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Ophiostomatoid fungi associated with pines infected by Bursaphelenchus xylophilus and Monochamus alternatus in China, including three new species
expand article infoHuiMin 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
Open Access

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.

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
ITS β-tubulin
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
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.

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

Species Strain No. Host/insect Country Genbank No. Reference
ITS β-tubulin
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
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
CBS 124564 Soil Spain FN546958 FN547384 Madrid et al. 2010
S. dentifunda CMW13016 Quercus wood Hungary AY495434 AY495445 Aghayeva et al. 2005
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
CMW12527 wheat-field soil Germany AY495426 AY495437 Aghayeva et al. 2005
S. nebularis CMW27319 Orthotomicus erosus Spain DQ674375 N/A Romón et al. 1900
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
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
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
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
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
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
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
C1086 Soil Sweden AF198231 N/A Gorton et al. 2004
O. fumeum CMW26813 Eucalyptus cloeziana South Africa HM051412 HM041878 Nkuekam et al. 2012
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
C1306 Ulmus India AF198234 N/A Harrington et al. 2001
O. ips CMW7075 N/A USA AY546704 N/A Zhou et al. 2004
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
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
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
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
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
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
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
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
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
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
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

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 Ophiostoma s. 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 Raffaelea s. 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–l Sporothrix-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–e Hyalorhinocladiella-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–e Hyalorhinocladiella-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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