoodiana	CMW13936T	Tib.granulosa	South Africa	DQ267131	DQ267143	DQ267137	JQ824071	N/A
	CMW13937	Tib.granulosa	South Africa	DQ267132	DQ267144	DQ267138	JQ824072	N/A
Chrysomorbuslagerstroemiae	CERC 8780	Lagerstroemiaspeciosa	China	KY929330	KY929340	KY929350	N/A	N/A
	CERC 8810T	Lag.speciosa	China	KY929338	KY929348	KY929358	N/A	N/A
Chrysoportheaustroafricana	CMW 62	Euc.grandis	South Africa	AF292041	AF273458	AF273063	N/A	N/A
	CMW 9327	Tib.granulosa	South Africa	AF273473	AF273455	AF273060	N/A	N/A
	CMW 2113T	Euc.grandis	South Africa	AF046892	AF273462	AF273067	N/A	N/A
Chrysoporthecubensis	CMW 10453	Euc.saligna	Democratic Republic of the Congo	AY063476	AY063480	AY063478	N/A	N/A
	CMW 10669 = CRY864	Eucalyptus sp.	Republic of the Congo	AF535122	AF535126	AF535124	N/A	N/A
Chrysoporthedeuterocubensis	CMW 11290	Eucalyptus sp.	Indonesia	AY214304	AY214268	AY214232	N/A	N/A
	CMW 8651	S.aromaticum	Indonesia	AY084002	AY084014	AY084026	N/A	N/A
Chrysoporthedoradensis	CMW 11287T	Euc.grandis	Ecuador	AY214289	AY214253	AY214217	N/A	N/A
	CMW 11286	Euc.grandis	Ecuador	AY214290	AY214254	AY214218	N/A	N/A
Chrysoporthehodgesiana	CMW 10625	Mic.theaezans	Colombia	AY956970	AY956980	AY956979	N/A	N/A
	CMW 9995	Tib.semidecandra	Colombia	AY956969	AY956978	AY956977	N/A	N/A
	CMW 10641T	Tib.semidecandra	Colombia	AY692322	AY692325	AY692326	N/A	N/A
Chrysoportheinopina	CMW 12727T	Tib.lepidota	Colombia	DQ368777	DQ368807	DQ368806	N/A	N/A
	CMW 12729	Tib.lepidota	Colombia	DQ368778	DQ368809	DQ368808	N/A	N/A
Chrysoporthesyzygiicola	CMW 29940T	S.guineense	Zambia	FJ655005	FJ805236	FJ805230	N/A	N/A
	CMW 29942	S.guineense	Zambia	FJ655007	FJ805238	FJ805232	N/A	N/A
Chrysoporthezambiensis	CMW29928T	Euc.grandis	Zambia	FJ655002	FJ805233	FJ858709	N/A	N/A
	CMW29930	Euc.grandis	Zambia	FJ655004	FJ805235	FJ858711	N/A	N/A
Corticimorbussinomyrti	CERC3629T	Rhodomyrtustomentosa	China	KT167169	KT167189	KT167189	N/A	N/A
	CERC3631	Rho.tomentosa	China	KT167170	KT167190	KT167190	N/A	N/A
Cryphonectriacitrina	CBS 109758	Punicagranatum	USA	MN172407	N/A	N/A	MN271843	EU219342
Cryphonectriadecipiens	CMW 10436	Quercussuber	Portugal	AF452117	AF525710	AF525703	N/A	N/A
	CMW 10484	Castaneasativa	Italy	AF368327	AF368349	AF368349	N/A	N/A
Cryphonectriajaponica	CMW13742	Q.grosseserrata	Japan	AY697936	AY697962	AY697961	N/A	N/A
Cryphonectriamacrospora	CMW10463	Cas.cuspidata	Japan	AF368331	AF368350	AF368351	N/A	N/A
	CMW10914	Cas.cuspidata	Japan	AY697942	AY697974	AY697973	N/A	N/A
Cryphonectrianaterciae	C0612	Q.suber	Portugal	EU442657	N/A	N/A	MN271844	MN271796
Cryphonectrianeoparasitica	CFCC 52146	Cas.mollissima	China	MH514029	MH539692	MH539682	MH539693	N/A
Cryphonectriaparasitica	CMW 7048	Q.virginiana	USA	AF368330	AF273470	AF273076	MF442684	N/A
	CMW 13749	Cas.mollisima	Japan	AY697927	AY697944	AY697943	N/A	N/A
Cryphonectriaquercicola	CFCC 52140T	Q.wutaishansea	China, Shaanxi	MG866026	MG896113	MG896117	N/A	N/A
	CFCC 52141	Q.wutaishansea	China, Shaanxi	MG866027	MG896114	MG896118	N/A	N/A
Cryphonectriaquercus	CFCC 52138T	Q. aliena var. acuteserrata	China, Shaanxi	MG866024	MG896111	MG896115	MN271849	N/A
	CFCC 52139	Q. aliena var. acuteserrata	China, Shaanxi	MG866025	MG896112	MG896116	N/A	N/A
Cryphonectriaradicalis	CMW10455	Q.suber	Italy	AF452113	AF525712	AF525705	N/A	N/A
	CMW 10477	Q.suber	Italy	AF368328	AF368347	AF368347	N/A	N/A
	CMW 13754	Fagusjaponica	Japan	AY697932	AY697954	AY697953	N/A	N/A
Cryptometrionaestuescens	CMW18793	Euc.grandis	Indonesia	GQ369459	GQ369456	GQ369456	N/A	N/A
	CMW28535T	Euc.grandis	North Sumatra, Indonesia	GQ369457	GQ369454	GQ369454	N/A	N/A
Diversimorbusmetrosiderotis	CMW37321	Metrosiderosangustifolia	South Africa	JQ862870	JQ862952	JQ862911	N/A	N/A
	CMW37322T	Met.angustifolia	South Africa	JQ862871	JQ862953	JQ862912	N/A	N/A
Endothiacerciana	CSF 15398	Quercus sp.	China	OM801201	OM685050	OM685038	N/A	N/A
	CSF 15420	Quercus sp.	China	OM801208	OM685033	OM685045	N/A	N/A
Endothiachinensis	CFCC 52144	C.mollissima	China	MH514027	MH539690	MH539680	MN271860	N/A
	CMW2091	Q.palustris	USA	AF368325	AF368336	AF368337	N/A	N/A
	CMW10442	Q.palustris	USA	AF368326	AF368338	AF368339	N/A	N/A
Holocryphiacapensis	CMW37887T	Met.angustifolia	South Africa	JQ862854	JQ862936	JQ862895	JQ863051	N/A
	CMW37329	Met.angustifolia	South Africa	JQ862859	JQ862941	JQ862900	JQ863056	N/A
Holocryphiaeucalypti	CMW7033T	Euc.grandis	South Africa	JQ862837	JQ862919	JQ862878	JQ863034	N/A
	CMW7035	Euc.saligna	South Africa	JQ862838	JQ862920	JQ862879	JQ863035	N/A
Holocryphiagleniana	CMW37334T	Met.angustifolia	South Africa	JQ862834	JQ862916	JQ862875	JQ863031	N/A
	CMW37335	Met.angustifolia	South Africa	JQ862835	JQ862917	JQ862876	JQ863032	N/A
Holocryphiamzansi	CMW37337T	Met.angustifolia	South Africa	JQ862841	JQ862923	JQ862882	JQ863038	N/A
	CMW37338	Met.angustifolia	South Africa	JQ862842	JQ862924	JQ862883	JQ863039	N/A
Holocryphia sp.	CMW6246	Tib.granulosa	Australia	JQ862845	JQ862927	JQ862886	JQ863042	N/A
Holocryphia sp.	CMW10015	Euc.fastigata	New Zealand	JQ862849	JQ862931	JQ862890	JQ863046	N/A
Immersiportheknoxdaviesiana	CMW37314T	Rapaneamelanophloeos	South Africa	JQ862765	JQ862775	JQ862785	N/A	N/A
	CMW37315	Rap.melanophloeos	South Africa	JQ862766	JQ862776	JQ862786	N/A	N/A
Latruncellusaurorae	CMW28274	Galpiniatransvaalica	Swaziland	GU726946	GU726958	GU726958	N/A	N/A
	CMW28276T	G.transvaalica	Swaziland	GU726947	GU726959	GU726959	N/A	N/A
Luteocirrhusshearii	CBS130775	Banksiabaxteri	Australia	KC197024	KC197009	KC197015	N/A	N/A
	CBS130776T	B.baxteri	Australia	KC197021	KC197006	KC197012	N/A	N/A
Microthiahavanensis	CMW11301	Myr.faya	Azores	AY214323	AY214287	AY214251	N/A	N/A
	CMW14550	E.saligna	Mexico	DQ368735	DQ368742	DQ368741	N/A	N/A
Myrtonectriamyrtacearum	CMW46433T	Heteropyxisnatalensis	South Africa	MG585736	MG585734	MG585720	N/A	N/A
	CMW46435	S.cordatum	South Africa	MG585737	MG585735	MG585721	N/A	N/A
Parvosmorbuseucalypti	CERC2060	Eucalyptus hybrid clone	China	MN258787	MN258801	MN258815	MN258829	N/A
	CERC2061T	Eucalyptus hybrid clone	China	MN258788	MN258802	MN258816	MN258830	N/A
Parvosmorbusguangdongensis	CERC10459	E.urophylla hybrid clone	China	MN258798	MN258812	MN258826	MN258840	N/A
	CERC10460T	E.urophylla hybrid clone	China	MN258799	MN258813	MN258827	MN258841	N/A
Pseudocryphonectriaelaeocarpicola	CFCC 57515	Elaeocarpus spp.	China	ON489048	N/A	N/A	ON456916	ON456918
	CFCC 57516	Elaeocarpus spp.	China	ON489049	N/A	N/A	ON456917	ON456919
Rostraureumtropicale	CMW9972	Ter.ivorensis	Ecuador	AY167436	AY167431	AY167426	N/A	N/A
	CMW10796T	Ter.ivorensis	Ecuador	AY167438	AY167433	AY167428	N/A	N/A
Ursicollumfallax	CMW18119T	Coccolobauvifera	USA	DQ368755	DQ368759	DQ368758	N/A	N/A
	CMW18115	Coc.uvifera	USA	DQ368756	DQ368761	DQ368760	N/A	N/A
Diaportheambigua	CMW5587	Malusdomestica	South Africa	AF543818	AF543822	AF543820	N/A	N/A

^a Designation of isolates and culture collections: CMW = Tree Protection Co-operative Program, Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa; ATCC = American Type Culture Collection, Manassas, USA; MES, CTS represent isolates in Beier et al. 2015; CBS = Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands; CBL represent isolates in Ferreira et al. 2019; CERC = China Eucalypt Research Centre (CERC), Chinese Academy of Forestry (CAF), ZhanJiang, GuangDong, China; CFCC = China Forestry Culture Collection Center, Beijing, China. ^b “T” following isolate number means isolates are ex-type or from samples that have been linked morphologically to type material of the species. ^c N/A = not available.

The taxonomic positions of two methods were used for phylogenetic analyses. Maximum parsimony (MP) analyses were performed using PAUP v. 4.0 b10 (Swofford 2003) and maximum likelihood (ML) analyses were conducted with PhyML v. 3.0 (Guindon and Gascuel 2003).

For MP analyses, gaps were treated as a fifth character, and characters were unordered and of equal weight with 1,000 random addition replicates. A partition homogeneity test (PHT) using PAUP v. 4.0 b10 (Swofford 2003) was conducted to determine whether data for the four genes could be combined. The most parsimonious trees were obtained using the heuristic search option with stepwise addition, tree bisection, and reconstruction branch swapping. MAXTREES was set to 5,000 and zero-length branches collapsed. A bootstrap analysis (50% majority rule, 1,000 replicates) was carried out to determine statistical support for internal nodes in trees. Tree length (TL), consistency index (CI), retention index (RI) and homoplasy index (HI) were used to assess phylogenetic trees (Hillis and Huelsenbeck 1992).

For ML analyses, the best nucleotide substitution model for each dataset was established using jModeltest v. 2.1.5 (Posada 2008). In PhyML, the maximum number of retained trees was set to 1,000 and nodal support was determined by non-parametric bootstrapping with 1,000 replicates. For both MP and ML analyses, the phylogenetic trees were viewed using MEGA v. 6.0.

Morphology

The representative isolates identified as the new species by DNA sequence analysis were grown on 2% water ager (WA), to which sterilized freshly cut branch sections (0.5–1 cm diam. 4–5 cm length) of Eucalyptusurophylla × E.grandis (CEPT53) branch sections were added. These fungi with branch sections on 2% WA were incubated at room temperature for 6–8 wks until fruiting structures emerged. Representative cultures are maintained in the China General Microbiological Culture Collection Centre (CGMCC), Beijing, China. Isolates linked to the type specimens connected to representative isolates were deposited in the mycological fungarium of the Institute of Microbiology, Chinese Academy of Sciences (HMAS), Beijing, China, and the Collection of Central South Forestry Fungi of China (CSFF), Guangdong Province, China.

The structures that emerged on the surface of the Eucalyptus branches were mounted in one drop of 85% lactic acid on glass slides under a dissecting microscope and then embedded in Leica Bio-systems Tissue Freezing Medium (Leica Biosystems nussloch GmbH, Nussloch, Germany) and sectioned (6 μm thick) using a Microtome Cryostat Microm HM550 (Microm International GmbH, Thermo Fisher Scientific, Walldorf, Germany) at -20 °C. Conidiophores, conidiogenous cells, and conidia were measured after crushing the sporocarps on microscope slides in sterilized water. For the holotype specimens, 50 measurements were performed for each morphological feature, and 30 measurements per character were made for the remaining specimens.

Measurements were recorded using an Axio Imager A1 microscope (Carl Zeiss Ltd., Munchen, Germany) and an AxioCam ERc 5S digital camera with Zeiss Axio Vision Rel. 4.8 software (Carl Zeiss Ltd., Munchen, Germany). The results are presented as (minimum–) (mean – standard deviation) – (mean + standard deviation) (–maximum).

Isolates identified as new species were selected for studying culture characteristics. After the isolates were grown for 7 days on 2% MEA, a 5 mm plug was removed from each culture and transferred to the central of 90 mm MEA Petri dishes. The cultures were incubated in the dark under temperatures ranging from 5 °C to 35 °C at 5 °C intervals. Five replicate plates for each isolate at each temperature condition were prepared. Two diameter measurements, perpendicular to each other, were taken daily for each colony until the fastest-growing culture had covered the 90 mm Petri dishes. Averages of the diameter measurements at each of the seven temperatures were computed with Microsoft Excel 2016 (Microsoft Corporation, Albuquerque, NM, USA). Colony colors were determined by incubating the isolates on fresh 2% MEA at 25 °C in the dark after 7 days. The color descriptions of the sporocarps and colonies were according to the color charts of Rayner (1970).

Pathogenicity tests

In this study, inoculations were conducted on two different Eucalyptus hybrid genotypes (CEPT46 and CEPT53) and T.neotaliala to understand the pathogenicity on Eucalyptus plantations and to fulfill Koch’s postulates. The selected isolates were grown on 2% MEA at 25 °C for 10 days before inoculation. Each selected isolate was inoculated on 10 seedlings or branches of each inoculated tree, and 10 additional seedlings or branches were inoculated with sterile MEA plugs to serve as negative controls. The inoculations were conducted in August 2019, and the results were evaluated after 7 weeks by measuring the lengths of the lesions on the cambium.

Inoculations were conducted on T.mantaly and two widely planted E.grandis hybrid genotype (CEPT46, CEPT53) to fulfill Koch’s postulates and understand the pathogenicity on Eucalyptus plantations. The selected isolates were grown on 2% MEA at 25 °C for 10 d before inoculation. Each of the selected isolates was inoculated on 10 seedlings or branches of each selected tree variety, and 10 additional seedlings or branches were inoculated with sterile MEA plugs to serve as negative controls. The inoculations on seedlings of two 1-year-old Eucalyptus hybrid genotypes were conducted in the glasshouse, and the inoculations on branches of 10-year-old T.mantaly were conducted in the field. The inoculations method followed Chen et al. (2010, 2013b).

Inoculations were conducted in August 2019 and the results were evaluated after 7 weeks by measuring the lengths (mm) of the lesions on the cambium. For re-isolations, small pieces of discolored xylem from the edges of the resultant lesions were cut and placed on 2% MEA at room temperature. Re-isolations of all seedlings/branches inoculated as negative controls and from four randomly selected trees per isolate were conducted. The identities of the re-isolated fungi were confirmed by morphological comparisons. The inoculation results were analyzed using SPSS Statistics 26 software (BM Corp., Armonk, NY, USA) by one-way analysis of variance (ANOVA).

Results

Isolation

Diseased samples from 14 trees were collected from three sites (20190523-1, 20190525-2, 20190525-3) of T.neotaliala (Fig. 1A) nurseries, and two sites (20190525-1, 20190525-4) of T.mantaly (Fig. 1B) nurseries (Table 2). In the surveyed sites, 10%–25% of Terminalia trees were infected. Cankers with stromata on the main stem bark surface, which often resulted in tree death, were observed on two to five-year-old T.neotaliala trees (Fig. 1C). Obvious orange conidiomata were observed on the branches and twigs of three-year-old T.mantaly trees (Fig. 1D, E). Developing lesions were observed on the main stem of T.neotaliala and resulting in bark depression (Fig. 1F) and xylem necrosis (Fig. 1G). Orange fruiting structures even presented on the barks of the main stem base (Fig. 1H) and roots (Fig. 1I). The fruiting structures on T.neotaliala and T.mantaly displayed the typical morphological characteristics of Cryphonectriaceae (Gryzenhout et al. 2009; Wang et al. 2020). Isolates obtained from the asexual fruiting structures on MEA were white when young and turned yellow with age, and the isolates on MEA exhibited typical morphological characteristics of Cryphonectriaceae. Twenty isolates from both T.neotaliala and T.mantaly in the five sampled nurseries were isolated and sequenced for further studies (Table 2).

Figure 1.

Disease symptoms on Terminalia trees associated with infection by Aurifilum spp. ATerminalianeotaliala in the field BTerminaliamantaly in a nursery C the main stems and branches of T.neotaliala infected by Aurifilum species and resulted in tree death D, E sporocarps of Aurifilum species on the main stem of T.neotaliala (D), and branch of T.mantaly (E) F, G lesions developing on the branches of T.neotalialaH, I Sporocarps of Aurifilum species on the base of main stem (H) and roots of T.neotaliala (I).

Table 2.

Isolates sequenced and used for phylogenetic analyses, morphological studies and pathogenicity tests in the current study.

Identity	Isolate Number	Genotypea	Host	Nursery No.	Location	GPS iformation	Collector	GenBank accession No.					References
								ITS	tub2	tub1	tef1	rpb2
A.terminali	CSF16295	AAAAA	T.neotaliala	20190523-1	ChaTing, LingBei, SuiXi	21°16′06.97"N, 110°5′16.8432"E	S.F.Chen & W. Wang	OQ912905	OQ921705	OQ921623	OQ921643	OQ921663	This study
A.terminali	CSF16309	AAAAA	T.mantaly	20190525-1	DaJia, SuiCheng, SuiXi	21°18′44.19"N, 110°11′46.7268"E	S.F.Chen & W. Wang	OQ912906	OQ921706	OQ921624	OQ921644	OQ921664	This study
A.terminali	CSF16310d	AAAAA	T.mantaly	20190525-1	DaJia, SuiCheng, SuiXi	21°18′44.19"N, 110°11′46.7268"E	S.F.Chen & W. Wang	OQ912907	OQ921707	OQ921625	OQ921645	OQ921665	This study
A.terminali	CSF16356d	AAAAA	T.neotaliala	20190525-3	DiaoLou, LingBei, SuiXi	21°15′57.006"N, 110°12′26.5824"E	S.F.Chen & W. Wang	OQ912908	OQ921708	OQ921626	OQ921646	OQ921666	This study
A.terminali	CSF16377	AAAAA	T.mantaly	20190525-4	DiaoLou, LingBei, SuiXi	21°15′57.006"N, 110°12′26.5824"E	S.F.Chen & W. Wang	OQ912909	OQ921709	OQ921627	OQ921647	OQ921667	This study
A.terminali	CSF16380	AAAAA	T.mantaly	20190525-4	DiaoLou, LingBei, SuiXi	21°15′57.006"N, 110°12′26.5824"E	S.F.Chen & W. Wang	OQ912910	OQ921710	OQ921628	OQ921648	OQ921668	This study
A.terminali	CSF16343d	AABAA	T.neotaliala	20190525-2	DuHao, MaZhang, MaZhang	21°14′16.4076"N, 110°17′23.9964"E	S.F.Chen & W. Wang	OQ912911	OQ921711	OQ921629	OQ921649	OQ921669	This study
A.terminali	CSF16387	AABAA	T.mantaly	20190525-4	DiaoLou, LingBei, SuiXi	21°15′57.006"N, 110°12′26.5824"E	S.F.Chen & W. Wang	OQ912912	OQ921712	OQ921630	OQ921650	OQ921670	This study
A.terminali	CSF16388d	AABAA	T.mantaly	20190525-4	DiaoLou, LingBei, SuiXi	21°15′57.006"E 110°12′26.5824"E	S.F.Chen & W. Wang	OQ912913	OQ921713	OQ921631	OQ921651	OQ921671	This study
A.cerciana	CSF16384c, d = CGMCC3.20108	BBCBB	T.mantaly	20190525-4	DiaoLou, LingBei, SuiXi	21°15′57.006"N, 110°12′26.5824"E	S.F.Chen & W. Wang	OQ912914	OQ921714	OQ921632	OQ921652	OQ921672	This study
A.cerciana	CSF16250	BBCBB	T.neotaliala	20190523-1	ChaTing, LingBei, SuiXi	21°16′06.97"N, 110°5′16.8432"E	S.F.Chen & W. Wang	OQ912915	OQ921715	OQ921633	OQ921653	OQ921673	This study
A.cerciana	CSF16251	BBCBB	T.neotaliala	20190523-1	ChaTing, LingBei, SuiXi	21°16′06.97"N, 110°5′16.8432"E	S.F.Chen & W. Wang	OQ912916	OQ921716	OQ921634	OQ921654	OQ921674	This study
A.cerciana	CSF16261b, c, d = CGMCC3.20107	BBCBB	T.neotaliala	20190523-1	ChaTing, LingBei, SuiXi	21°16′06.97"N, 110°5′16.8432"E	S.F.Chen & W. Wang	OQ912917	OQ921717	OQ921635	OQ921655	OQ921675	This study
A.cerciana	CSF16262	BBCBB	T.neotaliala	20190523-1	ChaTing, LingBei, SuiXi	21°16′06.97"N, 110°5′16.8432"E	S.F.Chen & W. Wang	OQ912918	OQ921718	OQ921636	OQ921656	OQ921676	This study
A.cerciana	CSF16267	BBCBB	T.neotaliala	20190523-1	ChaTing, LingBei, SuiXi	21°16′06.97"N, 110°5′16.8432"E	S.F.Chen & W. Wang	OQ912919	OQ921719	OQ921637	OQ921657	OQ921677	This study
A.cerciana	CSF16268	BBCBB	T.neotaliala	20190523-1	ChaTing, LingBei, SuiXi	21°16′06.97"N, 110°5′16.8432"E	S.F.Chen & W. Wang	OQ912920	OQ921720	OQ921638	OQ921658	OQ921678	This study
A.cerciana	CSF16273	BBCBB	T.neotaliala	20190523-1	ChaTing, LingBei, SuiXi	21°16′06.97"N, 110°5′16.8432"E	S.F.Chen & W. Wang	OQ912921	OQ921721	OQ921639	OQ921659	OQ921679	This study
A.cerciana	CSF16385	BBCBB	T.mantaly	20190525-4	DiaoLou, LingBei, SuiXi	21°15′57.006"N, 110°12′26.5824"E	S.F.Chen & W. Wang	OQ912922	OQ921722	OQ921640	OQ921660	OQ921680	This study
A.cerciana	CSF16351c, d	BBCBC	T.neotaliala	20190525-3	DiaoLou, LingBei, SuiXi	21°15′57.006"N, 110°12′26.5824"E	S.F.Chen & W. Wang	OQ912923	OQ921723	OQ921641	OQ921661	OQ921681	This study
A.cerciana	CSF16352c, d	BBCBC	T.neotaliala	20190525-3	DiaoLou, LingBei, SuiXi	21°15′57.006"N, 110°12′26.5824"E	S.F.Chen & W. Wang	OQ912924	OQ921724	OQ921642	OQ921662	OQ921682	This study

^a Genotype determined by sequence of ITS, tub2, tub1, tef1, and rpb2 four regions. ^b Isolates ex-type. ^c Isolates used for culture growth. ^d Isolates used in pathogenicity.

Phylogenetic analysis

Phylogenetic analyses indicated that all of the Cryphonectriaceae genera formed independent phylogenetic clades with high bootstrap values (ML > 77%, MP > 100%) both in the ML and MP analyses, with the exception of Aurifilum, and strains sequenced in this study formed sub-clades (Fig. 2). The partition homogeneity test (PHT), comparing the combined ITS and BT2/BT1 loci dataset generated a value of P was 0.68, indicating some incongruence in the dataset of the four loci, and the accuracy of the combined data suffered relative to the individual partitions (Huelsenbeck et al. 1996; Cunningham 1997).

Figure 2.

Phylogenetic trees based on maximum likelihood (ML) analyses of combined DNA sequence dataset of combination of ITS and BT2/BT1 regions for species in Cryphonectriaceae. combination of, TEF-1α and rpb2 regions. Bootstrap values ≥ 70% for ML and MP (maximum parsimony) analyses are presented at branches as ML/MP. Bootstrap value lower than 70% are marked with *, and absent analysis value are marked with –. Isolates representing Aurifilumcerciana are in shade, and isolates obtained in this study are in bold and blue. Diaportheambigua (CMW55887) was used as outgroup taxon.

Further species analyses selected twenty-four Aurifilum isolates (Table 2). Based on the sequences of ITS, BT2/BT1, TEF-1α, rpb2 sequences, four genotypes were generated for the 20 isolates sequenced in this study (Table 2). Sequences for two ex-type specimen strains and other of two Aurifilum species related to isolates obtained in this study were downloaded from GenBank (Table 1). Celoporthecerciana (CERC9128) was used as an outgroup taxon. The partition homogeneity test (PHT), comparing the combined ITS, BT2/BT1, TEF-1α and rpb2 loci dataset generated a value of P was 1, indicating some incongruence in the dataset of the four loci, and the accuracy of the combined data suffered relative to the individual partitions (Huelsenbeck et al. 1996; Cunningham 1997). Although the P value was high, the sequence of four loci was combined and subjected to phylogenetic analyses. All sequences obtained for the isolates of Aurifilum in this study were deposited in GeneBank (Table 2). The number of taxa and characters in each of the datasets, and the summary of the most important parameters applied in the MP and ML analyses, are presented in Table 3. The six datasets were deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S30284?x-access-code=cf2a0ef843604b8fa4301eced72cec7f&format=html,30284).

Table 3.

Datasets used and statistics resulting from phylogenetic analyses.

Dataset	No. of taxa	No. of bp a	Maximum parsimony
			PIC b	No. of trees	Tree length	CI c	RI d	RC e	HI f
ITS+BT	116	1465	4	1	6	1.000	1.000	1.000	0
ITS	25	558	3	1	3	1.000	1.000	1.000	0
BT	25	907	12	1	12	1.000	1.000	1.000	0
TEF	23	266	1	1	1	1.000	1.000	1.000	0
rpb2	23	1058	6	1	6	1.000	1.000	1.000	0
ITS+BT+TEF+rpb2	25	2789	22	1	22	1.000	1.000	1.000	0
Dataset	Maximum likelihood
	Subst. model g	NST h	Rate matrix					Ti/Tv ratio i	p-inv	Gamma	Rates
ITS+BT	TPM2uf+I+G	6	1.428	4.552	1.428	1.000	4.526	4.525	0.445	1.107	gamma
ITS	TrNef	6	1.000	1.389	1.000	1.000	3.247	–	0	–	equal
BT	TrN	6	1.000	2.380	1.000	1.000	5.893	–	0	–	equal
TEF	TrN	6	1.000	1.989	1.000	1.000	4.887	–	0	–	equal
rpb2	TrN+G	6	1.000	4.377	1.000	1.000	233.189	–	0	0.055	gamma
ITS+BT+TEF+rpb2	TrN	6	1.000	2.257	1.000	1.000	7.842	–	0	–	equal

^abp = base pairs. ^bPIC = number of parsimony informative characters. ^cCI = consistency index. ^dRI = retention index. ^eHI = homoplasy index. ^fRC = rescaled consistency index. ^gmodel = best-fit substitution model. ^hNST = number of substitution rate categories. ^ITi/Tv ratio = transition/transversion ratio.

For each of the six datasets, the MP and ML analyses generated trees with generally consistent topologies and phylogenetic relationships among taxa. Among the trees generated by the Aurifilum spp. single loci dataset, the BT2/BT1, TEF-1α, rpb2 show that 20 isolates obtained in this study mainly grouped into two clades, one clade contained nine isolates cluster into a lineage with A.terminali, the other 11 isolates clade formed a novel monophyletic lineage that was distinct from any known Aurifilum sp., and was supported by high bootstrap values in these gene trees (Fig. 3B–D).

Figure 3.

Phylogenetic trees based on maximum likelihood (ML) analyses for species in AurifilumAITS region B two regions of β-tublin (BT2/BT1) CTEF-1α gene region Drpb2 gene region E combination of ITS, BT2/BT1, TEF-1α and rpb2 regions. Bootstrap values ≥ 70% for ML and MP (maximum parsimony) analyses are presented at branches as ML/MP. Bootstrap value lower than 70% are marked with *, and absent analysis value are marked with –. Isolates representing A.cerciana are in shade, and isolates obtained in this study are numbered followed CSF. Celoporthecirciana (CERC9128) was used as outgroup taxon.

Among the BT2/BT1 trees, isolate CSF16343, CSF16387, CSF16388 grouped into the lineage with A.terminali, and among the rpb2 tree, isolates CSF16351, CSF16352 grouped into the novel lineage, formed a single independent branch but the bootstraps value within the clades were not significant (Fig. 3B, D), which suggests that these differences reflect intraspecific rather than interspecific variation. The combined ITS, BT2/BT1, TEF-1α and rpb2 tree (Fig. 3E) indicated that the isolates grouped into novel lineage are putative undescribed species of Aurifilum (bootstrap values of the combined dataset, ML and MP: 96 and 100%).

Morphology and taxonomy

Based on phylogenetic analyses and morphology characteristics, the isolates from Terminalia trees in southern China represent two distinct species in Aurifilum. Isolates CSF16295, CSF16309, CSF16310, CSF16343, CSF16356, CSF16377, CSF16380, CSF16387, CSF16388 in phylogenetic cluster with A.terminali (Fig. 3B–E), and isolates CSF16343, CSF16387, CSF16388 appear a branch in BT2/BT1, rpb2, and combine trees (Fig. 3B, D, E) in this cluster, was finally identified as A.terminali. The isolates in the other cluster present a novel species in Aurifilum, here named as Aurifilumcerciana sp. nov. (Fig. 3); this unknown species was described as follows:

. Aurifilum cerciana

W. Wang & S.F. Chen sp. nov.

D4F3F5AD-9AEF-509E-B4E4-E43FA218912D

MycoBank No: 848235

Fig. 4

Figure 4.

Morphological characteristics of AurifilumcercianaA, B conidiomata on the bark C longitudinal section through conidioma showing umber stroma D prosenchymatous stromatic tissue of the conidia E paraphyses F conidiophores and conidiogenous cells G conidia H, I colony of A.cerciana on MEA after 7 days at 25 °C H front I reverse. Scal bars: 200 µm (A); 100 µm (B, C); 10 µm (D, E, F); 5 µm (G); 1 cm (H, I).

Etymology.

the name refers to China Eucalypt Research Centre (CERC), the former institution of the Research Institute of Fast-Growing Trees (RIFT), which served as the identification site for this study on Terminalia trees disease caused by Aurifilum spp.

Stromata.

No ascostromata were observed on inoculated Eucalyptus branch tissue, the conidiomata on the inoculated Eucalyptus branch tissue were superficial to slightly immersed, pulvinate, globose pyriform to various shapes without necks, blight yellow when young, orange to brown when mature (Fig. 4A, B), unilocular, 46–236 μm (av. 142 μm) diameter (Fig. 4C). Stromatic tissue prosenchymatous (Fig. 4D). Stromatic conidiomatal base was 119 – 678 μm (av. 428 μm) high above the level of the bark and 58 – 269 μm (av. 158 μm) wide. Conidiomatal necks absent. Conidiomatal locules unilocular. Conidiophores, hyaline, branched irregularly at the base or above into cylindrical cells, with or without separating septa, (11.2–)23.8–28.6(–70.2) μm (av. 26.2 μm) long, (1.7–)2.3–3.7(–6.5) μm (av. 3 μm) wide (Fig. 4F). Conidiogenous cells phialidic, cylindrical, without attenuated apices, (0.8–)1.0 – 1.8(–2.6) μm (av. 1.4 μm) wide (Fig. 4F). Paraphyses or cylindrical sterile cells, occur among conidiophores, up to 99 μm (av. 51.4 μm) long (Fig. 4E). Conidia hyaline, non-septate, oblong to cylindrical, occasionally allantoid, extend through on opening at stromatal surface as orange droplets, (3.6–)4.3–4.5(–5.7) × (1.5–)1.8(–2.2) (av. 4.4 × 1.8 μm) (Fig. 4G).

Culture characteristics.

Colonies on MEA are fluffy with an uneven margin, white when young, turning pale luteous to luteous after 10 days, and reverse yellow to orange-white. Optimal growth temperature 35 °C, reaching the edge of the 90 mm plates after 7 days. No growth at 5, 10 °C. After 7 days, colonies at 15, 20, 25, 30, and 35 °C reached 15.8, 45.9, 49, 50.5, and 74.4 mm, respectively.

Substrate.

Bark of Terminalianeotaliala.

Distribution.

Guangdong Province, China.

Additional materials examined.

China, Guangdong Province, Zhanjiang Region, Suixi District, Chating Town (21°16′06.97″N, 110°5′16.8432″E) from branch bark of T.neotaliala tree, 23 May 2019, S. F. Chen & W. Wang, holotype, CSFF2078, HMAS350333, ex-type culture CSF16261 = CGMCC3.20107; Guangdong Province, Zhanjiang Region, Suixi District, Diaolou Town (21°15′57.006″N, 110°12′26.5824″E) from twigs of T.mantaly tree, 25 May 2019, S. F. Chen & W. Wang, CSFF2079, HMAS350334, culture CSF16384 = CGMCC3.20108.

Notes.

Three species were described in the genus Aurifilum, including A.marmelostoma, A.terminali, A.cerciana. Aurifilumcerciana morphologically differs from A.terminali by the absence of conidiomatal necks (Wang et al. 2020), and differs from A.marmelostoma by longer paraphyses (Begoude et al. 2010). A.cerciana could also be distinguished from A.terminali and A.marmelostoma by growth characteristics in culture. The optimal growth temperature of A.cerciana is 35 °C, whereas A.terminali grows relatively slowly at this temperature and no growth is observed for A.marmelostoma (Begoude et al. 2010; Wang et al. 2020).

Pathogenicity tests.

Eight isolates representing the two species of Aurifilum identified in this study were used to inoculate seedlings of two Eucalyptus hybrid genotypes, and branches of T.neotaliala. These include four isolates in A.terminali and A.cerciana, respectively (Table 2). Seedling stems or tree branches inoculated with Aurifilum isolates exhibited lesions, whereas the control group only showed wounds without any lesions. (Fig. 5). The lesions produced by Aurifilum species on T.neotaliala and Eucalyptus clones CEPT53 were significantly longer than the wounds on the controls (P < 0.05), whereas for the Eucalyptus clones CEPT46, the lesions produced by Aurifilum species were not significantly different (Fig. 5). The overall data revealed that A.cerciana and A.terminali have similar pathogenicity (Fig. 5). The overall data further showed that CEPT53 is more susceptible than CEPT46 to Aurifilum spp. (Fig. 5B). Yellow or orange fruiting structures and cankers were produced on the bark of inoculated trees within 7 weeks; these structures displayed similar characteristics of conidiomata on the Terminalia trees in the field and the re-isolated fungi from lesions share the same culture morphology with the Aurifilum fungi originally from the Terminalia trees in the nursery. The inoculated Aurifilum fungi were successfully re-isolated from the lesions but not from the control, indicating that the Koch’s postulates had been fulfilled.

Figure 5.

Column chart showing average lesion lengths (mm) produced by each isolate of Aurifilum on the branches of T.neotaliala (left) and two Eucalyptus hybrid genotypes (right). Eight isolates of Aurifilum were used. Vertical bars represent the standard error of the means. Different letters above the bars indicate treatments that were statistically significantly different (P = 0.05).

Discussion

In this study, many Aurifilum isolates were obtained from diseased Terminalia trees in Southern China, and two species of four genotypes belonging to Aurifilum were identified from two species of Terminalia. Including the new taxon, A.cerciana sp. nov., there are fifty-seven taxa in the Cryphonectriaceae.

In the genus Aurifilum, A.marmelostoma was the first described species, which was isolated from the bark of native T.ivorensis and the dead branches of non-native T.mantaly in Cameroon (Begoude et al. 2010), and the A.terminali, the second identified species, was isolated from non-native T.neotaliala in southern China (Wang et al. 2020). In the present study, a new species, A.cerciana was isolated from non-native T.neotaliala and T.mantaly, and a previously known species, A.terminali was isolated from T.mantaly too. The species T.mantaly was a newly reported host for A.terminali. Our results indicated that the Aurifilum species are widely distributed on non-native Terminalia trees in southern China, which is consistent with the previous hypothesis of Wang et al. (2020).

Members of the Cryphonectriaceae are well known to occur on Myrtales in Southern China. Prior to this study, six genera, including Aurifilum, Celoporthe, Chrysoporthe, Chrysomorbus, Corticimorbus, Parvosmorbus were reported infecting trees in Combretaceae, Lythraceae, Melastomataceae and Myrtaceae (All Myrtales) in southern China (Chen et al. 2010, 2011, 2016, 2018; Wang et al. 2020). Although the diversity of Cryphonectriaceae in Myrtales has been extensively studied in recent years (Chen et al. 2010, 2011, 2013a, b, 2016a, b, 2018; Wang et al. 2018, 2020; Huang et al. 2022), there is still a need for further investigation into its diversity, geographical distribution, and host range in China (Wingfield et al. 2015).

Pathogenicity test showed that all tested Aurifilum isolates were pathogenic to mature T.neotaliala and E.grandis hybrid genotypes of CEPT53 and CEPT46 seedlings. To clarify the threat of these pathogens to Eucalyptus plantations, further inoculations on mature Eucalyptus in the field should be conducted. Variations in pathogenicity among different individuals of the same species have been observed, with some strains showing stronger pathogenicity in different hosts. This phenomenon has also been observed in previous studies (Chen et al. 2010, 2011, 2013a, b; 2016a, b, 2018; Wang et al. 2018, 2020), and further comparison of the genetic features of these individual exhibiting differences in pathogenicity may help reveal the pathogenic mechanisms of the pathogen.

Citation

Wang W, Chen S (2023) Identification and pathogenicity of Aurifilum species (Cryphonectriaceae, Diaporthales) on Terminalia species in Southern China. MycoKeys 98: 37–58, https://doi.org/10.3897/mycokeys.98.104719

Funding Statement

This study was initiated through the bilateral agreement between the Governments of South Africa and China and supported by the National Key R&D Program of China (China-South Africa Forestry Joint Research Centre Project; project No. 2018YFE0120900)

Additional information

Conflict of interest

No conflict of interest was declared.

Ethical statement

No ethical statement was reported.

Funding

No funding was reported.

Author contributions

Conceptualization: SC, WW. Data curation: SC, WW. Formal analysis: WW. Funding acquisition: SC. Investigation: WW. Methodology: WW. Project administration: SC. Software: WW. Supervision: WW, SC. Writing - original draft: WW. Writing - review and editing: WW.

Author ORCIDs

ShuaiFei Chen https://orcid.org/0000-0002-3920-9982

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.