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Research Article
Diversity of fungi associated with Monochamus alternatus larval habitats in Bursaphelenchus xylophilus-infected Pinus massoniana and identification of two new ophiostomatalean species (Ascomycota, Ophiostomatales)
expand article infoGuiheng Zheng, Minqi You§, Xuening Li|, Qinzheng Zhou, Zheng Wang, Huimin Wang, Quan Lu
‡ Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, China
§ Agriculture and Rural Affairs Bureau of Huangyan District, Zhejiang, China
| Research Institute of Desertification, Chinese Academy of Forestry, Beijing, China
Open Access

Abstract

Bursaphelenchus xylophilus, a pathogenic pine wood nematode (PWN), is responsible for pine wilt disease (PWD), which has caused significant economic and ecological damage worldwide, particularly in East Asia. Multiple biological factors, such as the beetle vector Monochamus, symbiotic bacteria and associated fungi, are involved in the disease infection cycle. This study isolated and identified the fungal communities of Monochamus alternatus larval galleries and pupal chambers from different instars through field investigation, morphological observation and multi-locus DNA sequence analyses in Zhejiang Province, China. A total of 255 and 454 fungal strains were isolated from M. alternatus galleries and pupal chambers infected with PWN, from the 2nd–3rd and 4th–5th instar larvae, respectively. A total of 18 species of fungi were identified, 14 species were isolated from the 2nd–3rd instar larval galleries and six species from the galleries and pupal chambers of the 4th–5th instar larvae. Amongst them were six species belonging to four genera of ophiostomatalean fungi, including two novel species, Graphilbum xianjuensis sp. nov. and Ophiostoma taizhouense sp. nov. and four known species, Ceratocystiopsis weihaiensis, Ophiostoma ips, Sporothrix zhejiangensis and S. macroconidia. The findings revealed that the fungal diversity and abundance of the 2nd–3rd instar larvae differed markedly from those of the 4th–5th instar larvae. This difference could be the result of fungal succession. This study provides a thorough understanding of the fungi associated with PWD and lays the groundwork for future research.

Keywords

Ceratocystiopsis, fungal succession, Graphilbum, Ophiostoma, pine wilt disease, Sporothrix, two new species

Introduction

The pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, is a pathogenic nematode that is responsible for the devastating epidemic of pine wilt disease (PWD) worldwide (Mota and Vieira 2008; Robertson et al. 2011), particularly throughout Japan, Korea and China (Togashi and Jikumaru 2007; Jung et al. 2010; Abelleira et al. 2011; Foit et al. 2019). Since the first report in Nanjing, China, in 1982, PWD has spread through more than 700 counties in 19 provinces (State Forestry Administration of the People’s Republic of China 2021), killing over one billion pine trees (Zhu et al. 2021). Economic and ecological losses have totalled thousands of billions of Chinese Yuan. PWN has diverse carriers and hosts. Carriers include more than eight beetle species and at least 25 hosts are susceptible under natural conditions (Zheng et al. 2021). In China, the primary PWN vector is the sawyer beetle Monochamus alternatus Hope (Coleoptera, Cerambycidae), while Pinus massoniana is one of the earliest and most susceptible hosts (Linit et al. 1983; Kobayashi et al. 1984; Ye 2019; Ji et al. 2021).

The PWN is the predominant pathogen in this complex ecosystem (Zhao et al. 2014). The symbiotic interaction between the PWN-vector and fungi is a key biological factor that promotes PWN pathogenicity and invasiveness (Zhao et al. 2014; Zhao and Sun 2017). Molecular analysis has repeatedly demonstrated that the fungus and the PWN have a close symbiotic relationship, as evidenced by the draft genome sequence of a PWN inbred line which revealed that all PWN cellulases were most likely acquired independently from fungi (Kikuchi et al. 2011). Metagenomic analysis of the PWN microbiome indicates that the PWN and its microbiome have established a potentially mutualistic symbiotic relationship with complementary pathways in detoxification metabolism (Kikuchi et al. 2011; Cheng et al. 2013).

Current research has shown that PWN has an important mycetophagous phase in its life history (Ryss et al. 2005; Espada et al. 2016). Many airborne fungi, including endophytes (Botrytis cinerea and Cladosporium herbarum) and pathogens (Sirococcus conigenus and Sphaeropsis sapinea), are positively correlated with the growth of the nematode population (Pimentel et al. 2021), with ophiostomatalean fungi (Ascomycota: Sordariomycetes: Ophiostomatales) particularly important in terms of their association with PWN-M. alternatus symbionts. The Ophiostomatales order includes one family (Ophiostomataceae) and twenty genera (Afroraffaelea, Aureovirgo, Ceratocystiopsis, Chrysosphaeria, Dryadomyces, Esteya, Fragosphaeria, Graphilbum, Grosmannia, Hawksworthiomyces, Harringtonia, Heinzbutinia, Intubia, Jamesreidia, Leptographium, Masuyamyces, Ophiostoma, Paleoambrosia, Raffaelea and Sporothrix) (de Beer et al. 2014, 2016, 2022; Hyde et al. 2020; Wijayawardene et al. 2022). Sporothrix sp.1, for example, induces the xylem tissue of the pine tree to produce diacetone alcohol, which may increase PWN propagation and beetle larvae growth (Zhao et al. 2013). PWN produces ascarosides that promote fungal (Leptographium pini-densiflora and Sporothrix sp.1) growth and sporulation (Zhao et al. 2018). In addition, some fungi are detrimental to PWN. Esteya vermicola is an example of direct antagonism (Wang et al. 2011). The lunate conidia of E. vermicola are highly infectious to PWN (Liou et al. 1999).

The invasion of beetles altered the internal habitat and mycoflora of pine trees (Zhang et al. 2021). Fungal invasion patterns in beetle-infested hosts may have been successional. Ips typographus, for example, attacked Norway spruce with the virulent Ceratocystis polonica, followed by other beetle-diffused Graphium and Ophiostoma fungi (Solheim 1992a, 1992b); Tomicus minor invaded Pinus sylvestris with Hormonema dematioides first, followed by Ophiostoma tingens and O. canum (Jankowiak 2008). Ophiostoma ips was not isolated from the 2nd–3rd instar larvae galleries of M. alternatus, but the isolation rate from the 4th–5th instar larvae galleries was 92.5% (Lun et al. 2019). The diversity and abundance of fungi associated with M. alternatus larvae of different instars in PWN-infected pines, as well as the successional pattern of fungi in PWN-M. alteratus symbionts are unknown. To date, 14 ophiostomatalean fungi have been obtained from M. alternatus galleries and pupal chambers along with PWN (Zhao et al. 2013, 2014, 2018; Wang et al. 2018; Lun et al. 2019). However, these studies are sporadic reports and no systematic studies have been conducted.

This research aimed to compare the diversity of fungi in different instars of the PWN-infected M. alternatus larval galleries and pupal chambers in south-eastern China. Field surveys were used in conjunction with integrated morphological observation and multi-locus DNA sequence analysis to describe the diversity of fungi associated with PWN and M. alternatus. This study provides a scientifically reliable and theoretical foundation for effective PWD control from the fungal perspective.

Materials and methods

Collection of samples and fungal isolations

From October to November 2020, fungi were isolated from 380 and 510 samples of different instars from M. alternatus larvae galleries and pupal chambers in Pinus massoniana, respectively, in the Huangyan District (28°56'90"N, 121°17'56"E), Xianju County (28°75'28"N, 120°59'97"E), Zhejiang Province. All the trees used in this study showed signs of death and sap stains and PWNs were simultaneously isolated from galleries and pupal chambers by Behrman funnel. The samples were collected by hand saw, individually placed in sterile envelopes, stored at 4 °C and separated within a week. The surfaces of galleries and pupal chambers were disinfected with 1.5% sodium hypochlorite for 1 min, rinsed with sterile water three times and then cut into approximately 3 × 3 mm2 tissue blocks. They were then inoculated on to a 2% (w/v) water agar medium (20 g agar powder in 1 l of deionised water) and cultured in the dark at 25 °C (Seifert et al. 1993; Wang et al. 2019; Wang et al. 2020). Subsequently, all strains were purified by hyphal tip isolation (Eckhardt 2002) and transferred on to 2% (w/v) malt extract agar (MEA: 20 g malt extract powder and 20 g agar powder in 1 l of deionised water) for growth in the dark at 25 °C. All strains were deposited at the Chinese Academy of Forestry (Table 1). Representative cultures were deposited at the China Forestry Culture Collection Center (CFCC) (Table 2).

Table 1.

Species of the fungi isolated from Pinus massoniana infected by Monochamus alternates and Bursaphelenchus xylophilus in the current study.

Taxon Species 2nd–3rd instar larvae 4th~5th instar larvae
number isolation rate number isolation rate
1 Ceratocystiopsis weihaiensis 3 1.18% N/A N/A
Chaetomium globosum 1 0.39% N/A N/A
Colletotrichum gloeosporioides 8 3.14% N/A N/A
Cytospora sp. 11 4.31% N/A N/A
Diplodia sapinea 27 10.59% 76 16.74%
Fusarium sp. 10 3.92% N/A N/A
2 Graphilbum sp. N/A N/A 12 2.64%
3 Ophiostoma ips N/A N/A 231 50.88%
4 Ophiostoma sp. 62 24.31% N/A N/A
Penicillium sp. 2 0.78% 5 1.10%
Pestalotiopsis sp. N/A N/A 2 0.44%
Phialocephala sp. 45 17.65% N/A N/A
Pseudocosmospora sp. 14 5.49% N/A N/A
Schizophyllum sp. 8 3.14% N/A N/A
5 Sporothrix macroconidia 4 1.57% N/A N/A
6 S. zhejiangensis 8 3.14% N/A N/A
Trichoderma atroviride N/A N/A 107 23.57%
Xenoacremonium sp. 37 14.51% N/A N/A
Unidentified 15 5.88% 21 4.63%
The total number of strains 255 100% 454 100%

Culture and morphological studies

The growth of representative strains was monitored daily and the culture characteristics of the colonies were recorded. Microscopic features were observed using a BX51 Olympus microscope (Tokyo, Japan) with differential interference contrast. Fifty measurements were made for each microscopic taxonomical structure. The formula (min–) (mean–SD)–(mean+SD) (–max) was used to calculate averages, ranges, standard deviation (SD), minimum (min) and maximum (max) measurements, respectively. All relevant data pertaining to type specimens were deposited in MycoBank (www. MycoBank.org).

Table 2.

Strains of ophiostomatalean fungi isolated from Pinus massoniana infested by Monochamus alternatus and Bursaphelenchus xylophilus in the current study.

Taxon Species Strain no Location GenBank no
ITS TUB2 TEF1-α
1 Ceratocystiopsis weihaiensis CFCC 55742 CXY4012 Huangyan OK104016 OM103280 N/A
CFCC 55743 CXY4013 Huangyan OK104017 OM103281 N/A
CXY4019 Huangyan N/A N/A N/A
2 Graphilbum xianjuensis sp. nov. CFCC 55738T CXY4010 Xianju OK104014 OM103285 ON033177
CFCC 55739 CXY4011 Xianju OK104015 OM103286 ON033178
CXY4018 Xianju N/A N/A N/A
3 Ophiostoma ips CFCC 55735 CXY4005 Xianju OK104009 OM056673 N/A
CFCC 55736 CXY4006 Xianju OK104010 OM056674 N/A
CFCC 55732 CXY4007 Xianju OK104011 OM056675 N/A
4 Ophiostoma taizhouense sp. nov. CFCC 55740T CXC4001 Huangyan OK104005 OM103276 N/A
CFCC 55731 CXY4002 Huangyan OK104006 OM103277 N/A
CFCC 55733 CXY4003 Huangyan OK104007 OM103278 N/A
CFCC 55734 CXY4004 Huangyan OK104008 OM103279 N/A
5 Sporothrix macroconidia CFCC 55741 CXY4009 Huangyan OK104013 OL352730 N/A
CXY4016 Huangyan N/A N/A N/A
CXY4017 Huangyan N/A N/A N/A
6 S. zhejiangensis CFCC 55737 CXY4008 Huangyan OK104012 OM103282 N/A
CXY4014 Huangyan N/A OM103283 N/A
CXY4015 Huangyan N/A OM103284 N/A

A 7 mm diameter mycelium plug was taken from a flourishing fungal colony using a sterile puncher and placed at the centre of 90 mm diameter 2% MEA plates, with one side of mycelium in contact with the media. Five replicate plates for each strain were incubated in a dark incubator at 5–35 °C with a temperature interval of 5 °C. The diameter of the colonies on each dish was measured every day by the orthogonal method until the fastest-growing mycelium reached the edge of the dish. The colony colour was then described using the Rayner (1970) colour chart.

DNA extraction, amplification and sequencing

Mycelia of representative strains were scraped with a sterile blade from the edge of the medium and transferred to 2 ml Eppendorf tubes for DNA extraction. DNA extraction and purification were carried out using the Invisorb Spin Plant Mini Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The primer pairs ITS1/ITS4 (White et al. 1990), BT2a/BT2b (Glass and Donaldson 1995) and EF1/EF2 (Jacobs et al. 2004) were used for the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA operon, including spacers 1 and 2 and the 5.8S gene, the β-tubulin (BT) gene region and transcription elongation factor-1α (TEF-1α), respectively.

Polymerase chain reaction (PCR) amplification was performed using a Veriti 96-Well Fast Thermal Cycler (Applied Biosystems Veriti96, Foster City, CA, USA). PCR was carried out in a final volume of 25 μl (2.5 mM MgCl2, 1× PCR buffer, 0.2 mM dNTP, 0.2 mM of each primer and 2.5 U Taq-polymerase enzyme). The cycling conditions were the same as those described for primer design (White et al. 1990; Glass and Donaldson 1995; Jacobs et al. 2004). The PCR products were purified using the MSB Spin PCR Apace Kit (250) (Invitek, Berlin, Germany) in accordance with the manufacturer’s instructions.

Sequencing reactions were performed using a CEQ DTCS Quick Start Kit (Beckman Coulter, Brea, CA, USA) according to 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). Complementary and overlapping DNA electropherograms were checked and assembled using BioEdit v. 7.2.0. (Hall 1999).

Sequence alignment and phylogenetic analysis

Preliminary identification of the strains was conducted using the standard basic local alignment search tool (BLAST) searches in NCBI GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and sequences with the highest similarity were downloaded from GenBank. Alignment of the genes was performed using MAFFT 7.0 (https://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013), with the E-INS-I option with a gap-opening penalty of 1.53 and an offset value of 0.00 and edited manually using Molecular Evolutionary Genetic Analyses (MEGA) 7.0 software (Kumar et al. 2016). Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI) were used to infer the phylogenetic trees from each dataset. Two concatenated matrices of ITS and BT sequences were generated for Ceratocystiopsis and the O. ips complex.

MP analyses were implemented using PAUP* version 4.0b10 (Swofford 2003). The gaps were treated as the fifth base. Bootstrap analysis (1000 bootstrap repetitions) was used to determine the confidence level for inferring the nodes in the tree topology. Tree bisection and reconnection were selected as the branch swapping option. For each dataset of the 5000 most-parsimonious trees, the best tree that was automatically output by PAUP* v. 4.0b10 was selected for use in the figure.

ML analyses were carried out using RAxML-HPC (version 8.2.3; Stamatakis 2014) and the selected GTR-GAMMA model of site substitution included the estimation of gamma-distributed rate heterogeneity and a proportion of invariant sites. ML analysis included 1000 bootstrap analyses to evaluate the overall reliability of the node support value and tree topology.

BI analyses using Markov Chain Monte Carlo (MCMC) methods were implemented in MrBayes version 3.1.2 (Ronquist et al. 2012), from a random starting tree for 5,000,000 generations, to calculate posterior probability values for the nodes. When we run to 5,000,000 generations, the split frequencies of all datasets were less than 0.01. Chain convergence for all datasets was determined using Tracer 1.7 (Rambaut et al. 2018). No lack of convergence was detected. Trees were sampled every 100 generations and the first 25% of trees sampled were discarded as burn-in, while the remaining trees were used to calculate Bayesian posterior probabilities of the clades. Phylogenetic trees were edited in Figtree version 1.4.3 (http://tree.bio.ed.ac.uk/sosftware/figtree/) and Adobe Illustrator CS6.

Results

Fungal isolation and sequence comparison

A total of 709 strains of fungi were isolated from the M. alternatus larval galleries and pupal chambers (2nd–5th instars). The strains were divided into 18 taxa, based on colony morphology and multi-locus DNA sequence alignment (ITS and BT) analysis. A total of 255 fungal strains, representing 14 taxa, were isolated from the galleries of 2nd–3rd instar larvae. Taxon 4 was the dominant taxon accounting for 62 of the 255 strains. A total of 454 fungal strains were isolated from the galleries and pupal chambers of the 4th–5th instar larvae and divided into six taxa. The dominant taxon was O. ips, accounting for 231 out of the 454 strains (Table 1). Only two fungi (Diplodia sapinea and Penicillium sp.) could be isolated from both 2nd–3rd instar larval galleries and 4th–5th instar larval galleries and pupal chambers, except for unidentified species. In this study, 320 ophiostomatalean fungi strains (320 strains out of 709 fungal strains) were isolated, including six tentative species. Four of these six species were obtained from the galleries of the 2nd–3rd instar larvae and two species were isolated from the galleries and pupal chambers of the 4th–5th instar larvae (Table 1).

Phylogenetic analyses

There were 709 strains obtained in this study, but some strains have a small number of strains. In this study, we selected 2–4 representative strains from each Taxon and nineteen representative strains of Ophiostomatales belonging to six tentative species (Taxa 1–6) were selected for phylogenetic analyses (Table 2). All the sequences used for the phylogenetic trees were submitted to GenBank. The three phylogenetic approaches yielded similar topologies, with statistical support varying slightly for each sequence dataset. Phylograms derived from ML analysis were presented for each individual dataset, along with nodal supports derived from the MP and BI analyses.

The ITS phylogenetic tree showed that six representative taxa (Table 2) belonged to six phylogenetic clades (Fig. 1). Taxa 1–2 nested within the Ceratocystiopsis and Graphilbum lineages, respectively; Taxa 3–4 nested within the Ophiostoma lineage, Taxon 3 belonging to the Ophiostoma minus complex and Taxon 4 belonging to the O. ips complex (de Beer and Wingfield 2013); Taxa 5–6 belonged to the Sporothrix and were not placed in any complex defined by de Beer et al. (2016) (Fig. 1).

Figure 1. 

ML tree of the ITS region of Ophiostoma, Sporothrix, Graphilbum, Ceratocystiopsis. Bootstrap values of ML/MP ≥ 70% are recorded at nodes as ML/MP and bold branches indicate posterior probability values ≥ 0.9. ML and MP, Bootstrap values < 70% are indicated by the symbol *. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Strains representing ex-type sequences are marked with “T.” ML, Maximum Likelihood; MP, Maximum Parsimony; BI, Bayesian Inference and the final alignment of 734 positions, including gaps.

Taxon 1 included three isolates, all of which were included in the analyses (Tables 1, 2). Based on the phylogenetic analysis of the combined dataset (ITS+BT), this taxon forms a well-supported clade with Ceratocystiopsis weihaiensis (Fig. 2). Hence, the strains in Taxon 1 should be identified as C. weihaiensis.

Figure 2. 

ML tree of Ceratocystiopsis generated from the combined (ITS+BT) sequence data. Bootstrap values of ML/MP ≥ 70% are recorded at nodes as ML/MP and bold branches indicate posterior probability values ≥ 0.9. ML and MP, Bootstrap values < 70% are indicated by the symbol *. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Strains representing ex-type sequences are marked with “T.” Abbreviations: ML, Maximum Likelihood; MP, Maximum Parsimony; BI, Bayesian Inference and the final alignment of 1040 positions, including gaps.

Taxon 2 consisted of 12 isolates, three of which were used for phylogenetic analyses (Tables 1, 2). The phylograms of ITS, BT and TEF-1α datasets revealed that Taxon 2 was an independent clade closely related to Graphilbum acuminatum, G. anningense and G. translucens (Figs 1, 3, 4). As a result, Taxon 2 should be interpreted as belonging to a distinct, undescribed Graphilbum species.

Figure 3. 

ML tree of the BT region of Graphilbum. Bootstrap values of ML/MP ≥ 70% are recorded at nodes as ML/MP and bold branches indicate posterior probability values ≥ 0.9. ML and MP, Bootstrap values < 70% are indicated by the symbol *. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Strains representing ex-type sequences are marked with “T.” Abbreviations: ML, Maximum Likelihood; MP, Maximum Parsimony; BI, Bayesian Inference and the final alignment of 548 positions, including gaps.

Figure 4. 

ML tree of the TEF region of Graphilbum. Bootstrap values of ML/MP ≥ 70% are recorded at nodes as ML/MP and bold branches indicate posterior probability values ≥ 0.9. ML and MP, Bootstrap values < 70% are indicated by the symbol *. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Strains representing ex-type sequences are marked with “T.” Abbreviations: ML, Maximum Likelihood; MP, Maximum Parsimony; BI, Bayesian Inference and the final alignment of 725 positions, including gaps.

Taxon 3 was represented by three sequences that formed a well-supported clade with O. ips, based on the ITS tree (Fig. 1). Further phylogenetic analysis, based on combined datasets (ITS+BT) yielded similar results (Fig. 5). Based on ITS and BT phylogenetic analysis, Taxon 4, represented by three sequences, has a well-supported independent clade with Ophiostoma sp.1 (CFCC52628) (Wang et al. 2019), which is closely related to O. allantosporum, O. pseudotsugae and O. wuyingensis (Figs 6, 7). Thus, Taxon 3 should be identified as a known species of O. ips, whereas Taxon 4 should be interpreted as a distinct, undescribed Ophiostoma species.

Figure 5. 

ML tree of the O. ips complex generated from the combined (ITS+BT) sequence data. Bootstrap values of ML/MP ≥ 70% are recorded at nodes as ML/MP and bold branches indicate posterior probability values ≥ 0.9. ML and MP, Bootstrap values < 70% are indicated by the symbol *. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Strains representing ex-type sequences are marked with “T.” Abbreviations: ML, Maximum Likelihood; MP, Maximum Parsimony; BI, Bayesian Inference and the final alignment of 953 positions, including gaps.

Figure 6. 

ML tree of the ITS region of O. minus complex. Bootstrap values of ML/MP ≥ 70% are recorded at nodes as ML/MP and bold branches indicate posterior probability values ≥ 0.9. ML and MP, Bootstrap values < 70% are indicated by the symbol *. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Strains representing ex-type sequences are marked with “T.” Abbreviations: ML, Maximum Likelihood; MP, Maximum Parsimony; BI, Bayesian Inference and the final alignment of 537 positions, including gaps.

Figure 7. 

ML tree of the BT region of O. minus complex. Bootstrap values of ML/MP ≥ 70% are recorded at nodes as ML/MP and bold branches indicate posterior probability values ≥ 0.9. ML and MP, Bootstrap values < 70% are indicated by the symbol *. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Strains representing ex-type sequences are marked with “T.” Abbreviations: ML, Maximum Likelihood; MP, Maximum Parsimony; BI, Bayesian Inference and the final alignment of 495 positions, including gaps.

Taxon 5 consisted of four isolates, three of which were used for the phylogenetic analyses. Based on the ITS and BT phylogenetic trees, Taxon 5 grouped with Sporothrix zhejiangensis (Figs 1, 8). Thus, it should be identified as S. zhejiangensis.

Figure 8. 

ML tree of Sporothrix generated from the BT sequence data. Bootstrap values of ML/MP ≥ 70% are recorded at nodes as ML/MP and bold branches indicate posterior probability values ≥ 0.9. ML and MP, Bootstrap values < 70% are indicated by the symbol *. The tree is drawn to scale (see bar) with branch length measured in the number of substitutions per site. Strains representing ex-type sequences are marked with “T.” Abbreviations: ML, Maximum Likelihood; MP, Maximum Parsimony; BI, Bayesian Inference and the final alignment of 313 positions, including gaps.

Taxon 6 consisted of eight isolates, three of which were selected for analysis. Taxon 6 grouped with Sporothrix macroconidia, based on the ITS and BT phylogenetic trees (Figs 1, 8). As a result, Taxon 6 was designated as S. macroconidia.

Taxonomy

According to the ITS and BT phylogenetic analyses, six different taxa (Taxon 1–6) were identified in this study. They represent four known species, Ceratocystiopsis weihaiensis, O. ips, S. macroconidia and S. zhejiangensis (Lun et al. 2019; Wang et al. 2019; Chang et al. 2021), in addition to two novel species. They are described as follows:

Graphilbum xianjuensis G. H. Zheng & Q. Lu, sp. nov.

MycoBank No: 842387
Fig. 9

Etymology

The epithet xianju (Latin) refers to the type locality.

Type

China, Zhejiang, Xianju County, from Monochamus alternatus galleries and pupal chambers of Pinus massoniana infested by Bursaphelenchus xylophilus, December 2020, collected by G. H. Zheng, culture ex-holotype CFCC55738 = CXY4010.

Figure 9. 

Morphological characteristics of Graphilbun xianjuensis sp. nov. (CFCC = 55738, Taxon 2). a, b thirty-day-old cultures on 2% MEA c–f Hyalorhincladiella-like asexual morph: conidiogenous cells and conidia. Scale bars: 10 μm (c–f).

Description

Sexual morph: not observed.

Asexual form: Hyalorhincladiella-like. Conidiogenous cells were simple or loosely branched, (9.12–) (15.44) – (48.64) (–62.49) × (1.25–) (1.53) – (2.21) (–2.45) μm. Conidia hyaline, smooth, cylindrical, aseptate, (4.76–) (6.07) – (9.87) (–13.41) × (0.99 –) (1.32) – (2.1) (–2.65) μm.

Culture characteristics

Colonies on 2% MEA reaching 44.9 mm diameter, after incubation in the dark at 25 °C for 3 d, growth rate up to 14.98 mm/d at the fastest and colony margin irregular. Mycelium superficial to flocculose or floccose, hyaline, reverse grey-white. The optimal temperature for growth at 30 °C; no growth was observed at 5 °C.

Habitat and distribution

Larval galleries and pupal chambers of Monochamus alternatus in Pinus massoniana, infested by Bursaphelenchus xylophilus, in Zhejiang Province, China.

Additional specimens examined

China, Zhejiang, from Monochamus alternatus galleries and pupal chambers of Pinus massoniana infested by Bursaphelenchus xylophilus, December 2020, collected by G. H. Zheng, CFCC55739 = CXY4011, CXY4018.

Note

Only the Hyalorhincladiella-like asexual form was observed in Graphilbum xianjuensis. This is closely related to the G. acuminatum, G. anningense and G. translucens, based on the ITS, BT and TEF1-α phylogenetic trees (Figs 1, 3, 4). Four species differed according to the size of their conidia. The conidia of G. xianjuensis (6.07–9.87 μm) are longer than those of G. anningense (4.5–6.4 μm), G. acuminatum (3.5–6 μm) and G. translucens (2.4–3.5 μm) (Wang et al. 2019; Jankowiak et al. 2020). Besides, G. xianjuensis was found to be associated with M. alternatus and PWN-infested P. massoniana, whereas G. anningense was reported in galleries of T. yunnanensis and T. minor associated with P. yunnanensis in southwest China (Wang et al. 2019), G. acuminatum has been reported in galleries of Ips acuminatus and Pityogenes bidentatus associated with P. sylvestris in Europe (Jankowiak et al. 2020) and G. translucens was first reported in Cryphalus piceae associated with P. densiflora. In conclusion, four species of Graphilbum differ not only in geographical distribution, but also in hosts and vectors. The optimum growth temperature of G. xianjuensis, G. anningense and G. translucens is 30 °C and only G. acuminatum had an optimum growth temperature of 25 °C (Wang et al. 2019; Jankowiak et al. 2020).

Ophiostoma taizhouense G. H. Zheng & Q. Lu, sp. nov.

MycoBank No: 842388
Fig. 10

Etymology

taizhou’ (Latin) refers to the type locality.

Type

China, Zhejiang Province, Taizhou City, from Monochamus alternatus galleries of Pinus massoniana infested by Bursaphelenchus xylophilus, October 2020, collected by G. H. Zheng and Q. Lu, culture ex-holotype CFCC55740 = CXY4001.

Figure 10. 

Morphological characteristics of Ophiostoma taizhouense sp. nov. (CFCC = 55740, Taxon 4). a, b twenty-day-old cultures on 2% MEA c–e Hyalorhincladiella-like asexual morph: conidiogenous cells and conidia. Scale bars: 10 μm (c–e).

Description

Sexual morph: not observed.

Asexual form: Hyalorhincladiella-like. Conidiophores abundant, conidiogenous cells single, disposed in a dense rachis (3.08–) (6.6) – (15.63) (–23.07) × (1.11–) (1.44) – (2.23) (–2.9) μm. Conidia hyaline, smooth, lunate, ellipsoid to ovoid, curvulate, aseptate, (3.24–) (4.27) – (7.42) (–10.08) × (1.17–) (1.6) – (2.39) (–2.86) μm.

Culture characteristics

Colonies on 2% MEA reaching 62.5 mm diameter, after incubation in the dark at 25 °C for 3 d, growth rate up to 22.83 mm/d at the fastest, colony margin smooth, hyphae are superficial on agar. Some white mycelium was produced early during growth and became black after 8–15 d, transitioning from brown to dark brown. The optimal temperature for growth at 30 °C; no growth was observed at 5 °C.

Habitat and distribution

Larval galleries of Monochamus alternatus in Pinus massoniana, infested by Bursaphelenchus xylophilus, in Zhejiang Province, China.

Additional specimen examined

China, Zhejiang, Taizhou City, from Monochamus alternatus galleries of Pinus massoniana infested by Bursaphelenchus xylophilus, October 2020, collected by G. H. Zheng and Q. Lu, CFCC55731 = CXY4002, CFCC55733 = CXY4003, CFCC55734 = CXY4004.

Note

Only the Hyalorhincladiella-like asexual form was observed in Ophiostoma taizhouense. According to ITS and BT phylogenetic analysis, it has a well-supported independent clade with Ophiostoma sp.1 (CFCC52628) and is closely related to O. allantosporum, O. pseudotsugae and O. wuyingensis (Figs 1, 5, 6). Only one strain of Ophiostoma sp.1 was isolated in our laboratory from P. yunnanensis infested with T. yunnanensis in Yunnan Province, so this strain was not officially named before this study. Although the geographical location and host of O. taizhouense and Ophiostoma sp.1 are different, their culture characteristics and gene sequences (ITS and BT) are identical (Figs 1, 5, 6) (Wang et al. 2019). In general, the conidia of O. taizhouense (4.27–7.42 μm) are longer than those of O. minus (2.5–6 μm) (Upadhyay 1981) and O. pseudotsugae (2.7–5 μm) (Rumbold 1936). The optimal growth temperature of O. allantosporum and O. pseudotsugae was 25 °C, that of O. wuyingensis was 25–30 °C and that of O. taizhouense was 30 °C (Gorton and Webber 2000, Chang et al. 2019). Both O. wuyingensis and O. taizhouense showed pigmentation on 2% MEA medium, whereas O. allantosporum has mid-brown hyphae, O. pseudotsugae has white-grey to snuff-brown, both showed no agar pigmentation (Rumbold 1936; Villarreal et al. 2005). Ophiostoma wuyingensis was first isolated from the gallery of Ips typographus on P. koraiensis in Heilongjiang Province (Chang et al. 2019). Ophiostoma allantosporum and O. pseudotsugae were isolated from P. resinosa in the USA and P. menziesii were infected with Dendroctonus frontalis in North America (Gorton and Webber 2000). Strains of O. taizhouense in this study were isolated from P. massoniana infected with PWN and M. alternatus.

Discussion

In the current study, 255 (containing 14 species) and 454 (containing six species) strains were obtained from M. alternatus larval galleries and pupal chambers of 2nd–3rd and 4th–5th instar, respectively, in P. massoniana infested with PWN in the Zhejiang Province, south-eastern China (Table 1). A total of 320 ophiostomatalean fungal strains out of overall 709 strains were obtained. The fungal diversity of 2nd–3rd instar larvae was higher than that of 4th–5th instar larvae (Table 1; Fig. 11). Ophiostoma taizhouense is the dominant species in the 2nd–3rd instar and O. ips is the primary species at the 4th–5th instar. This is both similar and distinct from the previous research. Some studies found Trichoderma sp. or Sporothrix sp.1 to be the most common fungus associated with PWD (Zeng et al. 2006; Zhao et al. 2013), while others found the same to be O. ips (Lun et al. 2019), as was found here. The phenomenon could be caused by fungal succession, which occurs when PWN and M. alternatus select fungal companions that are more conducive to their own growth and reproduction at different life cycle stages. Therefore, future research on fungal diversity and abundance will necessitate a more comprehensive sampling analysis.

Figure 11. 

Diagram showing the species of fungi were isolated from the galleries and pupae chambers of different instar larvae of Monochamus alternates.

Only two common fungal species were obtained from both 2nd–3rd instar larval galleries, 4th–5th instar larval galleries and pupal chambers. The abundance of D. sapinea (103 out of 709) was higher than that of Penicillium sp. (7 out of 709). Diplodia sapinea is commonly isolated from P. nigra tip blight, P. halepensis and P. pinaster branch cankers worldwide (Luchi et al. 2014). It is an important pathogen of the Pinus spp. In addition, research has shown that D. sapinea can promote PWN reproduction and settlement (Kobayashi et al. 1974; Sriwati et al. 2007). Penicillium sp. is a common fungus in nature that also serves as an important biocontrol fungus (Sartaj et al. 2011; Win et al. 2021). However, there are no reports of Penicillium sp. affecting PWN, either negatively or positively.

Ophiostoma ips was first reported in pine trees associated with bark beetles in the south-eastern United States (Rumbold 1936) and it has since been confirmed, using microsatellite markers, to be distributed worldwide (Zhou et al. 2007). Unfortunately, the study did not include Chinese strains. However, O. ips is regarded as one of the most stable natural associates of PWN in the wild of China (Zhao et al. 2013; Lun et al. 2019). According to Lun et al. (2019), O. ips was the dominant strain associated with PWN and was frequently isolated at the late stage of PWD. In a study by Zhao et al. (2013), O. ips was one of the three dominant ophiostomatoid fungi associated with PWN, with an isolation rate of 36%. Although O. ips was not found in the galleries of M. alternatus 2nd–3rd instar larvae in this study, it was the primary species at the 4th–5th instar, with an isolation rate of 50.88% and fungal abundance was much higher than that of other fungi during this period. Experiments with nematode propagation revealed that O. ips could breed nematodes, but not as effectively as Botrytis cinerea (Pimentel et al. 2021). In addition, biochemical analysis results revealed that O. ips could produce a wide range of volatile chemical substances (Cale et al. 2019). The 4th–5th instar larvae of M. alternatus are closely related to pinewood nematode dispersal stage JIV (the fourth-stage dispersal juvenile). However, the mechanism underlying the interaction between O. ips and dispersal nematode juveniles is still lacking.

Ophiostoma taizhouense was the second most frequently isolated species of ophiostomatalean fungi in our study (62 out of the 255 strains); nevertheless, it was only associated with 2nd–3rd instar larvae. The association between O. taizhouense and PWD needs further experimental verification as a new species. Although the isolation rate of Phialocephala sp. and Xenoacremonium sp. is relatively high in 2nd–3rd instar larvae, these two fungi are both endophytes and there are no reports relating them to PWD.

In addition, three known ophiostomatalean fungi (C. weihaiensis, S. macroconidia and S. zhejiangensis) and one new species (G. xianjuensis) were revealed with low isolation rates during the survey. Simultaneously, some common endophytic and saprophytic fungi were isolated from the galleries and pupal chambers of M. alternatus larvae. The relationship between these fungi and PWN has not yet been reported.

In this study, a relatively large diversity of fungal species was obtained and identified as associated with PWN and M. alternatus in south-eastern China. The results showed that the fungal diversity and abundance of the 2nd–3rd instar larvae differed from those of the 4th–5th instar larvae. Fungi play an important role during the successful survival, reproduction and spread of PWN (Zhao et al. 2014; Zhao and Sun 2017). Hence, it is vital to explore the relationship between fungi and PWDs. This study provides a research basis for the fungi-PWN-M. alternatus symbiosis.

Acknowledgements

This work was supported by Zhejiang Science and Technology Program (2020C02007) and the National Natural Science Foundation of China (Project Nos. 32071769, 31770682). We thank Prof. Jiafu Hu of the Zhejiang A&F University for sampling in the fieldwork. We thank Editage (www.editage.cn) for its linguistic assistance during the preparation of this manuscript.

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