FungiOphiostomatalesOphiostomataceaeWangHuiMinLunYingYingLuQuanLiuHuiXiangDecockConyZhangXingYaoOphiostomatoid fungi associated with pines infected by Bursaphelenchusxylophilus and Monochamusalternatus in China, including three new speciesMycoKeys49201820183912710.3897/mycokeys.39.27014 Sporothrix zhejiangensis MB825556 Wang & Lusp. nov.Figure 2Etymology.

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

Type.

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

Description.

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

Asexual morph: pesotum-like and sporothrix-like.

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

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

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

Culture characteristics.

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

Habitat and distribution.

Galleries of Monochamusalternatus in Pinusmassoniana infested by PWN; known hitherto from Zhejiang Province, China.

Additional specimens examined.

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

Note.

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

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

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

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

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