Ophiostomatoid fungi associated with pines infected by Bursaphelenchus xylophilus and Monochamus alternatus in China, including three new species

HuiMin Wang; YingYing Lun; Quan Lu; HuiXiang Liu; Cony Decock; XingYao Zhang

doi:10.3897/mycokeys.39.27014

Research Article

Ophiostomatoid fungi associated with pines infected by Bursaphelenchus xylophilus and Monochamus alternatus in China, including three new species

HuiMin Wang^‡, YingYing Lun^‡§|, Quan Lu^‡, HuiXiang Liu^¶, Cony Decock^#, XingYao Zhang^‡

‡ Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing, China

§ Shandong Agricultural University, Taian, China

| Longju Ecological Forest Farm, Dongying, China

¶ College of Plant Protection of Shandong Agricultural University, Taian, China

# Mycothèque de l’Université Catholique de Louvain, Louvain-la-Neuve, Belgium

Corresponding author: Quan Lu ( luquan@caf.ac.cn )

Corresponding author: HuiXiang Liu ( hxliu@sdau.edu.cn )

Academic editor: Marc Stadler

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: Wang HM, Lun YY, Lu Q, Liu HX, Decock C, Zhang XY (2018) Ophiostomatoid fungi associated with pines infected by Bursaphelenchus xylophilus and Monochamus alternatus in China, including three new species. MycoKeys 39: 1-27. https://doi.org/10.3897/mycokeys.39.27014

Abstract

The activity of the pine wood nematode Bursaphelenchus xylophilus leads to extremely serious economic, ecological and social losses in East Asia. The nematode causes pine wilt disease, which is currently regarded as the most important forest disease in China. The pathogenic nematode feeds on dendrocola fungi to complete its cycle of infection. As the vector of the nematode, the Japanese pine sawyer (Monochamus alternatus) also carries dendrocola fungi. Pine woods, infected by B. xylophilus and tunnelled by M. alternatus, are also inhabited by ophiostomatoid fungi. These fungi are well known for their association with many bark and ambrosia beetles. They can cause sapstain and other serious tree diseases. The aims of our study were to investigate and identify the ophiostomatoid communities associated with the epidemic pine wood nematode and the pine sawyer in Pinus massoniana and P. thunbergii forests, which are the main hosts of the pine wood nematode in China. Two hundred and forty strains of ophiostomatoid fungi were isolated from nematode and sawyer–infected trees in the coastal Shandong and Zhejiang Provinces, representing newly and historically infected areas, respectively. Six ophiostomatoid species were identified on the basis of morphological, physiological and molecular data. For the latter, DNA sequences of the internal transcribed spacer (ITS1–5.8S–ITS2) region and partial b-tubulin gene were examined. The ophiostomatoid species included one known species, Ophiostoma ips, three novel species, viz. Ophiostoma album sp. nov., Ophiostoma massoniana sp. nov. and Sporothrix zhejiangensis sp. nov. and two species whose identities are still uncertain, Ophiostoma cf. deltoideosporum and Graphilbum cf. rectangulosporium, due to the paucity of the materials obtained. The ophiostomatoid community was dominated by O. ips. This study revealed that a relatively high species diversity of ophiostomatoid fungi are associated with pine infected by B. xylophilus and M. alternatus in China.

Keywords

Ophiostoma , taxonomy, Sporothrix , Ophiostoma minus complex, Ophiostoma ips complex

Introduction

The pathogenic pine wood nematode (PWN) Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle (Aphelenchida, Parasitaphelenchidae), presumably native to North America (Steiner and Buhrer 1934, Robbins 1982, Ryss et al. 2005, Zhao et al. 2014), is a mild threat to pine trees in its native area. Nevertheless, this species and the concomitant systematic wilt symptom are responsible for pine tree deaths affecting many trees in eastern Asia, notably in Japan and China (Evans et al. 1996, Mota and Vieira 2008, Mamiya and Shoji 2009, Jung 2010, Futai 2013). Since the first report in China, in Nanjing City in 1982, the disease has spread through more than 300 counties in the provinces of Jiangsu, Zhejiang, Shandong and others, which are currently listed as PWN epidemic areas (State Forestry Administration of the People’s Republic of China 2018). The wilt disease has caused enormous losses not only to the economy and ecology, but also to society, becoming one of the most serious ecological devastation events in Chinese forests.

Bursaphelenchus xylophilus infects many species of coniferous trees, mainly from the genus Pinus (Yan et al. 2003). Pinus armandii, P. kesiya var. langbianensis, P. koraiensis, P. massoniana, P. tabuliformis, P. taiwanensis, P. thunbergii and P. yunnanensis are naturally infected by PWN in China (Zhao and Sun 2017). During the infection cycle, the nematode needs vector beetles for dispersal and inoculation into new hosts. The Japanese pine sawyer, Monochamus alternatus Hope (Coleoptera, Cerambycidae), is considered to be the primary PWN vector indigenous to Asia. At the initial stage of infection, PWN feeds on epithelial cells of the host pine (Mota and Vieira 2008, Zhao et al. 2008, Futai 2013). Upon tree death, it feeds on the dendrocola fungi to maintain its population and propagate (Suh et al. 2013, Zhao et al. 2013, 2014).

The ophiostomatoid fungi are one of the most common fungal groups inhabiting wood infected by B. xylophilus. Further, many ophiostomatoid reproduction structures are detected in the tunnels of M. alternatus, suggesting a relationship between the fungi and the occurrence and development of the disease. For instance, O. ips has been found in the PWN vector beetles in North America, China and Korea (Wingfield 1987, Suh et al. 2013, Zhao et al. 2014). There is some evidence that the fungi adhere to the body surface of adult M. alternatus and thus are transmitted to the twigs of healthy trees (Suh et al. 2013).

The association of PWN with ophiostomatoid fungi and bacteria likely contributes to the nematode’s pathogenicity (Zhao et al. 2013, Zhao and Sun 2017). Ophiostoma minus and Sporothrix sp. can stimulate the reproduction of PWN and, consequently, the numbers of PWN carried by the emerging beetles (Maehara and Futai 1997, Zhao et al. 2013, Zhao and Sun 2017). Moreover, the fragrant diacetone alcohol released from wood infected by Sporothrix sp. 1 can induce B. xylophilus to produce greater number of offspring and promotes beetle growth and survival (Zhao et al. 2013).

Thus far, the association with PWN and Monochamus spp. has been documented for only five species of ophiostomatoid fungi worldwide (Wingfield 1987, Maehara and Futai 1997, Hyun et al. 2007, Suh et al. 2013, Zhao et al. 2013, Zhao and Sun 2017). Determination of the identities of these species is mainly based on morphology and sequence comparisons of a single DNA locus. Given the diversity of ophiostomatoid fungi associated with other beetles, the serious impact of the nematode and sawyers on wood and the potential importance of these fungi in the disease infection cycle, studies of the diversity and occurrence of the ophiostomatoid fungi involved in the pine wilt disease should be intensified. Such studies will enable understanding of the interaction between the disease system and the fungi, ultimately helping to redress the current situation of the ceaseless outbreaks and rapid expansion of the disease.

The aims of the current study were to investigate and identify the ophiostomatoid mycobiota associated with the nematode and sawyer in the epidemic forests of Shandong and Zhejiang Provinces in eastern China to facilitate the understanding of pine wilt disease infection and prevalence mechanisms. The two coastal provinces, Shandong and Zhejiang, represent new and historic epidemic areas, with P. thunbergii and P. massoniana as hosts, respectively.

Materials and methods

Collection of samples and fungus isolations

Fungi were isolated from 98 samples of M. alternatus galleries or pupal chambers in P. massoniana and P. thunbergii in the Zhejiang and Shandong Provinces (Table 1), in November 2012. All host trees used for sample collection in this study were exhibiting weak or dying symptoms, blue stain and 4–5 instar larvae residing inside after dissecting the stems. The nematodes were also isolated from these galleries and pupal chambers by Behrman funnel. The fungi were isolated on the surface of 2% (w/v) water agar (20 g agar powder in 1000 ml of deionised water) in 9 cm wide Petri dishes and incubated at 25 °C (Seifert et al. 1993, Zhao et al. 2013, Chang et al. 2017). Subsequently, all strains were purified by hyphal tip isolation, using the procedure described by Jacobs and Wingfield (2001) and routinely grown on 2% (w/v) malt extract agar (MEA; 20 g malt extract powder and 20 g agar powder in 1000 ml of deionised water). Representative cultures were deposited in the China Forestry Culture Collection Center (CFCC), culture collection of the Chinese Academy of Forestry (CXY) and part of the Belgian Coordinated Collections of Microorganisms (MUCL), culture collection at Université Catholique de Louvain, Belgium.

Table 1.

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Strains of ophiostomatoid fungi isolated from pines infested by Monochamus alternatus and pine wood nematode in the current study.

Group	Species	Strain No.	Host	Origin (Latitude, Longitude)	Genbank No.		Collector
Group	Species	Strain No.	Host	Origin (Latitude, Longitude)	ITS	β-tubulin	Collector
A	Sporothrix zhejiangensis sp. nov.	MUCL 55181 (CFCC52167, CXY1612)	Pinus massoniana	Yuyao, Zhejiang (29°58'10.2"N, 121°05'57.1"E)	KY094069	MH397728	Q. Lu, YY Lun
		MUCL 55182 (CFCC52164, CXY1613)	P. massoniana	Yuyao, Zhejiang (29°58'10.2"N, 121°05'57.1"E)	KY094070	MH397729
		MUCL 55183 (CFCC52165, CXY1614)	P. massoniana	Yuyao, Zhejiang (29°58'10.2"N, 121°05'57.1"E)	KY094071	MH397730
		MUCL 55184 (CFCC52166, CXY1615)	P. massoniana	Yuyao, Zhejiang (29°58'10.2"N, 121°05'57.1"E)	KY094072	MH397731
B	Ophiostoma album sp. nov.	MUCL 55189 (CFCC52168, CXY1622)	P. massoniana	Yuyao, Zhejiang (29°58'10.2"N, 121°05'57.1"E)	KY094073	MH360979
		MUCL 55190 (CFCC52169, CXY1642)	P. massoniana	Yuyao, Zhejiang (29°58'10.2"N, 121°05'57.1"E)	KY094074	MH360980
		CFCC52170 (CXY1643)	P. massoniana	Yuyao, Zhejiang (29°58'10.2"N, 121°05'57.1"E)	KY094075	MH360981
C	Ophiostoma ips	CXY1628	P. thunbergii	Changdao, Shandong (37°59'13.5"N, 120°42'18.1"E)	KY593324	MH324804
		CXY1631	P. thunbergii	Zhoushan, Zhejiang (29°52'51.33"N, 122°24'14.13"E)	MH324811	MH324805
		CXY1635	P. massoniana	Yuyao, Zhejiang (29°58'10.2"N, 121°05'57.1"E)	MH324812	MH324808
		CXY1638	P. thunbergii	Fuyang, Zhejiang (30°05'15.1"N, 119°58'55.1"E)	MH324813	MH324809
		CXY1639	P. massoniana	Weihai, Shandong (37°23'23.6"N, 122°32'33.1"E)	MH324814	MH324810
D	Ophiostoma massoniana sp. nov.	MUCL 55179 (CFCC51648, CXY1610)	P. massoniana	Fuyang, Zhejiang (30°05'15.1"N, 119°58'55.1"E)	KY094067	MH370810
D	Ophiostoma massoniana sp. nov.	MUCL 55180 (CFCC51649, CXY1611)	P. massoniana	Yuyao, Zhejiang (29°59'36.87"N, 121°09'09.90"E)	KY094068	MH370811
E	Graphilbum cf. rectangulosporium	CXY1623	P. massoniana	Yuyao, Zhejiang (29°59'36.87"N, 121°09'09.90"E)	MH324816	–
F	Ophiostoma cf. deltoideosporum	MUCL 55191 (CXY1640)	P. thunbergii	Weihai, Shandong (37°23'23.6"N, 122°32'33.1"E)	MH324815	–

Culture and morphological studies

The ophiostomatoid fungal strains were incubated on 2% MEA and 2% potato dextrose agar (PDA; 200 g potato and 20 g dextrose, 20 g agar powder in 1000 ml of deionised water: the dextrose was obtained from American Amresco) in the dark at 25 °C in an incubator. Fungal growth on MEA plates was monitored daily. Hyphal tips of emerging colonies were transferred to fresh MEA plates to purify the fungi. Slides were made to observe the sexual/asexual state structures; these were mounted in lactic acid cotton blue on glass slides and examined under a BX51 OLYMPUS microscope. Fifty measurements were made of each microscopic taxonomically informative structure. The measurements are presented in the form: (minimum–) mean minus standard deviation–mean plus standard deviation (–maximum).

A 5-mm mycelium disc was cut from an actively growing fungal colony using a sterile cork borer and placed at the centre of MEA plates, with the aerial mycelium side in contact with the medium. Three replicate plates were prepared for each strain and were incubated at temperatures ranging from 5–40 °C at five-degree intervals. The colony diameters on each Petri dish were determined along two perpendicular axes every day until the entire dish was covered. The colour descriptions were provided according to Rayner (1970).

DNA extraction, PCR and sequencing reactions

DNA was extracted from freshly collected mycelia grown in liquid malt medium (20g malt extract in 1000 ml of deionised water) at 25 °C in the dark for 7 d using an Invisorb Spin Plant mini kit (Invitek, Berlin, Germany), following the manufacturer’s instructions. The internal transcribed spacer (ITS) regions and partial β–tubulin (tub2) genes were amplified using primer pairs ITS1/ITS4 (White et al. 1990) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively.

PCR reactions were performed in 25 ml volumes (2.5 mM MgCl₂, 1X PCR buffer, 0.2 mM dNTP, 0.2 mM of each primer and 2.5 U of Taq polymerase). The conditions for ITS and tub2 PCR amplifications were as described earlier (White et al. 1990, Glass and Donaldson 1995). PCR products were purified using an MSB Spin PCRapace kit (250) (Invitek), following the manufacturer’s instructions.

Sequencing reactions were performed using CEQ DTCS Quick Start KitH (Beckman Coulter, American), following the manufacturer’s instructions, with the same PCR primers as above. Nucleotide sequences were determined using a CEQ 2000 XL capillary automated sequencer (Beckman Coulter).

Phylogenetic analyses

Contigs were subjected to BLAST searches of the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/); published sequences of closely related species were retrieved. Alignments of the related genes (most up-to-date sequence regions deposited in the GenBank) were conducted online using MAFFT v 7.0 (https://mafft.cbrc.jp/alignment/server/index.html) (Katoh and Standley 2013) and the E-INS-i strategy. Subsequently, the datasets were checked manually by using MEGA v 5.2 (Tamura et al. 2011). Gaps were treated as a fifth base. Phylogenetic analyses were performed using maximum parsimony (MP), as implemented in PAUP* v 4.0b10 (Swofford 2003); Bayesian Inference (BI), as implemented in MrBayes v 3.1.2 (Huelsenbeck and Ronquist 2001); and Maximum Likelihood (ML), using PhyML v 3.0 (Guidon and Gascuel 2003).

The most parsimonious trees generated by MP analyses were identified by heuristic searches with a random addition sequence (1000); max trees were set to 200 and further evaluated by bootstrap analysis, retaining clades compatible with the 50% majority rule in the bootstrap consensus tree. The analysis was based on tree bisection reconnection branch swapping (TBR). The tree length (TL), consistency index (CI), retention index (RI), homoplasy index (HI) and rescaled consistency index (RC) were recorded for each dataset after tree generation.

The general-time-reversible (GTR) model for ML analyses was selected using the Akaike Information Criterion (AIC) in ModelTest v 3.7 (Posada and Crandall 1998). ML runs performed using the CIPRES cluster at the San Diego Supercomputing Center (USA). Node support was estimated from 1000 bootstrap replicates.

For BI analyses, the most appropriate substitution models were also selected using the general-time-reversible model (GRT) with AIC in ModelTest v 3.7. BI was carried out with MrBayes using the Markov Chain Monte Carlo (MCMC) approach with 5,000,000 generations, to estimate posterior probabilities.

Results

Fungal isolation and sequence comparison

In total, 240 strains belonging to Ophiostomatales were obtained from PWN-infected galleries and pupal chambers of M. alternatus. The strains were sorted into six morphological groups (groups A–F in Table 1), tentatively identified as Sporothrix, Ophiostoma and Graphilbum. After preliminary ITS sequence comparisons of all these strains, 11 strains were clearly disparate to any known species and the remaining 229 strains possessed > 99% similarity with type strain of O. ips (GenBank no. AY546704).

Phylogenetic analyses

ITS and tub2 sequences were generated for 16 strains and deposited in GenBank (Table 1). The ITS alignment matrix contained 110 sequences (Tables 1 and 2) and 651 characters, including gaps, following the preliminary determination of strain affinities using the BLAST search engine (GenBank). Due to the presence or absence in intron in the tub2 sequence in the Sporothrix and Ophiostoma lineage species (Zipfel et al. 2006, de Beer et al. 2016), three separate datasets were built for the tub2 sequences. These were Sporothrix, Ophiostoma minus complex and Ophiostoma tenellum complex datasets (Linnakoski et al. 2010, de Beer et al. 2013, 2016). The Sporothrix dataset contained 8 species, 17 sequences and 403 characters, including gaps. The O. minus dataset contained 5 species, 17 sequences and 447 characters, including gaps. The O. tenellum dataset contained 8 species, 14 sequences and 280 characters, including gaps.

Table 2.

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The information of references sequences used for phylogenetic analyses in this study.

Species	Strain No.	Host/insect	Country	Genbank No.		Reference
Species	Strain No.	Host/insect	Country	ITS	β-tubulin	Reference
Sporothrix abietina	CBS125.89	Abies vejari	Mexico	AF484453	KX590755	de Beer et al. 2003
S. aurorae	CMW19362	Pinus eliottii	South Africa	DQ396796	DQ396800	Francois et al. 2006
S. bragantina	CBS 474.91	Soil	Brazil	FN546965	FN547387	Madrid et al. 2010
S. bragantina	CBS 430.92	Soil	Brazil	FN546964	FN547386	Madrid et al. 2010
S. brasiliensis	Ss383	Felis catus	Brazil	KP890194	FN547387	Araujo et al. 2015
S. brunneoviolacea	CBS 124562	Soil	Spain	FN546959	FN547385	Madrid et al. 2010
S. brunneoviolacea	CBS 124564	Soil	Spain	FN546958	FN547384	Madrid et al. 2010
S. dentifunda	CMW13016	Quercus wood	Hungary	AY495434	AY495445	Aghayeva et al. 2005
S. dentifunda	CMW13017	Quercus wood	Poland	AY495435	AY495446	Aghayeva et al. 2005
S. epigloea	CBS 573.63	Tremella fusiformis	Argentina	KX590817	KX590760	de Beer et al. 2016
S. eucalyptigena	CPC 24638	Eucalyptus marginata	Western Australia	KR476721	N/A	Crous et al. 2015
S. gemella	CMW23057	Protea caffra	South Africa	DQ821560	DQ821554	Roets et al. 2008
S. inflata	CMW12529	Soil	Canada	AY495428	AY495438	Aghayeva et al. 2005
S. inflata	CMW12527	wheat-field soil	Germany	AY495426	AY495437	Aghayeva et al. 2005
S. nebularis	CMW27319	Orthotomicus erosus	Spain	DQ674375	N/A	Romón et al. 1900
S. nebularis	CMW27900	O. erosus	Spain	DQ674376	N/A	Romón et al. 1900
S. pallida	CBS131.56	Stemonitis fusca	Japan	EF127880	EF139110	de Meyer et al. 2008
S. pallida	CBS150.87	S. fusca	Japan	EF127879	EF139109	de Meyer et al. 2008
S. palmiculminata	CMW23049	Protea repens	South Africa	DQ316191	DQ821543	Francois et al. 2006
S. phasma	CMW20676	P. laurifolia	South Africa	DQ316219	DQ821541	Francois et al. 2006
S. proteara	CMW1103	P. caffra	South Africa	DQ316203	DQ316165	Francois et al. 2006
S. schenckii	MITS2474	N/A	Mexico	KP132783	N/A	Irinyi et al. 2015
S. schenckii	CBS 938.72	Human	Franch	KP017094	N/A	Irinyi et al. 2015
S. fusiforis	CMW9968	Populus nigra	Azerbaijan	AY280481	AY280461	Aghayeva et al. 2004
S. lunata	CMW10563	Carpinus betulus	Austria	AY280485	AY280466	Zhou et al. 2006
S. narcissi	CBS138.50	N/A	Canada	AY194510	KX590765	Jacobs et al. 2003
S. splendens	CMW872	Protea repens	South Africa	DQ316215	DQ316177	Francois et al. 2006
S. stenoceras	CMW2524	Acacia mearnsii	South Africa	AF484459	AY280473	de Beer et al. 2003
S. stenoceras	CBS237.32	pine pulp	Norway	AF484462	N/A	de Beer et al. 2003
S. thermara	CMW38930	Euphorbia ingens	South Africa	KR051115	KR051103	Ja et al. 2016
S. thermara	CMW38929	E. ingens	South Africa	KR051114	KR051102	Ja et al. 2016
S. stylites	CMW14543	Pine utility poles	Australia	EF127883	EF139096	de Meyer et al. 2008
Ophiostoma adjuncti	CMW135	Pinus ponderosa	USA	AY546696	N/A	Zhou et al. 2004
O. allantosporum	CBS185.86	P. pinaster	Europe	AY934506	N/A	Villarreal et al. 2005
O. angusticollis	Zoq16	N/A	N/A	EU109671	N/A	de Beer et al. 2016
O. angusticollis	CBS186.86	Pinus banksiana	USA	AY924383	KX590757	Villarreal et al. 2005
O. bicolor	CBS492.77	Picea glauca/Ips sp.	USA	DQ268604	DQ268635	Massoumi et al. 2007
O. candidum	CMW26484	Eucalyptus cloeziana	South Africa	HM051409	HM041874	Nkuekam et al. 2012
O. candidum	CMW26483	E. cloeziana	South Africa	HM051408	HM041873	Nkuekam et al. 2012
O. catonianum	C1084	Pyrus	Italy	AF198243	N/A	Gorton et al. 2004
O. coronatum	CBS 497.77	Pinus pinaster	Iberian Peninsula	AY924385	KX590758	Villarreal et al. 2005
O. cupulatum	C1194	Pseudotsuga	USA	AF198230	N/A	Uzunovic et al. 2000
O. deltoideosporum	WIN(M)41	N/A	N/A	EU879121	N/A	Mullineux and Hausner 2009
O. fasciatum	UM56	Pseudotsuga menziesii	CanadaCanada	EU913720	EU913759	Plattner et al. 2009
O. floccosum	C01-021	Girdled Picea rubens	Canada	AY194504	N/A	Jacobs et al. 2003
O. floccosum	C1086	Soil	Sweden	AF198231	N/A	Gorton et al. 2004
O. fumeum	CMW26813	Eucalyptus cloeziana	South Africa	HM051412	HM041878	Nkuekam et al. 2012
O. fumeum	CMW26818	E. cloeziana	South Africa	HM051415	HM041877	Nkuekam et al. 2012
O. fuscum	CMW23196	Picea abies	Finland	HM031504	HM031563	Linnakoski et al. 2010
O. himai ulmi	C1183	Ulmus	India	AF198233	N/A	Harrington et al. 2001
O. himai ulmi	C1306	Ulmus	India	AF198234	N/A	Harrington et al. 2001
O. ips	CMW7075	N/A	USA	AY546704	N/A	Zhou et al. 2004
O. ips	CMW22843	Orthotomicus erosus	N/A	DQ539549	N/A	Romón et al. 2007
O. japonicum	YCC099	N/A	N/A	GU134169	N/A	Yamaoka et al. 2009
O. kryptum	DAOM 229701	Picea abies/Tetropium sp.	Austria	AY304436	AY305685	Jacobs and Kirisits 2013
	DAOM 229702	Larix decidua/T. gabrieli	Austria	AY304434	AY305686	Jacobs and Kirisits 2013
	K6/3/2	Picea abies/Tetropium sp.	Austria	AY304428	AY305687	Jacobs and Kirisits 2013
O. minus	PIR 18S	N/A	N/A	AY934509	N/A	Villarreal et al. 2005
	CMW22802	Dryocoetes autographus	N/A	DQ539507	N/A	Romón et al. 2005
	RJ-T144	Tetropium sp.	Poland	AM943886	N/A	Jankowiak and KolařÍk 2010
	CMW28117	Picea abies/Tomicus minor	Russia	HM031497	HM031535	Linnakoski et al. 2010
	AU58.4	Lodgepole pine	Canada	AF234834	N/A	Gorton et al. 2004
	DAOM 212686	N/A	Canada	AY304438	AY305690	Jacobs and Kirisits 2013
O. micans	CMW:38903	Picea crassifolia	China	KU184432	KU184303	Yin et al. 2016
O. montium	CMW13221	Pinus ponderosa/ Dendroctonus ponderosae	USA	AY546711	N/A	Zhou et al. 2004
O. montium	CMW13222	P. contorta/D. ponderosae	Canada	AY546712	N/A	Zhou et al. 2004
O. nigrocarpum	CMW 560	Abies sp.	USA	AY280489	AY280479	Aghayeva et al. 2004
O. nigrocarpum	CMW651	Pseudotsuga menziesii	USA	AY280490	AY280480	Aghayeva et al. 2004
O. nitidum	CMW:38907	Picea crassifolia	China	KU184437	KU184308	Yin et al. 2016
O. novo ulmi	C1185	Ulmus	Russia	AF198235	N/A	Harrington et al. 2001
O. novo ulmi	C510	Ulmus	USA	AF198236	N/A	Harrington et al. 2001
O. olgensis	CXY1404	Larix gmelini/Ips subelongatus	China	KU551299	KU882938	Wang et al. 2016
	CXY1405	L. gmelini/I. subelongatus	China	KU551300	KU882939	Wang et al. 2016
	CXY1410	L. gmelini/I. subelongatus	China	KU551303	KU882942	Wang et al. 2016
O. pallidulum	CMW23279	Pinus sylvestris/Hylastes brunneus	Finland	HM031509	N/A	Linnakoski et al. 2010
O. pallidulum	CMW23278	P. sylvestris/ H. brunneus	Finland	HM031510	HM031566	Linnakoski et al. 2010
O. piceae	C1087	N/A	Germany	AF198226	N/A	Uzunovic et al. 2000
O. piceae	C1246	Pseudotsuga	USA	AF198227	N/A	Uzunovic et al. 2000
O. pseudotsugae	92-634/302/6	Pinus menziesii/Dendroctonus frontalis	Canada	AY542502	AY548744	Gorton et al. 2004
O. pseudotsugae	D48/3	N/A	Canada	AY542501	AY542511	Gorton et al. 2004
O. proteasedis	CMW 28601	Protea caffra	Zambia	EU660449	EU660464	Roets et al. 2009
O. pulvinisporum	CMW9022	Pinus pseudostrobus/Dendroctonus mexicanus	Mexico	AY546714	DQ296100	Zhou et al. 2004
O. qinghaiense	CMW:38902	Picea crassifolia	China	KU184445	KU184316	Yin et al. 2016
O. querci	C970	Quercus	United Kingdom	AF198239	N/A	Gorton et al. 2004
	C969	Quercus	United Kingdom	AF198238	N/A	Gorton et al. 2004
	C1085	Fagus	Germany	AF198237	N/A	Gorton et al. 2004
O. rostrocoronatum	CBS434.77	Woodpulp	USA	AY194509	KX590771	Jacobs et al. 2003
O. saponiodorum	CMW29497	Picea abies/Ips typographus	Finland	HM031507	HM031571	Linnakoski et al. 2010
O. saponiodorum	CMW28135	P. abies	Russia	HM031508	N/A	Linnakoski et al. 2010
O. sejunctum	Ophi 1B	N/A	N/A	AY934520	N/A	Villarreal et al. 2005
O. sejunctum	Ophi 1A	N/A	N/A	AY934519	N/A	Villarreal et al. 2005
O. setosum	AU160-38	Pseutotsugae menziesii	North America	AF128929	N/A	Uzunovic et al. 2000
O. setosum	CMW12378	Tsuga sp.	China	FJ430485	FJ430515	Grobbelaar et al. 2009
O. tenellum	CBS189.86	Pinus banksiana	USA	AY934523	KX590772	Villarreal et al. 2005
O. tetropii	C00-027a	Tetropium fuscum	Canada	AY194482	NA	Jacobs et al. 2003
O. tetropii	C00-003	T. fuscum	Canada	AY194485	AY305701	Jacobs et al. 2003
O. ulmi	C1182	Ulmus	Netherlands	AF198232	N/A	Harrington et al. 2001
Graphilbum crescericum	CMW 22829	Hylastes ater	Spain	DQ539535	N/A	Romón et al. 2007
Gra. fragrans	C1224	Pinus sylvestris	Sweden	AF198248	N/A	Harrington et al. 2001
Gra. microcarpum	YCC612	Japanese larch logs	Japan	GU134170	N/A	Yamaoka et al. 2009
Gra. rectangulosporium	MAFF 238951	N/A	Japan	AB242825	N/A	Ohtaka et al. 2006
Raffaelea canadensis	CBS 168.66	N/A	N/A	GQ225699	N/A	Kyunghee et al. 2009
Leptographium lundbergii	DAOM 64746	N/A	N/A	EU879151	AY534943	Mullineux and Hausner 2009
L. truncatum	WIN(M)1435	Pinus taeda	South Africa	AY935626	N/A	Hausner et al. 2005

ITS = internal transcribed spacer regions 1 and 2 of the nuclear ribosomal DNA operon, including the 5.8S region; tub2 = beta-tubulin;

N/A= represents information that are not available.

CMW = Culture Collection of the Forestry and Agricultural Biotechnology Institute; CBS = The culture collection of Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; MAFF = Ministry of Agriculture, Forestry, and Fisheries, Genetic Resource Centre, Culture Collection of National Institute of Agrobiological Resources, Japan; CXY (Culture Xingyao): Culture collection of the Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry.

For each phylogenetic tree, MP, ML and BI analyses yielded trees with very similar topologies. Phylograms, generated by the MP analysis, are presented for all the datasets, with nodal support obtained from ML indicated at the nodes (Figure 1). In addition, posterior probabilities (above 90%), obtained from BI, are indicated by bold lines at the relevant branching points. Analyses of the ITS1–5.8S–ITS2 region revealed that the analysed strains formed six distinct clades (Figure 1).

Figure 1.

Phylograms of fungal associates of pine infected by PWN and Monochamus alternatus in China. The phylograms were generated after MP analysis of the ITS1–5.8S–ITS2 rDNA and partial tub2 sequences. Novel sequences obtained in the current study are indicated in bold type. MP bootstrap values (10,000 replicates) and ML bootstrap support values (1000 replicates) (normal type) above 70% are indicated at the nodes. Values below 70% are indicated by asterisk (*). Posterior probabilities (above 90%) obtained from BI are indicated by bold lines at the relevant branching points. Scale bar, total nucleotide differences between taxa; ML, maximum likelihood; MP, maximum parsimony; BI, Bayesian inference.

According to the ITS sequence analysis, strains of the morphological group A nested in the Sporothrix lineage, as defined by de Beer et al. (2016). They form a well-supported independent clade, closely related to S. nebularis, S. epigloea and S. eucalyptigena. Strains exhibiting morphotypes B, C and D formed three clades in the Ophiostomas. str lineage (de Beer and Wingfield 2013). Group B strains nested in the O. minus complex, with O. olgensis forming a well-supported clade, which closely related to O. kryptum (Linnakoski et al. 2010, de Beer and Wingfield 2013, Wang et al. 2016). Group C strains nested within the well-supported O. ips clade. Group D strains nested within the Ophiostoma lineage and closely related to O. saponiodorum and O. pallidulum. Finally, strains exhibiting morphotypes E and F nested in the Graphilbum and Raffaeleas. l. lineages, respectively (de Beer and Wingfield 2013) (TL=821, CI=0.5445, RI=0.8046, HI=0.4555, RC=0.4381 in the MP phylogenetic tree).

Phylogenetic inferences based on tub2 sequences revealed that clade A, B and D strains formed three well-supported independent clades within the Sporothrix and Ophiostoma lineages, respectively. Clade C strains nested within the well-supported O. ips clade (Suppl. material 1).

Considering morphological differences, strains in groups A, B and D represent three undescribed species of Sporothrix or Ophiostoma. We concluded that group C strains belong to O. ips; group E and F strains clustered together with the well-supported Graphilbum rectangulosporium and O. deltoideosporum clades, respectively. However, because of a limited number of strains, further analysis of this potential species will need to be postponed until a sufficient amount of material obtained.

Taxonomy

Based on the phylogenetic signals of the ITS and tub2 and morphological characteristics, all strains analysed in the current study were assigned to six different groups (A–F). They represent one known species, O. ips (Rumbold 1931, Upadhyay 1981, Benade et al. 1995, Rane and Tattar 1987, Suh et al. 2013, Zhao et al. 2013) and two uncertain species (Gra. cf. rectangulosporium and O. cf. deltoideosporum) and the three species are hereby described as new species.

Sporothrix zhejiangensis Wang & Lu, sp. nov.

MycoBank No: MB825556

Figure 2

Etymology

The epithet reflects Zhejiang Province in China where the species was first collected.

Type

CHINA, Zhejiang, Yuyao City, from Monochamus alternatus gallery in Pinus massoniana infested by numerous PWN, November 2012, collected by Q Lu and YY Lun, culture ex-holotype MUCL 55183 = CFCC52165 = CXY1614.

Description

Sexual morph perithecial: Perithecia occasional on 2% MEA, emerging from the superficial mycelium or partly iμmersed, with a globose base, (75–)80–108(–120) μm in diameter, with some basal hyphal ornamentation, black; extending progressively into a straight, brown to black neck, (127–)156–550(–631) μm long, (26–)32–58.5(–65) μm wide at the base, (7–)7.5–10.7(–12) μm wide at the apex; ending in a crown of hyaline, (6–)9–19.5(–24) μm long ostiolar hyphae; ascospores reniform in side view, without sheath, aseptate, hyaline, (2–)2.2–3.4(–4) × (0.6–)0.74–2(–2.5) μm.

Asexual morph: pesotum-like and sporothrix-like.

Pesotum-like: Conidiophores macronematous, synnematous, abundant in 2% MEA. Synnemata occurring singly, enlarging towards both the apex and the base, dark brown at base, becoming paler toward the apex, (100–)120–260(–290) μm long including the conidiogenous apparatus, (56–)63–145(–158) μm wide at base, rhizoids present; conidiogenous cells (7–)9.5–29(–45.5) × 1–2(–1.7) μm; conidia hyaline, aseptate, single-celled, smooth, cylindrical or obovoid, (2–)2.5–4.8(–6) × (0.5–)0.8–2.1(–2.6) μm.

Sporothrix-like: Conidiophores micronematous, single on aerial mycelia, unbranched, (4.5–)9.6–31.5(–51.5) × (1.0–)1.5–2(–2.4) μm; conidia hyaline, smooth, aseptate, ellipsoid to ovoid, (2.5–)3–4.8(–5) × (0.7–)1–2.1(–2.5) μm.

Figure 2.

Light micrographs of Sporothrix zhejiangensis. a–c Growth on 2% MEA and 2% PDA, 2 weeks after inoculation d Occasionally observed ostiolar hyphae (scale bar, 20 μm) e–f Perithecium (scale bar, 20 μm) g Pesotum-like anamorph, rhizoid, conidiophores, conidiogenous apparatus (scale bar, 20 μm), and conidia (bottom right corner) (scale bar, 10 μm) h, i Reniform ascospores without sheaths (scale bar, 10 μm) j–lSporothrix-like anamorph, conidiophores, and conidia (scale bar, 10 μm).

Culture characteristics

Colonies on 2% MEA medium are white, with colony edge thinning radially. Hyphae are superficial on agar. Diameter reaches 50 μm in the dark after 8 d at 25 °C, able to grow at 5 °C and 40 °C, with the optimal growth temperature of 30 °C. Growth characteristics on PDA medium are similar.

Habitat and distribution

Galleries of Monochamus alternatus in Pinus massoniana infested by PWN; known hitherto from Zhejiang Province, China.

Additional specimens examined

CHINA, Zhejiang, Yuyao City, from Monochamus alternatus galleries in Pinus massoniana infested by PWN, November 2012, collected by Q Lu and YY Lun, MUCL 55181 = CFCC 52167 = CXY1612, MUCL 55182 = CFCC 52164 = CXY1613, MUCL 55184 = CFCC 52166 = CXY1615.

Note

Sporothrix zhejiangensis is characterised by a sexual and two asexual forms (pesotum-like and sporothrix-like). It is phylogenetically related to S. nebulare, S. eucalyptigena and S. epigloea (Figure 1). Sporothrix zhejiangensis differs from S. nebulare in both ascomatal and conidial features. The perithecial neck of S. nebulare is shorter than that of S. zhejiangensis, respectively (140–)169–293(–365) μm and (127–)156–550(–631) μm. The conidia of S. nebulare also are smaller than those of S. zhejiangensis, mostly respectively 2.9–3.7 × 1.1–1.3 μm and 3–4.8 × 1–2.1 μm (Romón et al. 1900).

Sporothrix eucalyptigena and S. epigloea produce perithecia and ascospores similar to those of S. zhejiangensis (Crous et al. 2015, Upadhyay 1981). However, S. eucalyptigena has a slightly wider neck than S. zhejiangensis (20–35 vs. 9–19.5 μm) and longer ostiolar hyphae. Furthermore, S. eucalyptigena and S. epigloea only produce a sporothrix-like asexual state and their conidia differ from those of S. zhejiangensis either in size or in shape. Sporothrix eucalyptigena has drop-shaped (lacrymoid) conidia, differing from the ellipsoid to ovoid conidia in S. zhejiangensis. Conidia of S. epigloea are larger than those of S. zhejiangensis (2.5–9 × 1–3.5 vs. 3–4.8 × 1–2.1 μm) (Crous et al. 2015). Another conspicuous difference between S. zhejiangensis and S. eucalyptigena is the growth rate; the former grows much faster than the latter (50 μm in 8 d vs. 50 μm in 30 d at 25 °C) (Upadhyay 1981).

Sporothrix zhejiangensis is also closely related to S. bragantina and S. thermara (Figure 1) (Pfenning and Oberwinkler 1993, de Beer et al. 2016). These three species display the same optimal growth temperature (30 °C) and a similar conidial shape (ellipsoid to obovoid) of their sporothrix-like morph. However, the perithecial base of S. bragantina is larger than that of S. zhejiangensis [globose base: 130–220 μm vs. (75–)80–108(–120) μm and the neck also is longer, 700–1200 μm vs. (127–)156–550(–631) μm]. The sporothrix-like conidia of S. bragantina also are larger than those of S. zhejiangensis (4–6 × 2–2.5 μm vs. 3–4.8 × 1–2.1 μm). Sporothrix thermara, hitherto, has no known sexual state. It only known by sporothrix-like state; conidia of S. thermara are larger than those of S. zhejiangensis (4–6 × 2–3 μm vs. 3–4.8 × 1–2.1 μm).

Ophiostoma album Wang & Lu, sp. nov.

MycoBank No: MB825557

Figure 3

Etymology

The epithet reflects the white colour of the colonies.

Type

CHINA, Zhejiang, Yuyao City, from Monochamus alternatus gallery of Pinus massoniana infested by numerous PWN, November 2012, collected by Q Lu and YY Lun, culture ex-holotype MUCL 55189 = CFCC 52168 = CXY1622.

Description

Sexual form: Unknown. Asexual form: Hyalorhinocladiella-like. Conidiogenous cells micronematous, (4.2–)9.5–16.5(–20.5) × (0.5–)1–2(–2.5) μm; conidia hyaline, single-celled, aseptate, clavate or fusiform obovoid with pointed bases and (occasionally) rounded apices, slightly curved at the base (4–)4.2–14.5(–18) × (0.5–)1–2(–2.3) μm.

Figure 3.

Light micrographs of Ophiostoma album. a, b Growth on 2% MEA and 2% PDA, 2 weeks after inoculation c–eHyalorhinocladiella-like anamorph, conidiophores, and conidia (scale bar, 10 mm).

Culture characteristics

Colonies on 2% MEA white, with the mycelium edge thinning radially; Hyphae are superficial on agar, sporulation weak. Colonies slowly growing, reaching 18.5 μm in diameter at 8 d at 25 °C, able to grow at 40 °C but not at 5 °C, with the optimal growth temperature of 35 °C. Growth characteristics on PDA culture medium are similar but the growth rate is slower than on MEA.

Habitat and distribution

Galleries of Monochamus alternatus in Pinus massoniana, infested by PWN, in Zhejiang Province, China.

Additional specimens examined

CHINA, Zhejiang, Yuyao City, from Monochamus alternatus galleries of Pinus massoniana infested by numerous PWN, November 2012, collected by Q Lu and YY Lun, MUCL 55190 = CFCC 52169 = CXY1642, CXY1643 = CFCC 52170.

Note

Ophiostoma album only known in its asexual hyalorhinocladiella-like form. According to both ITS and tub2 based phylogenetic analysis, it is closely related to O. kryptum and O. olgensis in the O. minus complex (Figure 1). Ophiostoma album is easily distinguished from O. olgensis and O. kryptum based on their reproduction structure. Ophiostoma album only produces a hyalorhinocladiella-like asexual form in vitro, whereas the two other species produce both a sexual and asexual forms in vitro (Jacobs and Kirisits 2003, Wang et al. 2016). The conidial size and shape of the three species are obviously different. Ophiostoma album produces clavate or fusiform to obovoid and sometimes, slightly curved conidia; these are obovoid with pointed bases in both O. olgensis and O. kryptum. Furthermore, the conidia of O. album are much larger, 4.2–14.5 × 1.0–1.9 μm vs. 1.5–7 × 1.5–5 μm in the two other species.

Ophiostoma massoniana Wang & Lu, sp. nov.

MycoBank No: MB825558

Figure 4

Etymology

The epithet reflects the host tree, Pinus massoniana.

Type

CHINA, Zhejiang Province, Fuyang City, from Monochamus alternatus gallery in Pinus massoniana infested by numerous PWN, November 2012, collected by Q Lu and YY Lun, culture ex-holotype, MUCL 55179 = CFCC 51648 = CXY1610.

Description

Sexual form: Unknown. Asexual form: Hyalorhinocladiella-like. Conidiophores abundant, single, borne on aerial hyphae, (3.3–)10.5–27.5(–42.5) × (0.7–)1.3–2.0(–2.7) μm; conidia hyaline, single-celled, aseptate, obovoid or globose with pointed bases and rounded apices, (2–)2.2–3.9(–5) × (0.5–)0.7–1.7(–2) μm.

Figure 4.

Light micrographs of Ophiostoma massoniana. a, b Growth on 2% MEA and 2% PDA, 2 weeks after inoculation c–eHyalorhinocladiella-like anamorph, conidiophores, conidia (scale bar, 10 μm).

Culture characteristics

Colonies on 2% MEA brown, the marginal hyphae sparse and radiating; some white mycelium produced early during growth that becomes black after 3–5 d. Colonies slowly growing, reaching 37.5 μm in diameter over 8 d at 25 °C, able to grow at 5 °C and 40 °C, with an optimal growth temperature of 30 °C; sporulation weak. On PDA culture medium, the colonies are dark brown; the mycelium is white, long and dense, with a daily growth of 4 μm at 25 °C.

Habitat and distribution

Galleries of Monochamus alternatus in Pinus massoniana infested by PWN, in Zhejiang Province, China.

Additional specimens examined

CHINA, Zhejiang Province, Yuyao City, from Monochamus alternatus galleries in Pinus massoniana infested by numerous PWN, November 2012, collected by Q Lu and YY Lun, MUCL 55180 = CFCC 51649 = CXY1611.

Note

Ophiostoma massoniana, only known by its asexual, hyalorhinocladiella-like state, does not cluster in any of the 10 species complexes defined by de Beer and Wingfield (2013) in Ophiostoma s. l. According to the ITS and tub2 phylogenetic analysis, the species is related to O. saponiodorum and O. pallidulum (Figure 1). Ophiostoma pallidulum also only produces asexual hyalorhinocladiella-like morphs in vitro, whereas O. saponiodorum produces a sexual and two asexual morphs (pesotum-like and hyalorhinocladiella-like). In addition, O. massoniana differs from O. saponiodorum in producing smaller conidia [(2–)2.2–3.9(–5) × (0.5–)0.7–1.7(–2) μm vs. (3–)4–6(–7) × 1–1.5(–2) μm] (Linnakoski et al. 2010). Further, the colour of O. massoniana colonies is different from that of the other two species. Namely, O. massoniana forms brown to dark brown colonies, while the other two species form pale colonies (Linnakoski et al. 2010).

Discussion

In the current study, six ophiostomatoid species were found associated with pines infected by M. alternatus and PWN in the eastern provinces of Shandong and Zhejiang in China: O. ips, the newly described S. zhejiangensis, O. album, O. massoniana and two species whose identities are uncertain; O. cf. deltoideosporum and Gra. cf. rectangulosporium. Ophiostoma ips was the most frequently isolated species, accounting for over 90% of all Ophiostomatales strains.

Ophiostoma ips was originally reported in association with bark beetles infecting pines in south-eastern North America (Rumbold 1931). It has been since reported in Central and South America (Mexico and Chile), Europe (Austria and Sweden), Asia (China, Japan and Korea), Africa (South Africa) and Australasia (New Zealand) (Rumbold 1931, Benade et al. 1995, Rane and Tattar 1987, Zhou et al. 2002; Lu et al. 2009, Suh et al. 2013, Zhao et al. 2013; 2014). Furthermore, O. ips is a ubiquitous sapstain fungus associated with PWN and Monochamus spp. (Zhao et al. 2014).

In China, O. ips was reportedly associated with P. massoniana infected by PWN (Zhao 1992, Zhao et al. 2006, 3013) and with P. tabuliformis infected by Dendroctonus valens (Lu et al. 2009), two invasive pests of the local conifer ecosystems. Zhao et al. (2013) reported O. ips an isolation frequency of 37% in three ophiostomatoid fungal communities associated with PWN, much lower than that reported in the current study.

Ophiostoma ips appears to have travelled long-distances in wood materials presumably originating from North America (Zhou et al. 2007). The cited study did not consider any Asian population, however. Nevertheless, the high population density of O. ips in China suggests either indigenous origin or effective adaption after the invasion to local pine forests, with a long evolution history. To verify this hypothesis, it will be necessary to analyse the dispersal routes of PWN populations in different areas globally and of the fungus–including Asian populations.

Members of Sporothrix are reportedly associated with a wide range of habitats (De Hoog 1974, Kwon-Chung and Bennet 1992, Roets et al. 2006, Zhou et al. 2006, Madrid et al. 2009), e.g. wood (Aghayeva et al. 2004), human (de Beer et al. 2016) and the soil (De Meyer et al. 2008). The genus is characterised by reniform ascospores without a mucilaginous sheath and sporothrix- and pesotum-like asexual states (Linnakoski et al. 2010, de Beer et al. 2013). Genetically, the species of the Sporothrix lineages lack the intron 4 but have intron 5 in the BT gene (Zipfel et al. 2006).

Sporothrix zhejiangensis forms an independent lineage according to both ITS and tub2 based on phylogenetic inferences. It is closely related to S. nebulare, S. eucalyptigena, S. epigloea, S. bragantina and S. thermara (Madrid et al. 2010, Romón et al. 1900, Crous et al. 2015, de Beer et al. 2016, Van der Linde et al. 2016) (Figure 1). Sporothrix nebulare was first described after isolation from Hylastes attenuatus infesting P. radiata in Spain (Romón et al. 1900). Sporothrix eucalyptigena was recently isolated from Eucalyptus marginata (Myrtaceae) in Western Australia (Crous et al. 2015). Sporothrix epigloea was isolated from Tremella fuciformis in Argentina (Upadhyay 1981). S. bragantina was isolated from the rhizosphere soil in Brazil (Pfenning and Oberwinkler 1993) and S. thermara from Cyrtogenius africus galleries in diseased Euphorbia ingens trees in South Africa (Van der Linde et al. 2016). Hence, S. zhejiangensis and these five species differ with respect to their (known) hosts and geographic distributions.

Although S. zhejiangensis is unrelated to S. fusiforis, S. lunata and S. stenoceras (Figure 1), these strains exhibit a similar sexual state (Hsiau 1996, Yamaoka et al. 2000, Aghayeva et al. 2004, Zhou et al. 2004). For instance, they all develop one to two perithecial necks emerging from the globular base; occasionally, abnormal specimens of O. stenoceras develop up to five necks in vitro (Yamaoka et al. 2000).

In the current study, S. zhejiangensis was notably different from Sporothrix sp. 1 and Sporothrix sp. 2 (Zhao et al. 2013) with regard to colony characteristics (S. zhejiangensis has a white and radially thinning edge; Sporothrix sp. 1: dark, superficial mycelium; Sporothrix sp. 2: white, radially dense mycelium). Consequently, the role of S. zhejiangensis in PWN needs further research and analysis, ruling out the possibility that the species had been already discovered and its ecological role partially studied.

According to ITS phylogeny analysis, Ophiostoma album is related to O. olgensis (Wang et al. 2016) in a single but weakly supported clade (Figure 1). This clade nests within the O. minus complex, in which it is closely related to O. kryptum (Jacobs and Kirisits 2003). The tub2 dataset confirmed that O. album and O. olgensis formed two clades.

The O. minus complex currently includes O. minus, O. pseudotsugae, O. allantosporum, O. kryptum and O. olgensis (Jacobs and Kirisits 2003, Gorton et al. 2004, de Beer and Wingfield 2013, Wang et al. 2016). The tub2 gene of the O. minus complex members includes intron 4 but lacks intron 5 (Gorton et al. 2004). Ophiostoma album is phylogenetically closely related to O. olgensis and O. kryptum. Both O. olgensis and O. kryptum inhabit Larix spp. (Jacobs and Kirisits 2003; Wang et al. 2016), whereas O. album inhabits P. massoniana. Both O. olgensis and O. album occur in China, whereas O. kryptum is found in central Europe. Moreover, the three species are associated with different vectors (Jacobs and Kirisits 2003, Wang et al. 2016).

According to both ITS and tub2 phylogenetic trees, O. massoniana forms a separated well-supported clade (Figure 1). It groups with O. pallidulum and O. saponiodorum (Figure 1), which has been isolated from Pinus sylvestris in Finland and Picea abies in Russia in association with various bark beetles (Linnakoski et al. 2010). The three species produce a hyalorhinocladiella-like asexual form (Linnakoski et al. 2010; de Beer et al. 2013) and their tub2 genes lack intron 4 but contain intron 5 (Zipfel et al. 2006).

Conclusions

In the current study, a relatively large number of ophiostomatoid fungal species associated with B. xylophilus and M. alternatus in Shandong and Zhejiang Provinces in China was identified. Three novel species, O. album, O. massoniana and S. zhejiangensis were discovered and described. Fourteen additional provinces in China are currently also listed as PWN epidemic areas (State Forestry Administration of the People’s Republic of China 2018). Hence, additional ophiostomatoid fungi associated with B. xylophilus and M. alternatus should be discovered and described. Future in-depth studies of the biodiversity, biogeography and ecology of fungi associated with pine wilt disease will contribute to the understanding of disease mechanisms and provide information on effective management methods to alleviate the subsequent plant losses.

Acknowledgments

This work was supported by the National Key R&D Programme of China (2017YFD0600103) and the National Natural Science Foundation of China (Project No.: 31770682). Cony Decock gratefully acknowledges the financial support received from the Belgian State (Belgian Federal Science Policy through the BCCMTM research programme). We are grateful to Professor Yuichi Yamaoka and Hugo Madrid for their invaluable suggestions to improve the manuscript.

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Supplementary materials

Supplementary material 1

Figure S1. Phylogram of fungal associates of pine infected by PWN and Monochamus alternatus in China

HuiMin Wang, YingYing Lun, Quan Lu, HuiXiang Liu, Cony Decock, XingYao Zhang

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

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Supplementary material 2

Figure S2. Phylograms of fungal associates of pine infected by PWN and Monochamus alternatus in China

HuiMin Wang, YingYing Lun, Quan Lu, HuiXiang Liu, Cony Decock, XingYao Zhang

Download file (156.40 kb)

Supplementary material 3

Figure S3. Three ML phylogenetic threes based on tub2 after excluding introns

HuiMin Wang, YingYing Lun, Quan Lu, HuiXiang Liu, Cony Decock, XingYao Zhang

Download file (312.92 kb)