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Differential patterns of ophiostomatoid fungal communities associated with three sympatric Tomicus species infesting pines in south-western China, with a description of four new species
expand article infoWang HuiMin, Zheng Wang, Fu Liu, Cheng Xu Wu, Su Fang Zhang, Xiang Bo Kong, Cony Decock§, Quan Lu, Zhen Zhang
‡ Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing, China
§ Mycothèque de l’Université Catholique de Louvain, Louvain-la-Neuve, Belgium
Open Access

Abstract

Bark beetles and their associated fungi, which cause forest decline and sometimes high mortality in large areas around the world, are of increasing concern in terms of forest health. Three Tomicus spp. (T. brevipilosus, T. minor and T. yunnanensis) infect branches and trunks of Pinus yunnanensis and P. kesiya in Yunnan Province, in south-western China. Tomicus spp. are well known as vectors of ophiostomatoid fungi and their co-occurrence could result in serious ecological and economic impact on local forest ecosystems. Nonetheless, knowledge about their diversity, ecology, including pathogenicity and potential economic importance is still quite rudimentary. Therefore, an extensive survey of ophiostomatoid fungi associated with these Tomicus species infesting P. yunnanensis and P. kesiya was carried out in Yunnan. Seven hundred and seventy-two strains of ophiostomatoid fungi were isolated from the adult beetles and their galleries. The strains were identified based on comparisons of multiple DNA sequences, including the nuclear ribosomal large subunit (LSU) region, the internal transcribed spacer regions 1 and 2, together with the intervening 5.8S gene (ITS) and the partial genes of β-tubulin (TUB2), elongation factor 1α (TEF1-α) and calmodulin (CAL). Phylogenetic analyses were performed using maximum parsimony (MP) as well as maximum likelihood (ML). Combinations of culture features, morphological characters and temperature-dependent growth rates were also employed for species identification. Eleven species belonging to five genera were identified. These included six known species, Esteya vermicola, Leptographium yunnanense, Ophiostoma brevipilosi, O. canum, O. minus and O. tingens and four novel taxa, described as Graphilbum anningense, O. aggregatum, Sporothrix pseudoabietina and S. macroconidia. A residual strain was left unidentified as Ophiostoma sp. 1. The overall ophiostomatoid community was by far dominated by three species, representing 87.3% of the total isolates; in decreasing order, these were O. canum, O. brevipilosi and O. minus. Furthermore, the ophiostomatoid community of each beetle, although harbouring a diversity of ophiostomatoid species, was differentially dominated by a single fungal species; Ophiostoma canum was preferentially associated with and dominated the ophiostomatoid community of T. minor, whereas O. brevipilosi and O. minus were exclusively associated with and dominated the ophiostomatoid communities of T. brevipilosus and T. yunnanensis, respectively. Eight additional species, representing the remaining 12.7% of the total isolates, were marginal or sporadic. These results suggested that sympatric Tomicus populations are dominated by distinct species showing some level of specificity or even exclusivity.

Keywords

Esteya vermicola, Graphilbum, Leptographium, Ophiostoma, species-specific association, Sporothrix, taxonomy

Introduction

Associations between insects and microorganisms are increasingly recognised as one of the major issues in forest ecology and forest health around the world (Wingfield et al. 2016). Many bark beetles are well known as tree pests causing various levels of tree mortality and forest decline in large areas of the world, mostly in temperate areas (Jankowiak 2006, Wingfield et al. 2017). These bark beetles are well known vectors of variably pathogenic fungi, forming symbiosis-like relationships (Six 2003, Lu et al. 2009).

The pine shoot beetles, Tomicus Latreille (syn. Blastophagus Eichhoff, Myelophilus Eichhoff, Scolytidae, Coleoptera), are destructive insects with a range spanning the Eurasian pine forests, seriously affecting tree growth and causing a great threat to the forest ecosystems (Kirkendall et al. 2008, Lieutier et al. 2015). Currently, eight species are recorded worldwide, i.e. T. armandii Li and Zhang (Li et al. 2010), T. brevipilosus Eggers, T. destruens Wollaston, T. minor Hartig, T. pilifer Spessivtsev, T. piniperda L., T. puellus Reitter, and T. yunnanensis Kirkendall and Faccoli (Kirkendall et al. 2008). They all occur in China except T. destruens and five of them, viz. T. armandii, T. brevipilosus, T. minor, T. pilifer and T. yunnanensis, are sympatric in forests of the Yunnan Province (Li et al. 1997, 2010, Kirkendall et al. 2008; Ye 2011). Tomicus brevipilosus, T. minor and T. yunnanensis have overlapping geographical distribution, host range and infection periods. They aggregately infect branches and trunks of two indigenous pines, Pinus yunnanensis and P. kesiya (Li et al. 1997, 2006, Chen et al. 2009, 2010, Lu et al. 2012, 2014), causing locally extensive tree decline or mortality (Ye and Dang 1986, Ye 1991, 2011). Since the 1980s, damage caused by these bark beetles has resulted in losses of more than 93,000 m3 of pinewood (Ji et al. 2007).

Generally, two or three pine shoot beetles co-occur underneath the bark or in shoots of a single host tree, either simultaneously but with spatially isolated galleries or successively, during differential infesting peaks. Spatial and chorological differentiation would reduce competition between beetles, but their co-occurrence also could enhance cooperation (Lu et al. 2012, Chen et al. 2015). Tomicus yunnanensis is considered to be the most aggressive species in Yunnan, causing primary infestations of healthy P. yunnanensis trees and eventually tree death (Ye and Lieutier 1997, Kirkendall et al. 2008, Chen et al. 2010, 2015, Lu et al. 2014). Although T. brevipilosus is able to infect healthy trees, it preferably colonises trunks already infested by T. yunnanensis or both T. yunnanensis and T. minor (Chen et al. 2010, 2015). Tomicus minor is often regarded as a secondary, opportunist species infesting trees already weakened by T. yunnanensis or/and T. brevipilosus (Ye and Ding 1999, Lieutier et al. 2003, Chen et al. 2009).

Pine shoot beetles such as T. piniperda, T. minor and T. destruens are commonly associated with ophiostomatoid fungi (Masuya et al. 1999, Kim et al. 2005, Jankowiak 2006, 2008). Fifteen ophiostomatoid fungi were reported associated with T. piniperda in Europe (Mathiesen 1950, Lieutier et al. 1989, Gibbs and Inman 1991, Solheim and Långström 1991, Jankowiak 2006, Jankowiak and Bilański 2007) and 11 were documented in eastern Asia (Japan and Korea) (Masuya et al. 1999, Kim et al. 2005). Ophiostoma minus was shown to be the dominant species associated with T. piniperda in Europe and Japan (Mathiesen 1950, Lieutier et al. 1989, Gibbs and Inman 1991, Masuya et al. 1999, Jankowiak 2006). Leptographium wingfieldii was shown to be the strongest pathogenic one (Gibbs and Inman 1991) in Europe. Tomicus minor also infests various pines in Europe and Asia. Fifteen (Mathiesen-Käärik 1953, Masuya et al. 1999, Jankowiak 2008) and 11 (Masuya et al. 1999) ophiostomatoid species have been reported to be associated with this beetle species in Europe and Japan, respectively. Ophiostoma canum was recorded as a frequent/dominant species in association with T. minor, both in Europe and Japan (Mathiesen 1950, 1951, Rennerfelt 1950, Francke-Grosmann 1952, Masuya et al. 1999) but seems to represent a weak pathogen to P. sylvestris (Solheim et al. 2001). Additionally, six ophiostomatoid fungi were documented associated with T. destruens in Europe (Lieutier 2002, Sabbatini Peverieri et al. 2006, Ben Jamaa et al. 2007).

Despite the fact that Tomicus spp. have caused serious losses to forest ecosystems in south-western China, there are no systematic studies of their ophiostomatoid associates but only a few sporadic reports. So far, nine ophiostomatoid species have been reported as being associated with Tomicus spp. in Yunnan. Six species (Leptographium yunnanense, Ophiostoma ips, O. minus, O. quercus, S. abietina and S. nebularis) were recorded to be associated with T. yunnanensis (Ye et al. 2000, Zhou et al. 2000, 2013, Chang et al. 2017). Two species (Graphilbum fragrans and O. tingens) were recorded as being associated with T. minor (Zhou et al. 2013, Pan et al. 2017), whereas only a single species (O. brevipilosi) was recorded as being associated with T. brevipilosus (Chang et al. 2017). Amongst them, L. yunnanense was the first species newly described from the area (Zhou et al. 2000) and is likely the most virulent one (Liao and Ye 2004, Gao et al. 2017). Until now, the relative abundance with which these fungi occur, their host (pine and beetle) relationships, and their pathogenicity remain unknown.

The symbiosis between bark beetles and ophiostomatoid fungi enhances their pathogenicity. The fitness of bark beetle populations may depend in part on the degree of the fungal partners’ pathogenicity and the resulting weakening of the tree (Christiansen et al. 1987, Kirisits 2004, Linnakoski et al. 2012), although this has been questioned by some (Six and Wingfield 2011). Therefore, the question remains whether there is any link between the differential aggression of the pine shoot beetles and the differential virulence of their fungal associates, especially in circumstances where various beetle species co-exist.

The aim of this study was to describe the diversity of ophiostomatoid fungal communities associated with three pine shoot beetles and their galleries infesting P. yunnanensis and P. kesiya in forest ecosystems of Yunnan Province. We also analysed the degree of beetle/ophiostomatoid fungi specificity. Such studies will enable us to understand the aggressive nature of the beetles and the pathogenicity of the associated fungi and the interactions, ultimately helping to address the current situation of ceaseless outbreaks and rapid expansion of the pests.

Materials and methods

Sample collection and fungus isolation

Samples of galleries in bark and shoots and adults of Tomicus spp. were collected from P. yunnanensis and P. kesiya at five sites in Yunnan Province (Fig. 1, Table 1) from December 2016 to March 2017. Beetles were placed individually in sterilised Eppendorf tubes and their galleries were placed in sterile envelopes and stored at 4°C until processed within one week.

Figure 1. 

A Map showing the 11 species of ophiostomatoid fungi detected from Yunnan Province, China B, D disease symptoms on Pinus yunnanensis and P. kesiya trees infested by Tomicus spp. (T. yunnanensis, T. minor and T. brevipilosus) and ophiostomatoid fungi C, G, H exposed branches of Tomicus spp. on P. yunnanensis and P. kesiya E, F, I–K galleries of Tomicus spp. on P. yunnanensis and P. kesiya.

Basic information on the sample collection plots in China.

Location Host Insect vector longitude\latitude altitude(m) No. of examained samples
Xiangyun,Yunnan Pinus yunnanensis Tomicus yunnanensis, T. minor 25°21'25.8"N, 100°51'49"E 2255.4 447
Puer,Yunnan P. kesiya T. brevipilosus, T. minor 22°56'36.1"N, 101°14'36.7"E 1400.7 346
Qujing,Yunnan P. yunnanensis T. yunnanensis, T. minor, T. brevipilosus 25°28'51"N, 103°46'32"E 2068.2 102
Anning,Yunnan P. yunnanensis T. yunnanensis, T. minor, T. brevipilosus 24°53'32"N, 102°24'23"E 1939.9 138
Yuxi, Yunan P. yunnanensis T. yunnanensis, T. minor 24°18'23"N, 102°34'37"E 1908.1 85

Isolations from beetles and their galleries were carried out on 2% malt extract agar (MEA: 20 g Biolab malt extract, 20 g Biolab agar and 1 000 ml deionised water) with 0.05% NaClO added, in 9-cm Petri dishes as described by Seifert et al. (1993). Hyphal tips of emerging colonies were cut and transferred to MEA plates in order to obtain pure strains. The strains were grown routinely on 2% MEA at 25 °C. Representative cultures of each morphotype were deposited in the China Forestry Culture Collection Center (CFCC, part of the National Infrastructure of Microbial Resources) and the culture collection of the Chinese Academy of Forestry (CXY) (Table 2).

Representative strains of the ophiostomatoid fungi associated with three Tomicus spp. in Yunnan Province, China, and three E. vermicola strains used in this study.

Group Taxon Strain no. Host Location Beetle GenBank no.
LSU ITS\ ITS2–LSU5 BT EF CAL
A Esteya vermicola CFCC52625 (CXY1893) P. yunnanensis Xiangyun T. yunnanensis MH325143 MH697597 MH605999
ATCC74485 Japanese black pine Taiwan, China Bursaphelenchus xylophilus GQ995674
CNU120806 soil Korea saprophytic nematodes EU627684 FJ490553 GQ995671
CBS 115803 oak Czech Republic Scolytus intricatus FJ490552 GQ995672
B Graphilbum anningense CFCC52631 (CXY1939) P. yunnanensis Anning T. yunnanensis MH325162 MH555903 MH683595
CFCC52632 (CXY1940) P. yunnanensis Anning T. yunnanensis MH325164 MH555901 MH683596
CFCC52633 (CXY1944) P. yunnanensis Anning T. minor MH325163 MH555902 MH683597
C Leptographium yunnanense CFCC52619 (CXY1897) P. kesiya Ninger T. brevipilosus MH325138 MH487721 MH603933 MH606000
CFCC52620 (CXY1900) P. yunnanensis Xiangyun T. yunnanensis MH325139 MH487724 MH603934 MH606001
CFCC52621 (CXY1904) P. yunnanensis Xiangyun T. yunnanensis MH325140 MH487726 MH603935 MH606003
CFCC52622 (CXY1908) P. yunnanensis Xiangyun T. yunnanensis MH325142 MH487725 MH603938 MH606002
CFCC52623 (CXY1917) P. kesiya Puer T. brevipilosus MH325137 MH487723 MH603936 MH606004
CFCC52624 (CXY1925) P. yunnanensis Xiangyun T. yunnanensis MH325141 MH487722 MH603937 MH606005
D Ophiostoma brevipilosi CFCC52596 (CXY1828) Pinus kesiya Puer T. brevipilosus MH325134 MH555904 MH619527
(CXY1806) CFCC52597 P. kesiya Puer T. brevipilosus MH325135 MH555905 MH619528
CFCC52598 (CXY1808) P. kesiya Puer T. brevipilosus MH325136 MH555906 MH619529
E O. canum CFCC52601 (CXY1858) P. yunnanensis Xiangyun T. minor MH325151 MH555889 MH619521
CFCC52602 (CXY1848) P. yunnanensis Xiangyun T. minor MH325152 MH555890 MH619522
CFCC52603 (CXY1857) P. yunnanensis Xiangyun T. minor MH325153 MH555891 MH619523
F O. aggregatum CFCC52615 (CXY1876) P. yunnanensis Xiangyun T. yunnanensis MH325146 MH555894 MH603927
CFCC52616 (CXY1875) P. yunnanensis Xiangyun T. yunnanensis MH325145 MH555893 MH603929
CFCC52617 (CXY1874) P. kesiya Puer T. minor MH325147 MH555895 MH603928
G O. minus CFCC52606 (CXY1885) P. yunnanensis Xiangyun T. yunnanensis MH325154 MH578163 MH619524
CFCC52607 (CXY1877) P. yunnanensis Xiangyun T. yunnanensis MH325155 MH578164 MH619525
CFCC52608 (CXY1881) P. yunnanensis Xiangyun T. yunnanensis MH325156 MH578165 MH619526
H O. tingens CFCC52611 (CXY1866) P. yunnanensis Xiangyun T. minor MH325148 MH578166 MH603931
CFCC52612 (CXY1865) P. yunnanensis Xiangyun T. minor MH325149 MH578167 MH603932
CFCC52613 (CXY1868) P. yunnanensis Xiangyun T. yunnanensis MH325150 MH578168 MH603930
I Ophiostoma sp. 1 CFCC52618 (CXY1936) P. yunnanensis Xiangyun T. yunnanensis MH325144 MH555892 MH683600
J Sporothrix macroconidia CFCC52628 (CXY1894) P. yunnanensis Xiangyun T. yunnanensis MH325157 MH555898 MH697594 MH592598
CFCC52629 (CXY1895) P. kesiya Ninger T. brevipilosus MH325158 MH555899 MH697595 MH592599
CFCC52630 (CXY1896) P. kesiya Ninger T. brevipilosus MH325159 MH555900 MH697596 MH592600
K S. pseudoabietina CFCC52626 (CXY1937) P. yunnanensis Qujing T. minor MH325160 MH555896 MH683598 MH592601
CFCC52627 (CXY1938) P. yunnanensis Qujing T. minor MH325161 MH555897 MH683599 MH592602

Morphology and growth studies

Morphological characterisation of both the sexual and asexual reproduction forms was performed on 2% MEA media incubated 3–6 weeks at 25 °C in the dark. Slide cultures were made to observe all microscopic characters (sexual/asexual structures) using a BX51 OLYMPUS microscope with differential interference contrast. Fifty measurements were made of each relevant structure and the ranges were calculated. Standard deviation (SD), minimum (min) and maximum (max) measurements are presented as (min–) (mean–SD) – (mean+SD) (–max).

The optimal growth temperature of the various strains was determined by placing a 5-mm (diam.) plug from an actively growing fungal colony upside down at the centre of an MEA plate. For each strain, three replicates were incubated at temperatures ranging from 5 to 35 °C at five-degree intervals, for 8d. The diameter of each colony was measured daily. Culture characters were recorded on MEA incubated at 25 °C for 8 d and 20 d. Colour descriptions were made by reference to Rayner (1970).

DNA extraction and sequencing

DNA was extracted from actively growing mycelium scraped from seven-day-old cultures using sterile scalpels and transferred to 2 ml Eppendorf tubes. DNA extraction and purification were performed using the Invisorb Spin Plant Mini Kit (Invitek, Berlin, Germany), following the manufacturer’s protocols.

DNA sequences were determined for six gene regions: the nuclear ribosomal large subunit region (LSU), the internal transcribed spacer regions 1 and 2, including the intervening 5.8S gene (ITS), as well as segments of the β-tubulin (TUB2), elongation factor 1α (TEF1-α) and calmodulin (CAL) genes. DNA fragments were amplified using the primer pairs LROR/LR5 (Vilgalys and Hester 1990), ITS1/ITS4 (White et al. 1990), ITS3/LR3 (Vilgalys and Hester 1990, White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), EF1/EF2 (Jacobs et al. 2004) and CL1/CL2a (Zhang et al. 2015), respectively. PCR reactions were conducted in 25 μl volumes (2.5 mM MgCl2, 1× PCR buffer, 0.2 mM dNTP, 0.2 mM of each primer and 2.5 U Taq-polymerase enzyme). PCR amplifications were carried out in a thermocycler (Applied Biosystems, Foster City, California, USA). The reaction conditions for these six gene regions were similar to those described in the references used for primer design. PCR products were cleaned with an MSB Spin PCR apace Kit (250), following the manufacturer’s instructions.

Phylogenetic analyses

BLAST searches for the obtained sequences were performed in NCBI GenBank and published sequences of closely related species were downloaded. Alignments of the genes were made using MAFFT 7.0 (Katoh and Standley 2013) and the E-INS-i strategy and edited manually in MEGA 5.2 (Tamura et al. 2011). Phylogenetic analyses were performed using maximum parsimony (MP) as well as maximum likelihood (ML).

ML analyses were implemented using RAxML v. 7.0.3 (Stamatakis 2006), under the GTR-GAMMA model. Support for the nodes was estimated from 1 000 bootstrap replicates. The results were subsequently exported to Figtree v.1.4.2 to visualise the trees.

MP analyses were implemented in PAUP* 4.0b10 (Swofford 2003). The most parsimonious trees were identified by a heuristic search of 1 000 random addition sequence replicates, using the tree-bisection-recognition (TBR) algorithm for branch swapping. Branch support was assessed by 1 000 bootstrap replicates. Tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were used to evaluate the trees.

Results

Fungal isolation and sequence comparisons

Three Tomicus species occurred on P. yunnanensis and P. kesiya in the areas studied, either independently or concomitantly in individuals of the host trees (Fig. 1). In total, 772 strains of ophiostomatoid fungi (Hyalorhinocladiella-like, Ophiostoma, Pesotum-like, Leptographium-like and Sporothrix-like) were isolated from 223 adult beetles (20% of the strains) and 890 galleries (80% of the strains). Galleries or adults of T. yunnanensis yielded 297 strains whereas 247 strains were retrieved from galleries or adults of T. minor and 228 strains from galleries or adults of T. brevipilosus (Table 3).

The LSU sequence was used to search for preliminary affinities using the BLASTn search option in GenBank. As a result, these strains were found to be distributed over 5 genera and 11 tentative species/groups (A–K) (Table 2).

Phylogenetic analyses

The degrees of polymorphism of LSU, ITS, TUB2, TEF1-α and CAL make them variably suitable for genus or species discrimination amongst ophiostomatoid fungi. The LSU sequence is a suitable marker to infer the generic affinities (de Beer and Wingfield 2013, de Beer et al. 2013a, 2016); it allowed confirming the preliminary placement of our strains based on morphological characters (Fig. 2). The ITS region would be useful to place strains within the Ophiostoma s. l. complex, but the degree of polymorphism does not allow distinguishing species. Usually, TUB2, TEF1-α and CAL regions are better markers to identify and, where appropriate, to show the genetic diversity within ophiostomatoid fungi (Zipfel et al. 2006, de Beer and Wingfield 2013, de Beer et al. 2016).

Figure 2. 

Phylograms obtained from ML analysis of LSU sequences, showing fungal associates with pines infected by Tomicus yunnanensis, T. minor and T. brevipilosus in Yunnan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values ≥ 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *. Strains representing ex-type sequences are marked with ‘T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 743 positions, including gaps.

On the basis of the LSU blast searches, one to six strains of each tentative species (A–K) were selected for sequencing of five additional DNA markers (ITS, ITS2-LSU, TUB2, TEF1-α and CAL) to infer more accurate identification and phylogenetic affinities. Six sequence datasets (LSU, ITS, ITS2-LSU, TUB2, TEF1-α and CAL) were generated for a total of 31 representative strains (Table 2) and the sequences were deposited in GenBank. Resulting alignments were deposited in TreeBASE (submission no: 24032). The topologies generated by the ML and MP analyses were highly concordant and the ML phylograms are presented for all the individual genes, incorporating nodal supports of both the ML and MP analyses.

The LSU dataset consisted of 109 sequences, 11 sequences obtained in this study and 98 downloaded from GenBank. The phylogenetic analyses confirmed the morphology-based placement of our strains into Esteya, Graphilbum, Leptographium, Ophiostoma and Sporothrix (Fig. 2).

Group A consisted of a single strain. LSU-based phylogenetic analysis showed this strain to be close to E. vermicola (Fig. 2). TUB2 and TEF1-α data analysis confirmed the strain’s close affinities to E. vermicola (Fig. 3a, b), that could justify conspecificity.

Figure 3. 

Phylograms obtained from ML analysis of β-tubulin A and elongation factor B sequences of Esteya, showing fungal associates with pines infected by Tomicus yunnanensis in Yunnan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values ≥ 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *. Strains representing ex-type sequences are marked with ‘T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 320 (A), 856 (B) positions, including gaps.

Group B strains nested within the Graphilbum lineage in the LSU-based phylogenetic analysis (Fig. 2). Phylogenetic analysis based on LSU, ITS and TUB2 concordantly showed that the group B strains formed a single, well-supported clade related to but distinct from Gra. rectangulosporium and Gra. microcarpum (Fig. 4a, b); this would warrant its recognition as a distinct, undescribed species.

Figure 4. 

Phylograms obtained from ML analysis of ITS sequences A and β-tubulin sequences B of Graphilbum showing fungal associates with pines infected by Tomicus yunnanensis and T. minor in Yunnan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values ≥ 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *. Strains representing ex-type sequences are marked with ‘T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 515 (A), 481 (B) positions, including gaps.

Group C strains were shown to belong to the Leptographium lineage in the LSU-based phylogenetic analysis (Fig. 2). The ITS2-LSU dataset consisted of six of our own sequences and 49 reference sequences downloaded from GenBank. Within the Leptographium lineage, group C strains nested in the L. lundbergii-complex; they were related to L. yunnanense, L. lundbergii and L. conjunctum (Fig. 5a). TUB2- and TEF1-α based analysis confirmed their close affinities with L. yunnanense, although forming a slightly divergent clade (Fig. 5b, c). TUB2 and TEF1-α sequences of group C strains showed some polymorphisms, which could be considered as falling within the natural diversity of L. yunnanense.

Figure 5. 

Phylograms obtained from ML analysis of ITS2-28S A β-tubulin B and elongation factor C sequences of Leptographium, showing fungal associates with pines infected by Tomicus yunnanensis and T. brevipilosus in Yunnan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values ≥ 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *. Strains representing ex-type sequences are marked with ‘T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 641 (A), 358 (B), 639 (C) positions, including gaps.

The six strains from groups D to I nested within the Ophiostoma lineage based on the LSU phylogenetic tree (Fig. 2). The ITS dataset comprised species from all lineages discovered in this study. Analysis of this dataset yielded the phylograms shown in Fig. 6. Sixteen ITS sequences generated in this study were compared with 61 sequences retrieved from GenBank, representing the major groups of Ophiostoma (de Beer and Wingfield 2013, Linnakoski et al. 2016).

Figure 6. 

Phylograms obtained from ML analysis of ITS sequences of Ophiostoma, showing fungal associates with pines infected by Tomicus yunnanensis, T. minor and T. brevipilosus in Yunnan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values ≥ 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *. Strains representing ex-type sequences are marked with ‘T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 633 positions, including gaps.

The ITS- and TUB2-based phylogenetic inferences (Figs 6, 7a, b) showed that the strains of groups D and E nested within the O. clavatum- and O. piceae-complex (de Beer and Wingfield 2013, Yin et al. 2016, Linnakoski et al. 2016), in which they were positioned in the near vicinity of the O. brevipilosi and O. canum clades, respectively. From these results, and considering their morphological features, we concluded that the strains of groups D and E are conspecific with O. brevipilosi and O. canum, respectively.

Figure 7. 

Phylograms obtained from ML analysis of β-tubulin sequences of Ophiostoma A, B, D, E and ITS sequences of O. minus-complex C showing fungal associates with pines infected by Tomicus yunnanensis, T. minor and T. brevipilosus in Yunnan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values ≥ 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *. Strains representing ex-type sequences are marked with ‘T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 455 (A), 430 (B), 541 (C), 378 (D), 423 (E) positions, including gaps.

In the ITS-based phylogenetic analysis, strains of groups G and I were grouped with the O. minus complex (Fig. 6). ITS- and TUB2-based phylogenetic analyses consistently showed that group G strains formed a well-supported subclade between the North American and European subclades within the O. minus lineage (Fig. 7c, d). The strains of group G are therefore identified as O. minus. The ITS- and TUB2-based phylogenetic analyses consistently showed that the single strain of group I formed a branch that is related to, but distinct from the O. minus, O. kryptum and O. olgensis clades (Figs 6, 7d). Hence, this strain is interpreted as belonging to a distinct, undescribed Ophiostoma.

The remaining two groups (F and H) were not placed in any defined complex. Phylogenetic analyses, based on ITS and TUB2 sequences, consistently showed that the group H strains clustered in the near vicinity of the O. tingens clade whereas group F strains formed a clade related to, but distinct from the O. macrosporum and O. tingens clades (Figs 6, 7e). Thus, the strains in group H should be identified as O. tingens whereas the strains of group F represent an undescribed Ophiostoma.

Strains of groups J and K nested within the Sporothrix lineage in LSU-based phylogenetic analysis (Fig. 2). The phylograms resulting from the analyses of individuals are shown in Fig. 8 (ITS), Fig. 9a, c (TUB2) and Fig. 9b, d (CAL).

Figure 8. 

Phylograms obtained from ML analysis of ITS sequences of Sporothrix, showing fungal associates with pines infected by Tomicus yunnanensis, T. minor and T. brevipilosus in Yunnan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values ≥ 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *. Strains representing ex-type sequences are marked with ‘T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 546 positions, including gaps.

Figure 9. 

Phylograms obtained from ML analysis of β-tubulin A, C and calmodulin B, D sequences of Sporothrix, showing fungal associates with pines infected by Tomicus yunnanensis, T. minor and T. brevipilosus in Yunnan Province, China. Novel sequences obtained in this study are printed in bold type. Bootstrap values ≥ 70% for ML and MP are indicated above branches. Bootstrap values < 70% are indicated by the symbol *. Strains representing ex-type sequences are marked with ‘T’; ML, maximum likelihood; MP, maximum parsimony and the final alignment of 284(A), 622(B), 260(C), 675(D) positions, including gaps.

The ITS-based analyses showed that group K strains belonged to the S. gossypina-complex whereas the group J strains were not placed in any species complex as defined by de Beer et al. (2016) (Fig. 8). Both groups formed independent, well-supported clades in ITS-, TUB2- and CAL-based phylogenetic analyses (Figs 8, 9). It could be deduced from results of multiple phylogenies that both groups represent novel species.

Morphology and taxonomy

From a morphological perspective, strains of groups D, E and G appeared, overall, concordant with the descriptions or our own observations of reference strains, namely of O. brevipilosi, O. canum and O. minus, respectively. However, although strains of groups A, C, and H are phylogenetically close to E. vermicola, L. yunnanense and O. tingens, respectively, justifying, for the time being, conspecificity, their phenotype deviated slightly from published descriptions and/or our own observation of type material. The description of these species is extended. Strains of groups B, F, J and K revealed unique combinations of phenotypes, allowing morphological distinction from their closest phylogenetic relatives; consequently, they are described below as new species. The strain of the stand-alone group I also may represent an undescribed species; however, we refrain from describing it for the time being, waiting for more material to become available.

Taxonomy

Esteya vermicola J.Y. Liou, J.Y. Shih & Tzean, Mycol. Res. 103(2): 243. 1999.

MycoBank No: MB450702
Fig. 10

Description

Sexual form: unknown.

Asexual form: Hyalorhinocladiella-like. Conidiophores mononematous, micronematous; conidiophorous cells solitary, integrated, flask-shaped, with an inflated base (3.6–) 4.6–6.1 (–7.1) μm in diam., the fertile hyphoid part (9.1–) 12.2–19.0 (–22.5) × (1.4–) 1.9–3.1 (–4.7) μm, often crooked due to successive conidial development; conidia 1-celled, asymmetrically ellipsoidal in face view, concave, lunate in side view, with a layer of adhesive mucus on the concave surface, ending slightly apiculate, hyaline, smooth, (8.0–) 10–12 (–13.1) × (3.3–) 3.4–4.5 (–5.1) μm, containing an ovoid endospore-like structure.

Figure 10. 

A–H Morphological characters of Esteya vermicolaA, B upper and reverse of cultures on 2% MEA 20 d after inoculation C–H conidiogenous cells with lunate conidia I–M the cuticle of a nematode attached by many lunate conidia. Scale bars: 20 μm (I, K, L); 10 μm (C–H, J, M).

Culture characteristics

Colonies on 2% MEA in the dark reaching 31 mm in diam. in 8 days at 25 °C, growth rate up to 5 mm/day at the fastest; colony margin smooth. Mycelium compact, somewhat floccose in the margin, white at first, gradually discolouring to greyish-green, eventually dark green. Optimal growth temperature 25 °C, growth at 5 °C and 35 °C.

Known substrate and host

Galleries of Tomicus yunnanensis in Pinus yunnanensis.

Known insect vector

Tomicus yunnanensis.

Known distribution

Yunnan Province, China.

Specimen examined

CHINA, Yunnan, Tomicus yunnanensis galleries in Pinus yunnanensis, Dec. 2016, HM Wang, CFCC 52625 = CXY 1893.

Note

Esteya vermicola is known only from an asexual, Hyalorhinocladiella-like state producing lunate and bacilliform conidia (Liou et al. 1999, Kubátová et al. 2000, Wang et al. 2009, 2014) that we also observed in various strains of E. vermicola with a different origin (Taiwan, Korea, Czech Republic). Our strain was identified as E. vermicola based on phylogenetic inferences and morphological characters. However, our strain differed from previous descriptions (Liou et al. 1999) in having only lunate conidia in vitro. The size of the lunate conidia of our strains (mostly 10 - 12 × 3.4 - 4.5 μm) was similar to that reported for E. vermicola, viz. 9.9–11.9 × 3.4–4.5 μm vs 8.2-11.1 × 3.5–3.7 μm (Taiwan, Liou et al. 1999), 9.3–12.4 × 3.0–3.2 μm (Czech Republic, Kubátová et al. 2000), 7.7–12.1 × 3.0–3.8 μm (Korea, Wang et al. 2009) or 8.7–11.9 × 3.0–3.6 μm (Brazil, Wang et al. 2014).

This is the first report of E. vermicola from continental China. The species was originally isolated from Japanese black pine infected by the pinewood nematode Bursaphelenchus xylophilus, in Taiwan (Liou et al. 1999). Since then, its distribution range has been extended to Japan and Korea, Europe (Czech Republic, Italy) and both North (USA) and South America (Brazil) (Liou et al. 1999, Kubátová et al. 2000, Wang et al. 2009, 2014, Li et al. 2018). This species is associated with various vectors, including the pinewood nematode, Oxoplatypus quadridentatus and the bark beetle Scolytus intricatus. It was isolated also from wooden packaging material infested by Bursaphelenchus rainulfi.

Graphilbum anningense H. Wang, Q. Lu & Z. Zhang, sp. n.

MycoBank No: MB828884
Fig. 11

Etymology

anningense’ (Latin), referring to the type locality.

Type

CHINA, Yunnan, Tomicus yunnanensis galleries in Pinus yunnanensis, Apr. 2017, HM Wang, holotype CXY 1939, culture ex-holotype CFCC 52631 = CXY 1939.

Description

Sexual form: unknown.

Asexual forms: Pesotum-like and Hyalorhinocladiella-like. Pesotum-like conidiophores abundant on 2% MEA, macronematous, synnematous, (150–) 210–293 (–336) μm long including conidiogenous apparatus, the base dark brown, slightly widened, (6.7–) 7.9–18.8 (–29.0) μm wide anchored in the media by brown rhizoid-like hyphae, the apex slightly enlarging, fan-shaped; conidiogenous cells hyaline, thin-walled, aseptate, (15.3–) 21.0–35.5 (–42) × (0.7–) 1.1–1.9 (–2.3) μm; conidia 1-celled, clavate, ellipsoid to ovoid with truncate base and rounded apex, hyaline, smooth, (3.1–) 3.6–6.3 (–9.7) × (1.4–) 1.6–2.2 (–2.5) μm.

Figure 11. 

Morphological characters of Graphilbum anningense sp. n. A, B Upper and reverse of cultures on 2% MEA 8 d after inoculation C, D, G conidiogenous cells of Pesotum-like macronematal asexual state and conidia E, F, H conidiogenous cells of Hyalorhinocladiella-like asexual state and conidia. Scale bars: 50 μm (C); 20 μm (D); 10 μm (E–H).

Hyalorhinocladiella-like: conidiogenous cells macronematous or semi-macronematous, mononematous, hyaline, simple or loosely branched, thin-walled, aseptate, (4.5–) 10.8–29.0 (–47) × (1.5–) 1.7–2.3 (–2.6) μm; conidia hyaline, clavate to ellipsoid, with obtuse ends, 1-celled, aseptate, smooth, (3.7–) 4.5–6.4 (–9.0) × (1.4–) 1.7–2.3 (–2.9) μm.

Culture characteristics

Colonies on 2% MEA in the dark reaching 90 mm in diam. in 6 days at 25 °C, growth rate up to 19.5 mm/day at the fastest; colony margin smooth. Mycelium superficial to flocculose or floccose, hyaline; reverse hyaline to pale yellowish. Optimal growth temperature 30 °C, slow growth at 40 °C, no growth at 5 °C.

Known substrate and hosts

Galleries of Tomicus yunnanensis and T. minor in Pinus yunnanensis.

Known insect vectors

Tomicus yunnanensis, T. minor.

Known distribution

Yunnan Province, China.

Additional specimens examined

CHINA, Yunnan, Tomicus yunnanensis, T. minor galleries in Pinus yunnanensis, Apr. 2017, HM Wang, CFCC 52632 = CXY 1940, CFCC 52633 = CXY 1944.

Note

Graphilbum anningense is characterised by a Pesotum-like and a Hyalorhinocladiella-like asexual state. It is phylogenetically closely related to Gra. rectangulosporium. However, Gra. rectangulosporium produced a sexual state in vitro (Ohtaka et al. 2006) which has not been observed in Gra. anningense. Other morphologically similar species include Gra. fragrans, Gra. crescericum, Gra. kesiyae and Gra. puerense. Graphilbum kesiyae and Gra. puerense also produce a Pesotum-like and a Hyalorhinocladiella-like asexual state. Graphilbum anningense and Gra. kesiyae differ by the size of their synnemata, whose length ranges do not overlap, viz. 210–293 μm and 112.5–173 μm long (Harrington et al. 2001), respectively. They also differ by their optimal growth temperature, respectively 30°C and 25°C. The synnemata of Gra. puerense, 206–357 μm long (Chang et al. 2017), are marginally longer than those of Gra. anningense. Graphilbum fragrans and Gra. crescericum produce only a Leptographium-like and/or a Hyalorhinocladiella-like asexual state in vitro (Harrington et al. 2001, Chang et al. 2017).

Graphilbum anningense was isolated from galleries of T. yunnanensis and T. minor infesting P. yunnanensis. Previously, Gra. fragrans had been reported from T. yunnanensis infesting P. yunnanensis and from Pissodes spp. infesting Tsuga dumosa and P. armandii in China (Paciura et al. 2010, Zhou et al. 2013). Graphilbum kesiyae and Gra. puerense were isolated from galleries of Polygraphus aterrimus, Po. szemaoensis and Ips acuminatus infesting P. kesiya (Chang et al. 2017). Although the geographic distribution of these four Graphilbum species overlaps, their hosts and vectors are nevertheless, as far as it is known, different (Chang et al. 2017).

Leptographium yunnanense X.D. Zhou, K. Jacobs, M.J. Wingf. & M. Morelet, Mycoscience 41(6): 576. 2000.

MycoBank No: MB 466542
Fig. 12

Description

Sexual form: unknown.

Asexual form: Leptographium-like. Conidiophores occurring singly or in groups of up to three, arising from the superficial mycelium, erect, macronematous, mononematous, (93.5–) 159–412 (–544) μm long, without rhizoid-like structures; stipes simple, cylindrical, not constricted at septa, 1-6-septate, pale olivaceous at the base, (12–) 19.0–128 (–245) × (3.3–) 4.1–6.1 (–7.3) μm; conidiogenous apparatus (33.0–) 65.5–119.5 (–168.0) μm long (high), with 2 to 3 series of cylindrical branches; primary branches hyaline to pale olivaceous, smooth, cylindrical, 2–3 septate, (11.5–) 18.2–37.7 (–56.0) μm long and (3.0–) 3.7–5.9 (–7.7) μm wide; secondary branches hyaline, 0–2 septate, (10.3–) 14.5–30.0 (–50.1) μm long, (2.8–) 3.4–5.5 (–7.3) μm wide; conidiogenous cells discrete, 2–3 per branch, cylindrical, (10.2–) 13.2–29.6 (–57.4) × (2.2–) 2.9–3.9 (–4.4) μm; conidia 1-celled, oblong to obovoid with truncate bases, hyaline, (5.8–) 7.0–10.4 (–13.0) × (2.9–) 3.6–5.3 (–6.4) μm.

Figure 12. 

Morphological characters of Leptographium yunnanense A, B upper and reverse of cultures on 2% MEA 8 d after inoculation D, I conidiophore on 2% MEA C, E–H conidiogenous cells of Leptographium-like asexual state and conidia. Scale bars: 50 μm (D, I); 10 μm (C, E–H).

Culture characteristics

Colonies on 2% MEA medium fast growing in the dark, reaching 76 mm in diam. in 8 days at 25 °C, growth rate up to 20 mm/day at the fastest; colony margin smooth. Hyphae submerged in agar with aerial mycelium, greenish-olivaceous to olivaceous, smooth, straight; reverse hyphae umber-brown to dark olivaceous. Optimal growth temperature 25 °C, slow growth at 5 °C and 30 °C.

Known substrate and hosts

Tomicus yunnanensis and its galleries in Pinus yunnanensis, galleries of T. brevipilosus in P. kesiya.

Known insect vectors

Tomicus brevipilosus, T. yunnanensis.

Known distribution

Yunnan Province, China.

Specimens examined

CHINA, Yunnan, adults of Tomicus yunnanensis and their galleries in Pinus yunnanensis, Tomicus brevipilosus galleries in P. kesiya. Apr. 2017, HM Wang, CFCC 52619 = CXY 1897, CFCC 52620 = CXY 1900, CFCC 52621 = CXY 1904, CFCC 52622 = CXY 1908, CFCC 52623 = CXY 1917, CFCC 52624 = CXY 1925.

Note

The sole reproductive structure formed on MEA in L. yunnanense is a Leptographium-like state. Our strains were identified as L. yunnanense, based on phylogenetic evidence and secondarily, on morphological features. However, our strains slightly deviated from L. yunnanense in having longer conidiophores, mainly 159–412 μm vs mostly 74–227 (–233) μm (Zhou et al. 2000) or 80–240 μm (Yamaoka et al. 2008). Furthermore, our strains grew faster than reported for the species, 76 mm vs 44 mm in 8 days at 25 °C (Zhou et al. 2000).

Although our strains were slightly genetically and morphologically divergent, we are of the opinion that they enter into the current L. yunnanense species concept (e.g. sensu Zhou et al. 2000). Yamaoka et al. (2008) showed the genetic diversity of L. yunnanense in Yunnan to be higher than in other places, that which is confirmed by the present study.

Leptographium yunnanense was originally described from Yunnan Province with only an asexual state (Zhou et al. 2000). Subsequently, mating of strains from different origins (Thailand, China and Japan) yielded the sexual state, which is formed by neckless ascocarps and cucullate ascospores (Yamaoka et al. 2008).

Leptographium yunnanense was the third most abundant species associated with T. yunnanensis in our study. A few strains also were isolated from T. brevipilosus infesting P. kesiya and none from T. minor.

Ophiostoma aggregatum H. Wang, Q. Lu & Z. Zhang, sp. n.

MycoBank No: MB828885
Fig. 13

Etymology

aggregatum’ (Latin), reflects to the conidiophores aggregated in clusters.

Type

CHINA, Yunnan, from Tomicus minor galleries in Pinus yunnanensis, Dec. 2016, HM Wang, holotype CXY 1876, culture ex-holotype CFCC 52615 = CXY 1876.

Description

Sexual form: unknown.

Asexual form: Leptographium-like. Conidiophores macronematous, mononematous, gathered in groups up to 5, (28.5–) 34–45.5 (–52) μm long; stipes cylindrical, 1–2 septate, not constricted at septa, umber-brown to dark olivaceous, (6.3–) 7.3–14.5 (–18) μm long × (2.2–) 3.1–4.6 (–5.8) μm wide. Conidiogenous apparatus (22–) 26.5–31 (–34) μm long, with 2–3 series of cylindrical branches; primary branches olivaceous, smooth, cylindrical all over, (5.9–) 7.2–13.5 (–20.5) × (3–) 3.3–4.2 (–4.6) μm; conidiogenous cells discrete, 2–3 per branch, aseptate, cylindrical, hyaline to pale umber, (5.8) 7.2–12.1 (–18.5) × (2.1–) 2.8–4.0 (–4.7) μm; conidia 1-celled, globose, elliptical with truncate bases, hyaline to pale umber, (4.0–) 4.8–5.9 (–6.3) × (3.1–) 4.0–5.0 (–5.6) μm.

Figure 13. 

Morphological characters of Ophiostoma aggregatum sp. n. A, B upper and reverse of cultures on 2% MEA 8 d after inoculation C conidiomata on 2% MEA (bar = 50 μm) D–H conidiogenous cells of Leptographium-like asexual state and conidia. Scale bars: 20 μm (C); 10 μm (D–H).

Culture characteristics

Colonies on 2% MEA fast growing in the dark, reaching 90 mm in diam. in 8 days at 25 °C, growth rate up to 13 mm/day at the fastest; colony margin smooth. Hyphae submerged and aerial, umber-brown to dark olivaceous, flocculose or floccose; reverse hyphae umber-brown to dark olivaceous. Optimal growth temperature 25 °C, able to grow at 5 °C and 30 °C. No growth at 35 °C.

Known substrate and hosts

Galleries of Tomicus yunnanensis and T. minor in Pinus yunnanensis.

Known insect vectors

Tomicus minor, T. yunnanensis.

Known distribution

Yunnan Province, China.

Additional specimens examined

CHINA, Yunnan, from Tomicus yunnanensis and T. minor galleries in Pinus yunnanensis, Dec. 2016, Apr. 2017, HM Wang, CFCC 52616 = CXY 1875, CFCC 52617 = CXY 1874.

Note

Ophiostoma aggregatum produced a single asexual, Leptographium-like state in vitro. This species is phylogenetically closely related to O. macrosporum, O. tingens, O. floccosum, O. tapionis and O. piliferum in LSU-, ITS- and TUB2-based phylogenetic inferences. Ophiostoma aggregatum and O. tingens are shown to be sympatric in Yunnan pine forest; both taxa were isolated from galleries and adults of T. minor and T. yunnanensis infesting P. yunnanensis (Table 2). Ophiostoma tingens was also reported from T. minor infesting P. yunnanensis in Yunnan (Pan et al. 2017).

Ophiostoma aggregatum and O. tingens differ in their asexual state. Ophiostoma aggregatum only produces a Leptographium-like state. Inversely, the asexual states of O. tingens are variable. Our strains produced a Pesotum-like and a Sporothrix-like state whereas previously, Francke-Grosmann (1952) and de Beer et al. (2013b) reported a Hyalorhinocladiella- to Raffaelea-like state in European strains. The origin of this variability and its importance for taxonomy is uncertain.

Ophiostoma macrosporum, O. floccosum, O. tapionis and O. piliferum also differ from O. aggregatum by their asexual state. Ophiostoma macrosporum and O. floccosum produce a Pesotum-like asexual state, O. tapionis a Hyalorhinocladiella-like state and O. piliferum produces a Sporothrix-like state (Francke-Grosmann 1952, Upadhyay 1981, Yamaoka et al. 2004, Linnakoski et al. 2008).

Ophiostoma macrosporum and O. tingens were both originally described in Trichosporium as T. tingens var. macrosporum and T. tingens (Lagerberg 1927, Francke-Grosmann 1952). Batra (1967) transferred these two species into Ambrosiella. It is only recently that the morphological characteristics were found to agree with those of Ophiostoma (de Beer et al. 2013b). Ophiostoma macrosporum has been reported from various Pinus spp. (including P. sylvestris) infected by Ips acuminatus in Europe (Francke-Grosmann 1952, Batra 1967).

Ophiostoma tingens (Lagerb. & Melin) Z.W. de Beer & M.J. Wingf., Svensk Skogsvårdsförening Tidskr. 25:233. 1927.

MycoBank No: MB801091
Fig. 14

Description

Sexual form: unknown.

Asexual forms: Pesotum-like and Sporothrix-like. Pesotum-like: conidiophores macronematous, synnematous; synnemata simple, anchored into the substrate by brown rhizoid-like hyphae, (333–) 344–584 (–684) μm long including conidiogenous apparatus, the base dark brown, slightly widened, (16.7–) 17–50.5 (–65.5) μm wide, the apex cream-coloured or pale brown, slightly widening; conidia hyaline, globose to elliptical, 1-celled, smooth, (2.7–) 3.6–7.2 (–8.0) × (2.8–) 4.3–6.1 (–7.0) μm.

Sporothrix-like: conidiophores semi-macronematous, mononematous, hyaline, simple or loosely branched, smooth, bearing terminal denticulate conidiogenous cells (8.3–) 15.6–30.0 (–42.5) × (1.1–) 1.7–3.1 (–4.7) μm; conidia hyaline, globose to elliptical, obovoid with pointed bases and rounded apices, 1-celled, smooth, (2.6–) 4.0–6.8 (–8.7) × (2.2–) 3.5–5.5 (–7.4) μm.

Figure 14. 

Morphological characters of Ophiostoma tingens A, B upper and reverse of cultures on 2% MEA 20 d after inoculation C–G conidiogenous cells of Sporothrix-like asexual state and conidia H–J conidiogenous cells of Pesotum-like macronematal asexual state and conidia. Scale bars: 10 μm (C–H); 50 μm (I, J).

Culture characteristics

Colonies on 2% MEA medium slow growing in the dark, reaching 39 mm in diam. in 8 days at 25 °C, growth rate up to 5 mm/day at the fastest; colony margin anomalous. Hyphae appressed to flocculose, black; reverse hyphae also black. Optimal growth temperature 25 °C, no growth at 5 °C and 30 °C.

Known substrate and hosts

Galleries of Tomicus yunnanensis and T. minor in Pinus yunnanensis.

Known insect vectors

Tomicus yunnanensis, T. minor.

Known distribution

Yunnan Province, China; Europe.

Specimens examined

CHINA, Yunnan, from Tomicus minor and T. yunnanensis galleries in Pinus yunnanensis, Feb. 2017, Nov. 2016, HM Wang, CFCC 52611 = CXY 1866, CFCC 52612 = CXY 1865, CFCC 52613 = CXY 1868.

Note

Our strains of O. tingens were identified based on phylogenetic affinities and morphological features. (cf. above under note for O. aggregatum.)

Ophiostoma tingens has been reported from sapwood of various Pinus spp. (including P. sylvestris) infested by T. minor, T. piniperda and Ips sexdentatus in Europe (Francke-Grosmann 1952, Batra 1967, Jankowiak 2008). The species was recorded in Yunnan Province in China in 2017, associated with T. minor infesting P. yunnanensis (Pan et al. 2017).

Sporothrix macroconidia H. Wang, Q. Lu & Z. Zhang, sp. n.

MycoBank No: MB828886
Fig. 15

Etymology

macroconidia’ (Latin), referring to the large conidia of this fungus.

Type

CHINA, Yunnan, from Tomicus yunnanensis galleries in Pinus yunnanensis, Dec. 2016, collected by HM Wang, holotype CXY 1894, culture ex-holotype CFCC 52628 = CXY 1894.

Description

Sexual form: unknown.

Asexual form: Sporothrix-like. Conidiophores semi-macronematous, mononematous; conidiogenous cells hyaline, simple or loosely branched, thin-walled, aseptate, bearing denticles forming a rachis (4.1–) 11.0–24.5 (–36.5) × (1.4–) 2.1–3.4 (–4.9) μm; conidia hyaline, cylindrical, ellipsoid to ovoid, 1-celled, smooth, (3.6–) 4.8–7.4 (–9.9) × (2.5–) 3.2–4.9 (–9.9) μm, solitarily or aggregating in slimy masses.

Figure 15. 

Morphological characters of Sporothrix macroconidia sp. n. A, B Upper and reverse of cultures on 2% MEA 20 d after inoculation C–H conidiogenous cells of Sporothrix-like asexual state and conidia. Scale bars: 10 μm (C–H).

Culture characteristics

Colonies on 2% MEA medium slow growing in the dark, reaching 34 mm in diam. in 8 days at 25 °C, growth rate up to 5 mm/day at the fastest; colony margin smooth. Hyphae appressed to flocculose, white; reverse hyaline to pale yellowish. Optimal growth temperature 25 °C, little growth at 5 °C and 35 °C.

Known substrates and hosts

Galleries of Tomicus yunnanensis and T. brevipilosus in Pinus yunnanensis and P. kesiya.

Known insect vectors

Tomicus yunnanensis, T. brevipilosus.

Known distribution

Yunnan Province, China.

Additional specimens examined

CHINA, Yunnan, from Tomicus brevipilosus galleries in Pinus kesiya, Dec. 2016, Jan. 2017, HM Wang, CFCC 52629 = CXY 1895, CFCC 52630 = CXY 1896.

Note

Sporothrix macroconidia is closely related to O. valdivianum, S. bragantina, S. brunneoviolacea and S. fumea in phylogenetic analyses inferred from LSU, ITS, TUB2 and CAL DNA sequence data. It differs from these species by its conidia, which are larger than those of the other four species, mostly 4.8–7.4 × 3.2–4.9 μm and 4–6 × 2 μm in O. valdivianum (Butin and Aquilar 1984), 4–6 × 2–2.5 μm in S. bragantina (Pfenning and Oberwinkler 1993), 3–7 × 1.5–3 μm in S. brunneoviolacea (Madrid et al. 2010) and 1.5–2.0 × 0.5–1.0 μm in S. fumea (Nkuekam et al. 2012). In addition, a sexual state was observed in vitro for O. valdivianum, S. bragantina and S. fumea, which was not observed in S. macroconidia and S. brunneoviolacea.

Sporothrix macroconidia was found associated with T. yunnanensis infesting P. yunnanensis and with T. brevipilosus infesting P. kesiya. The other four similar species have very different ecology and known geographic distributions. Sporothrix fumea was isolated from Eucalyptus cloeziana infested by Phoracantha beetles in South Africa (Nkuekam et al. 2012), whereas O. valdivianum, S. bragantina and S. brunneoviolacea were obtained from soil or Nothofagus in Europe and South America (Butin and Aquilar 1984, Pfenning and Oberwinkler 1993, Madrid et al. 2010).

Sporothrix pseudoabietina H. Wang, Q. Lu & Z. Zhang, sp. n.

MycoBank No: MB828887
Fig. 16

Etymology

pseudoabietina’ (Latin), referring to the phylogenetic affinities to S. abietina.

Type

CHINA, Yunnan, from T. minor galleries in P. yunnanensis, Apr. 2017, HM Wang, holotype CXY 1937, culture ex-holotype CFCC 52626 = CXY 1937.

Description

Sexual form perithecial: on 2% MEA, perithecia superficial or partially immersed, with a globose base extending into a cylindrical neck, often terminated by ostiolar hyphae; bases (85–) 110–152 (–168) μm diam., black, the outer layer with dark brown hyphal ornamentation; apical neck mild to dark brown at the base, pale brown to pale yellow or hyaline toward the apex, straight or slightly curved, (172–) 560–985 (–1039) μm long, (37–) 41–62 (–78) μm wide at the base, (9.3–) 12.5–17.5 (–20) μm wide at the apex; ostiolar hyphae numerous, hyaline, divergent, (19.5–) 21.5–38.0 (–43) μm long; asci not seen; ascospores hyaline, 1-celled, orange-shaped in lateral view, ellipsoid in face view, circular in polar view, (2.9 –) 3.4–4.4 (–5.3) × (0.8–) 1.0–1.5 (–1.9) μm, without mucilaginous sheath.

Asexual form: Sporothrix-like. Conidiophores semi-macronematous to mononematous; conidiogenous cells hyaline, simple or loosely branched, smooth, bearing denticles disposed in a dense rachis (16.0–) 20.5–30.5 (–34.5) × (1.2–) 1.6–2.0 (–2.3) μm; conidia 1-celled, clavate, ellipsoid to ovoid, hyaline, (3.0–) 4.0–7.0 (–9.0) × (1.0–) 1.1–3.1 (–4.8) μm.

Figure 16. 

Morphological characters of Sporothrix pseudoabietina sp. n. A, B upper and reverse of cultures on 2% MEA 20 d after inoculation C, D ostiolar hyphae present E, F perithecium G ascospores of sexual state H–I conidiogenous cells of Sporothrix-like asexual state and conidia. Scale bars: 20 μm (C, D); 50 μm (E, F); 10 μm (G–I).

Culture characteristics

Colonies on 2% MEA slow growing in the dark, reaching 23 mm in diam. in 8 days at 25 °C, growth rate up to 2.5 mm/day at the fastest; colony margin smooth. Hyphae appressed to flocculose or floccose, white; reverse hyaline to pale yellowish. Optimal growth temperature 25 °C; very slow growth at 35 °C; no growth at 5 °C.

Known substrate and hosts

Galleries of Tomicus yunnanensis and T. minor in Pinus yunnanensis.

Known insect vectors

Tomicus yunnanensis, T. minor.

Known distribution

Yunnan Province, China.

Additional specimen examined

CHINA, Yunnan, Tomicus minor galleries in Pinus yunnanensis, Apr. 2017, HM Wang, CFCC 52627 = CXY 1938.

Note

Sporothrix pseudoabietina is characterised by a perithecial sexual form and a Sporothrix-like asexual state. Multiple phylogenetic inferences (LSU, ITS, TUB2 and CAL) showed that S. pseudoabietina belonged to the S. gossypina complex, in which it is closely related to S. abietina. However, it can be distinguished from this species, based on both morphological and physiological features. The conidia of S. pseudoabietina (4.0–7.0 × 1.1–3.1 μm) are wider than those of S. abietina (4–7.5 × 1–2 μm) (Marmolejo and Butin 1990). Perithecia are known from S. abietina but only on natural substrates and not in vitro on artificial media, contrary to those from S. pseudoabietina. The perithecial neck in S. pseudoabietina is much longer than that of S. abietina, viz. mostly 560–985 μm and 450–650 μm, respectively. Ostiolar hyphae of S. abietina and S. pseudoabietina also differ in number, numerous vs 7–10 and size, mostly 13–19 μm and in S. pseudoabietina 21.5–38.0 μm (Fig. 11c, d). In addition, no growth of S. abietina was observed at 35 °C, but S. pseudoabietina can grow at 35 °C.

The hosts and geographic distributions of S. pseudoabietina and S. abietina are also very different. Sporothrix pseudoabietina was found associated with T. minor and T. yunnanensis infecting P. yunnanensis, whereas S. abietina was reported from Abies vejari attacked by Pseudohylesinus sp. in Mexico (Marmolejo and Butin 1990).

Discussion

In this study, 772 strains of ophiostomatoid fungi were isolated from galleries and adults of three pine shoot beetles, T. brevipilosus, T. minor and T. yunnanensis, inhabiting P. yunnanensis and P. kesiya in forests in Yunnan Province, south-western China. Multiple phylogenetic analyses and morphological features allowed the identification of 11 species from 5 genera. Six species corresponded to known taxa (E. vermicola, L. yunnanense, O. brevipilosi, O. canum, O. minus and O. tingens), whereas four species are proposed as new, Gra. anningense, O. aggregatum, S. pseudoabietina and S. macroconidia. A single strain remained unnamed.

The global ophiostomatoid fungal communities, associated with these three Tomicus species in pine forest, were dominated by far by three species, which are, in decreasing order of isolates, O. canum, O. brevipilosi and O. minus. Furthermore, these three ophiostomatoid species are not equally associated with the three Tomicus species but show variable degrees of preference or specificity.

Overall, O. canum was the most frequently isolated species in our study (253 out of the 772 strains). It was preferably (79.4% of the O. canum strains) isolated from galleries and adults of T. minor, infesting both P. yunnanensis and P. kesiya (Table 3) and dominated the ophiostomatoid community associated with this beetle (81.4%, 201 strains of O. canum out of 247 strains in the community, Table 3).

This is the first report of this species in China. It was previously reported in eastern Asia but only in Japan (Masuya et al. 1999). Ophiostoma canum was also shown to be the dominant species associated with T. minor, both in Europe and Japan (Masuya et al. 1999, Jankowiak 2008). In addition, this species was found in association with other bark beetles in Finland and Russia, e.g. Hylastes brunneus, Hylurgops palliatus, Ips typographus, Pityogenes chalcographus and Trypodendron lineatum (Linnakoski et al. 2010). The close association between O. canum and T. minor appears stable over an extensive geographical distribution and tree host range, indicating likely intimate relationships.

Ophiostoma brevipilosi represented the second most frequently isolated species in our survey (224 out of 772 strains), occurring exclusively in galleries and adults of T. brevipilosus, dominating this beetle’s ophiostomatoid community (98.2%, 224 strains of O. brevipilosi out of 228 strains in the community, Table 3). The occurrence or fitness of O. brevipilosi is therefore strongly linked to the presence of T. brevipilosus.

Ophiostoma brevipilosi was described originally from Yunnan, based on six strains, all isolated from T. brevipilosus (Chang et al. 2017). It belongs to the recently defined O. clavatum complex (Linnakoski et al. 2016). It is only known from this area of south-western China.

Ophiostoma minus was the third most frequently isolated species overall (197 strains out of 772), occurring exclusively in galleries and adults of T. yunnanensis infesting P. yunnanensis, dominating this beetle ophiostomatoid community (66.3%, 197 strains of O. minus out of 297 strains in the community, Table 3).

Ophiostoma minus, first reported as a blue-stain agent in Europe (Munch 1907), is a widely distributed species, also recorded from North America and East Asia (Japan and China) (Hedgcock 1906, Gorton and Webber 2000, Gorton et al. 2004, Lu et al. 2009, Linnakoski et al. 2010). It infests various pines and is transported by various bark beetles. This species was predominantly associated with T. piniperda in Europe (Jankowiak 2006) and Japan (Masuya et al. 1999) and with the southern pine beetle, Dendroctonus frontalis, in the southern states of the USA (Klepzig 1998, Gorton and Webber 2000, Gorton et al. 2004).

Ophiostoma minus was deemed to have two allopatric populations, viz. a North American and a Eurasian population (Gorton et al. 2004). In ITS/TUB2 phylogenetic inferences, the North American and Eurasian populations of O. minus were resolved as two closely related clades (Gorton et al. 2004, Lu et al. 2009). ITS and TUB2-based phylogenetic inferences (Fig. 7c, d) also resolved our strains as a third distinct clade, which could thus be interpreted as a third allopatric population. The question of translating these populations into a Linnaean taxonomic rank, however, remains open.

Tomicus yunnanensis galleries and adult beetles harboured the highest diversity of ophiostomatoid fungi; ten of the 11 species identified were isolated from galleries and adults of this beetle. Three species were exclusively found with this beetle (O. minus, E. vermicola, Ophiostoma sp. 1). By comparison, galleries and adults of T. minor and of T. brevipilosus yielded less species; five species were isolated from T. minor, none of which was associated exclusively with this beetle and three species from T. brevipilosus, of which one was exclusive, O. brevipilosi. Five species are shared by both T. yunnanensis and T. minor and two species by both T. yunnanensis and T. brevipilosus, but none by T. minor and T. brevipilosus and also none by all three pine shoot beetles (Table 3, Fig. 17).

Strain numbers of various ophiostomatoid fungi obtained from three Tomicus spp. and their galleries collected in Yunnan Province.

Group Fungi species Tomicus yunnanensis T. minor T. brevipilosus Total no. strains\samples
A Ophiostoma brevipilosi 0 0 224 224
B O. canum 52 201 0 253
C O. minus 197 0 0 197
D O. tingens 4 26 0 30
E O. aggregatum 3 2 0 5
F Ophiostoma sp. 1 1 0 0 1
G Leptographium yunnanense 30 0 2 32
H Esteya vermicola 1 0 0 1
I Sporothrix pseudoabietina 4 15 0 19
J S. macroconidia 1 0 2 3
K Graphilbum anningense 4 3 0 7
Total no. strains 297 247 228 772
Total no. samples 455 324 339 1118
Figure 17. 

Venn diagram showing overlaps of the ophiostomatoid fungal communities associated with three pine shoot beetles.

The ectosymbiosis between bark beetles and fungi is widespread and diverse. Some fungi are highly specific and associated with a single beetle species, forming a ‘species-specific association’ (Six and Paine 1999, Six 2012), while others can be associated with many vectors (Kostovcik et al. 2014). The species-specific associations include, for instance, Ips typographus and Endoconidiophora polonica, I. cembrae and End. laricicola (Harrington et al. 2002) or I. subelongatus and End. fujiensis (Marin et al. 2005, Meng et al. 2015). The present study showed that species-specific associations might occur with various sympatric beetles that share the same niche. The association of T. brevipilosus and O. brevipilosi seems to be species-specific in the pine forest of Yunnan, where both taxa are, so far, endemic. In the pine forest of Yunnan, the Chinese ‘population’ of O. minus is also specifically associated with T. yunnanensis, whereas the two other O. minus ‘populations’ are associated, at least preferably, with Dendroctonus frontalis and T. piniperda (Gorton et al. 2004, Jankowiak 2006). The genetically distinct ‘populations’ might originate from both the allopatric distribution and vector specificity and both factors could support recognition of three distinct taxa. In the pine forest of Yunnan, the association of O. canum with T. minor is preferential but not exclusive.

Up to now, no data have been provided proving the pathogenicity of these ophiostomatoid species to both indigenous pines, except for L. yunnanense (Liao and Ye 2004, Gao et al. 2017). Pathogenicity tests have been done by artificial inoculation of the dominant species into seedlings of the two pines. The results preliminarily showed that the virulence of O. minus and O. brevipilosi was significantly stronger than that of O. canum. This is similar to the relative aggressive nature of the three Tomicus species. Thus, we suspect there might be some link between beetle aggression and fungus virulence (Christiansen et al. 1987, Kirisits 2004).

Conclusions

This study provides evidence for the diversity of ophiostomatoid fungi associated with T. yunnanensis, T. minor and T. brevipilosus in Yunnan pine forest in south-western China. Eleven species were identified, of which four were new to science. The diversity is the highest in the galleries and adults of T. yunnanensis and the poorest in the galleries and adults of T. brevipilosus.

Three species, namely O. brevipilosi, O. canum and O. minus, dominate the ophiostomatoid communities; each is associated predominantly with one species of Tomicus, namely T. brevipilosus, T. minor and T. yunnanensis, respectively. In this regard, this study has revealed differential associations between beetles living sympatrically, concomitantly or sequentially, in the same ecological niche, which indicates a certain level of specificity of the relationships between the fungi and the beetles. However, the parameters behind these (partial) species-specific relationships remain unknown.

Increased study of the biodiversity, biogeography and ecology of ophiostomatoid fungi in China, in particular of those associated with Tomicus spp., would facilitate comparison with well-known species associated with other Tomicus spp. in other neighbouring or distant geographical areas, e.g. in European countries, Japan and Korea and allow a better understanding of the occurrence and mechanisms behind the outbreak of infections, enabling the development of effective management methods to alleviate the subsequent plant losses.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Project No.: 31770693 and 31770682). We are very grateful to Shuangcheng Li and Hongxun Wang for their help in field survey and collection. Cony Decock gratefully acknowledges the financial support received from the Belgian State–Belgian Federal Science Policy through the BCCM programme.

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