Ophiostomatoid fungi including a new species associated with Asian larch bark beetle Ips subelongatus, in Heilongjiang (Northeast China).

Ips subelongatus (Coleoptera, Scolytinae) is an important bark beetle species that infests Larix spp. in Asia. Individuals of this beetle are vectors of ophiostomatoid fungi, on their exoskeletons, that are transmitted to infested trees. In this study, the symbiotic assemblage of ophiostomatoid fungi associated with I. subelongatus in Northeast China was studied. Fungal isolates were identified based on their morphological characters and sequences of ITS, beta-tubulin, elongation factor 1-alpha and calmodulin gene regions. In total, 48 isolates were collected and identified, residing in six taxa. These included a novel species, described here as Ophiostoma gmelinii sp. nov.

INTRODUCTION

The Asian larch bark beetle, Ips subelongatus (Coleoptera, Scolytinae), is the most important insect pest infesting various Larix (larch) species in China (Chen ). This beetle is also found in other Eurasian countries including Japan, Mongolia, North Korea, Russia, and South Korea (Cognato 2015). Ips subelongatus typically infests weakened, wind-thrown, and fire-damaged trees, but it is also able to kill healthy trees when outbreaks result in substantial population growth (Zhang ).

Ips subelongatus as well as other bark beetles and their associated phoretic mites are well-known vectors of ophiostomatalean and microascalean fungi (Six 2003, Hofstetter & Moser 2014). The best known genera are Ophiostoma and Ceratocystis respectively (De Beer & Wingfield 2013). Most of these fungi only cause discoloration of the sapwood of infested trees (Six 2003). However, some species such as the Dutch elm disease fungi (Ophiostoma ulmi and O. novo-ulmi) and species accommodated in Ceratocystis sensu lato are important tree pathogens (Brasier 2001, Roux & Wingfield 2009).

There have been several investigations considering the fungal associates of bark beetle I. subelongatus in China (Liu , Wang ) and these have revealed 21 species. Specifically, the recent study by Wang was the most extensive and included the description of eight new taxa. The present study arose from a collection of isolates made from the beetles and galleries of I. subelongatus infesting Larix gmelinii in Heilongjiang, China. Identification of these isolates based on DNA sequence comparisons showed that most were of known taxa. Three of isolates appeared to represent a novel taxon and the aim of this study was to test this hypothesis.

MATERIALS AND METHODS

Fungal collection

Samples for this study were collected in July 2011, during the flight season of I. subelongatus, from a natural forest in Heilongjiang (northeast China) where L. gmelinii was also present. A total of 44 beetles and 46 galleries were collected and used for fungal isolation. The isolation medium was 2 % malt extract agar [MEA: 20 g Difco agar, 20 g Difco BactoTM malt extract (Becton, Dickinson & Company), 1 L deionized water] supplemented with 0.05 % streptomycin. Fungal spore drops were picked up directly from the galleries and inoculated onto MEA plates. Living beetles were placed onto the surface of MEA plates and let crawling for several minutes and removed thereafter. Dead beetles were crushed and spread onto MEA plates. The MEA plates were incubated at room temperature (~ 25 °C) until fungal growth was observed. Tips of hyphae were picked and transferred onto new MEA plates. All the isolates collected in this study have been maintained in Culture Collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa. Ex-type cultures of a new species were deposited in the culture collection (CBS) of the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands. Herbarium specimens established as dried cultures were deposited to South African National Collection of Fungi (PREM), Roodeplaat, Pretoria, South Africa.

DNA extraction and PCR

DNA was extracted from fresh mycelium grown on 2 % MEA following the instructions provided by PrepMan™ Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA). The primers ITS1F and ITS4 (White ; Gardes & Bruns 1993) were used to amplify the internal transcribed spacer region (ITS), the primers BT2a and BT2b (Glass & Donaldson 1995) were used to amplify the beta-tubulin (BT) gene, the primers EF2F (Marincowitz ) and EF2R (Jacobs ) were used to amplify the translation elongation factor 1-alpha gene (EF). The primers CL2F and CL2R (Duong ) were used to amplify the calmodulin gene (CAL). PCR and sequencing were conducted using the methods described by Duong . All sequences were assembled and checked by forward and reverse sequence using Geneious v. 7.1.4 (Biomatters, Auckland, New Zealand).

DNA sequence analyses

The ITS sequences of all isolates were subjected to BLASTn searches against the NCBI GeneBank nucleotide database (http://blast.ncbi.nlm.nih.gov) for preliminary identification. Based on the BLAST results, datasets of BT, EF and CAL sequences for different genera and species complexes were compiled and analyzed separately. Sequence alignments were done using an online version of MAFFT v. 7.0 (Katoh & Standley 2013), and were further curated in MEGA v. 6 (Tamura ). Maximum likelihood (ML) and Bayesian inference (BI) analyses were conducted using RAxML-HPC2 v. 8.2.4 (Stamatakis 2014) and MrBayes v. 3.2.6 (Ronquist ) respectively; both are available from the CIPRES Science Gateway (Miller ). For the BI analysis, trees were sampled every 100th generation for 5 M generations and 25 % of trees were discarded as burn-in phase. All alignments have been deposited in TreeBASE (https://treebase.org/) under the study number S28534.

Morphology and culture characteristics

Depending on the number of isolates available, two to three isolates per species were chosen for morphological examination. Fresh mycelium was inoculated onto MEA plates to which sterilized pine twigs (with bark) had been added to induce the production of sexual or asexual structures. Fungal structures were examined using a Nikon Eclipse Ni light microscope (Nikon, Japan) and a Nikon SMZ 18 dissection microscope attached with a Nikon DS-Ri2 camera. Up to 50 measurements were made for characteristic structures whenever available using the NIS Elements BR software. Measurements are presented as “maximum – minimum” except for spores where “average ± standard deviation” was additionally presented.

Growth rates were determined at seven temperatures ranging from 5 to 35 °C at 5 °C intervals. Mycelial plugs (5 mm diam) were transferred from the margins of actively growing cultures to the centers of 90 mm Petri dishes containing 2 % MEA. Two diameter measurements perpendicular to each other were made of the colonies and the daily growth rate was calculated.

RESULTS

A total of 48 ophiostomatoid isolates were obtained. Twenty-one were obtained from the galleries of the beetle and the remaining isolates were from the beetles themselves. BLAST searches of the ITS sequences showed that the isolates collected in this study resided in three genera, Endoconidiophora, Ophiostoma, and Sporothrix (Table 1). Analyses of ITS (Fig. 1), BT, EF and CAL sequences suggested that these isolates resided in six taxa, five of which were known species i.e., O. hongxingense (11 isolates) (Fig. S1), O. peniculi (two isolates) (Fig. S1), O. pseudobicolor (one isolate) (Fig. S2), Sporothrix cf. abietina (two isolates) (Fig. S3), and Endoconidiophora fujiensis (five isolates) (Fig. S4), and three isolates belonging to a novel species (Fig. 2) which is described below.

Table 1

Isolates of ophiostomatoid fungi obtained from I. subelongatus on Larix gmelinii in Heilongjiang and used in phylogenetic analyses.

Species	Isolate number1,2		B/G[3]	GenBank number4
	CMW	CBS		ITS	BT	EF	CAL
Endoconidiophora	40450		B	MW581513	MW579724	MW579746	n/a
fujiensis	40453		B	MW581514	MW579725	MW579747	n/a
	40460		B	MW581515	MW579726	MW579748	n/a
	40465		G	MW581516	MW579727	MW579749	n/a
	40479		B	MW581517	MW579728	MW579750	n/a
Ophiostoma gmelinii sp. nov.	40447		B	MW581494	MW579705	MW579729	n/a
	40463 H	141909	B	MW581495	MW579706	MW579730	n/a
	40464	141911	B	MW581496	MW579707	MW579731	n/a
O. hongxingense	40448		G	MW581498	MW579709	MW579733	n/a
	40455		G	MW581499	MW579710	MW579734	n/a
	40466		B	MW581500	MW579711	MW579735	n/a
	40467		B	MW581501	MW579712	MW579736	n/a
	40469		B	MW581502	MW579713	MW579737	n/a
	40474		B	MW581503	MW579714	MW579738	n/a
	40477		B	MW581504	MW579715	MW579739	n/a
	40483		B	MW581505	MW579716	MW579740	n/a
	40485		B	MW581506	MW579717	n/a	n/a
	40486		B	MW581507	MW579718	MW579741	n/a
	40487		B	MW581508	MW579719	MW579742	n/a
O. peniculi	40444		G	MW581509	MW579720	MW579743	n/a
	40472		B	MW581510	MW579721	MW579744	n/a
O. pseudobicolor	40478		B	MW581497	MW579708	MW579732	n/a
Sporothrix cf. abietina	40475		B	MW581511	MW579722	n/a	MW579751
	40454		G	MW581512	MW579723	MW579745	MW579752

1 CMW: Culture Collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; CBS: Culture Collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands.

2 H = ex-holotype isolate.

3 B = beetle; G = gallery.

4 ITS = internal transcribed spacer regions 1 and 2 of the nuclear ribosomal DNA operon, including the 5.8S region; BT = beta-tubulin; EF = translation elongation factor 1-alpha; CAL = calmodulin.

Fig. 1.

Phylogram obtained from ML analyses of the ITS region of Ophiostoma and Sporothrix. Sequences obtained in this study are printed in bold type. Maximum-likelihood bootstrap support values (1 000 replicates) above 70 % are indicated at the nodes. Bayesian inference posterior probabilities (above 0.9) are indicated by bold lines at the relevant branches. T = ex-type cultures.

Fig. 2.

Phylogrammes obtained from ML analyses of the ITS region, and the partial BT and EF gene regions of the O. piceae species complex. Isolates of the novel species are printed in bold type. Maximum-likelihood bootstrap support values (1 000 replicates) above 70 % are indicated at the nodes. Bayesian inference posterior probabilities (above 0.9) are indicated by bold lines at the relevant branches. T = ex-type cultures.

Taxonomy

R.L. Chang, Z.W. de Beer & M.J. Wingf., MycoBank MB 840569. Fig. 3.

Fig. 3.

Microscopic features of Ophiostoma gmelinii sp. nov. (ex-holotype, CMW 40463 = CBS 141909). A. Colony grown at 25 °C for 7 and 34 d in the dark. B. String of pigmented hyphae found in aged culture. C. Conidiophore. D. Conidiogenous cell. E. Conidia. Scale bars: B = 25 μm; C, D = 10 μm; E = 5 μm.

Etymology: The epithet gmelinii refers to Larix gmelinii, a tree that Ips subelongatus infests in China.

Diagnosis: Ophiostoma gmelinii differed from its closest phylogenetic relatives, O. rufum and O. xinganense, in the size of conidia, growth rate, and lack of pesotum-like asexual morph.

Sexual morph not observed. Asexual morph sporothrix-like. Mycelium mostly submerged, densely compacted, hyaline, sometimes developing brown strings of hyphae with age. Conidiophores simple, upright, flexuous, rarely branched, septate, occasionally reduced to conidiogenous cells, 4–290 × 1–2 μm (4–30 μm long when reduced to conidiogenous cells). Conidiogenous cells blastic, denticulate, often showing sympodial growth, terminal, sometimes directly borne on vegetative hyphae. Conidia hyaline, mostly oblong, aseptate, the apex often slightly inflated, tapering abruptly to the pointed base, 4.5–8 × 2–3 μm (6.4 ± 0.96 × 2 ± 0.22 μm).

Culture characteristics: Colony shiny, smooth, colorless to whitish, developing pigmented zone or patches of hyphal strings when aged or damaged. Optimum growth at 25 °C (2.8 mm / d), followed by 20 °C (2.3 mm / d), 15 °C (1.7 mm / d), 10 °C (1 mm / d), 30 °C (0.9 mm / d), no growth at 5 and 35 °C.

Typus: China, Heilongjiang province, from gallery of Ips subelongatus on Larix gmelinii, 2011, coll. X.D. Zhou (holotype PREM 61562, culture ex-type CMW 40463 = CBS 141909).

Additional material examined: China, Heilongjiang province, from gallery of Ips subelongatus on Larix gmelinii, 2011, coll. X.D. Zhou, PREM 61564, culture CMW 40464 = CBS 141911.

Notes: The newly described O. gmelinii resides in the O. piceae species complex. Based on the analyses of BT sequences, all the isolates of this species formed a single clade together with O. rufum, which was recently described from the Czech Republic (Jankowiak ), and O. xinganense described from China (Wang ). This clade had a strong bootstrap support in the ML analysis but not in the case of the BI analysis (Fig. 2). However, based on the analyses of ITS sequences, O. gmelinii formed a well-resolved clade and this formed a sister clade with O. genhense and O. multisynnematum. Ophiostoma rufum grouped with O. piceae, O. micans, and other species. In the EF tree, all isolates of O. gmelinii formed a sister clade with O. flexuosum, O. multisynnematum and O. genhense. Collectively, these analyses resolved O. gmelinii as a distinct species in the O. piceae species complex as defined by Yin .

Morphological characters of O. gmelinii differed from its closest relatives O. rufum and O. xinganense. The conidia of O. gmelinii were larger than those of O. rufum (Jankowiak ) but similar to those of O. xinganense (Wang ). The growth rate of O. gmelinii was faster than in O. rufum, but much slower than in O. xinganense. In addition, O. gmelinii and O. xinganense are able to grow, albeit slowly at 30 °C, but O. rufum is not able to grow at this temperature (Jankowiak ). Only a sporothrix-like asexual state was found in O. gmelinii, which is different to most of the other species in the O. piceae complex. These fungi typically produce pesotum-like synnemata as well as a sporothrix-like asexual morph (Yin , Wang ). All the species in this complex, including the newly described O. gmelinii, are from conifer hosts (Yin , Jankowiak ).

DISCUSSION

In this study, 48 isolates of ophiostomatoid fungi were obtained from beetles of I. subelongatus or its galleries. Six taxa were identified, residing in either the Microascales (one species) or the Ophiostomatales, including one new species. All the known species had previously been reported from China. Sporothrix cf. abietina was isolated from I. subelongatus for the first time.

Previous studies have revealed 21 species associated with I. subelongatus in China with 14 were new to science (Paciura , Wang , Liu , Wang ). The present study adds two more species to this list. These results suggest that the fungal symbionts of I. subelongatus have now been relatively well sampled. As is true for many other ophiostomatoid fungi associated with conifer-infesting bark beetles, relatively little is known regarding their biology. In this regard, most are thought not to play a role in killing trees (Six & Wingfield, 2011). Although in the case of Endoconidiophora fujiensis (Yamaoka ), which is a sister species to the E. laricicola associate of Ips cembrae (Marin ) and the E. polonica associate of I. typographus (Krokene & Solheim, 1998), this question remains contentious (Lieutier , Biedermann )

Ips subelongatus has a wide distribution in China (Yang ), including Heilongjiang, Jilin, Liaoning, Xinjiang, Gansu, Shandong, Shanxi, Hebei, Beijing, Zhejiang and Yunnan provinces and in other parts of Asia (Cognato 2015). All the studies on the fungal associates of this beetle in China have been in the north eastern parts of this country. It would thus be valuable in future studies to compare the fungal associates of this insect in different parts of China, as well as in other areas of East Asia.