Introduction

The genus Trichoderma Pers. (Ascomycota, Sordariomycetes, Hypocreales) is cosmopolitan, often grows on decaying wood or exists as soil inhabitants, and occasionally lives as one of the most abundant endophytes in stems of woody plants (Evans et al. 2003; Mahesh et al. 2005; Crozier et al. 2006; Gond et al. 2007; Verma et al. 2007; Gazis and Chaverri 2010), as well as saprophytes and parasites of other fungi (Samuels et al. 2002; Samuels et al. 2006; Druzhinina et al. 2011). Trichoderma is noteworthy for its important roles in promoting plant growth (Bae and Knudsen 2005; Vargas-Garcia et al. 2005), producing useful secondary metabolites (Samuels 1996; dos Reis Almeida et al. 2007; Degenkolb et al. 2008; Cheng et al. 2012; Lopes et al. 2012; Mukherjee et al. 2013) and remediating soil contaminated by heavy metals (Chaverri et al. 2003a; Chaverri and Samuels 2003; Harman et al. 2004). In particular, as biocontrol agents of plant pathogens, some have been commercialised (Verma et al. 2007). However, several Trichoderma species are threatening to the food industry, mushroom production and human health (Kredics et al. 2003; Wiater et al. 2005; Oda et al. 2009; Schuster and Schmoll 2010; Kim et al. 2012, 2013).

Trichoderma is characterised by perithecia immersed in fleshy stromata of a different colour, shape and size, producing cylindrical asci containing usually 16 disarticulating hyaline or green part-ascospores which are uniseriately arranged, giving rise to conidiophores with several types of branch patterns on natural substrates or in culture, forming either hyaline or green conidia, and with or without chlamydospores. Monographic treatments of Trichoderma species were carried out by Jaklitsch (2009, 2011) and Jaklitsch and Voglmayr (2015). Ascospore colour has provided useful phylogenetic information based on morphology and multi-gene sequence analyses (Chaverri and Samuels 2003; Jaklitsch 2009, 2011). Five clades were established to accommodate species with green ascospores, which are named Ceramicum, Chlorosporum, Harzianum, Spinulosum and Strictipile. Nine clades contain mainly species with hyaline ascospores, i.e. Brevicompactum, Deliquescens, Hypocreanum, Longibrachiatum, Polysporum, Psychrophilum, Semiorbis, Stromaticum and Viride. Recognitions of clades in Trichoderma are generally based on phylogenetic analyses; however, the phenotypic features, such as stromatal shape and colour, ascospore colour, ostiole type, colony and conidiophore branch patterns, may be proved to be important. The size of clades varies significantly. Some contain only two or three species, while the largest one includes more than 60 species. For clades with a limited number of taxa, it is relatively easy to recognise morphological features shared by members within individual clades. But for complex clades, such as the Viride clade, morphology among species in a clade varies too much to establish a reasonable concept based solely on phenotypes. Integrated studies of morphology and sequence analysis are, thus, required.

Researches of Trichoderma in China have concentrated on its potential application and taxonomy (Chen 2014; Zhu and Zhuang 2014a). In taxonomic studies, the first Chinese record of the genus dates back to 1895 (Patouillard 1895), and 11 species were reported mainly from Southern China (Teng 1934, 1935, 1936, 1963). Later, two more species were described based on the Chinese materials, and five new records for China were added (Liu and Doi 1995; Liu et al. 2002, 2003; Zhang et al. 2007; Jaklitsch et al. 2013). Recently, Zhu and Zhuang (2014a) outlined the high species diversity of Trichoderma in China and summarised the 91 species currently known from the country. In the past two years, ten more new species were published, four new combinations were made and 23 species were found new to China upon examinations of collections from 18 provinces (Zhu and Zhuang 2014b, c, 2015a, b; Qin and Zhuang 2016a, b). This study updates our knowledge based on the more recent collections.

When the collections from central China were examined, four new species having hyaline ascospores, namely T. asterineum, T. henanense, T. odoratum and T. pseudobritdaniae, are discovered. Their morphology of sexual and asexual states were described, and their phylogenetic positions were ascertained based on sequence analyses of the combined RNA polymerase II subunit b (rpb2) and translation elongation factor 1 alpha (tef1) genes.

Materials and methods

Specimens and strains

Specimens examined were collected from Henan and Hubei provinces, China. They were deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS) and Cryptogamic Herbarium, Kunming Institute of Botany, Academia Sinica (HKAS). Strains were obtained from single ascospore isolation.

Morphological study

Dried stromata were rehydrated and longitudinal sections were made with a freezing microtome (YD-1508-III, Yidi Medical Appliance Factory, Zhejiang, China) at a thickness of 8–10 μm. Colonies on cornmeal dextrose agar (CMD), potato dextrose agar (PDA) and synthetic low nutrient agar (SNA) were incubated in 90-mm-diameter Petri dishes with alternating light/darkness (12/12 h) at 25 °C and the radius was measured daily until mycelium was covering the dishes. The morphology of sexual and asexual states was described following Jaklitsch (2009) and Jaklitsch and Voglmayr (2015). Photographs were taken with a Zeiss AxioCam MRc 5 digital camera (Jena, Germany) connected to a Zeiss Imager A2 microscope (Göttingen, Germany) for anatomical structures and a Leica DFC450 digital camera (Wetzlar, Germany) connected to a Leica M125 stereomicroscope (Milton Keynes, UK) for gross morphology. The nomenclature of fungi follows the Melbourne Code (McNeill et al. 2012) and the generic name used is according to Rossman et al. (2013).

DNA extraction, amplification and sequencing

Genomic DNA was extracted from mycelia following the methods of Wang and Zhuang (2004). Fragments of rpb2 were amplified using the primer pairs fRPB2-5f and fRPB2-7cr (Liu et al. 1999) or 5′-TTGKAAAGAARCGTCTGGAT-3′ and 5′-YRRCATACCTGGTTGTG-3′ (newly designed primers); fragments of tef1 were amplified using the primer pair EF1-728F (Carbone and Kohn 1999) and TEF1LLErev (Jaklitsch et al. 2005). Polymerase chain reaction (PCR) conditions were following Zhu and Zhuang (2015b). The PCR products were purified with the PCR Product Purification Kit (Biocolor BioScience and Technology Co., Shanghai, China) and cycle-sequenced using the primer pairs reported by Jaklitsch (2009) on an ABI 3730xl DNA Sequencer (Applied Biosciences, Foster City, CA, USA) at Beijing Tianyihuiyuan Bioscience and Technology, China. The sequences used in this study are provided in Table 1.

Table 1 Materials of Trichoderma species used in the phylogenetic analyses
Full size table

Phylogenetic analyses

The partition homogeneity test (PHT) was performed with PAUP 4.0b10 (Swofford 2002) to evaluate statistical congruence between the sequence data of rpb2 and tef1. To locate the phylogenetic positions of the four new Trichoderma species, 42 combined sequences of rpb2 and tef1 were analysed with Nectria berolinensis and N. eustromatica as outgroup taxa. Among them, ten species having green ascospores and 19 species producing hyaline ascospores were in the named clades. Eleven species were scattered as terminal branches which do not assign to any named clades. The sequences were checked and visually adjusted where necessary using BioEdit 7.0.5 (Hall 1999), alignment was carried out by ClustalX 1.83 and a NEXUS file was subsequently generated (Thompson et al. 1997).

Maximum parsimony (MP) analysis was performed via PAUP 4.0b10 using a heuristic search with tree-bisection-reconnection branch swapping, with settings as follows: all characters were treated as unordered and unweighted, gaps treated as missing data, Maxtrees = 1000 and auto-increased. Clade stability was assessed by maximum parsimony bootstrap proportion (MPBP) with 1000 replicates, each with ten replicates of random stepwise addition of taxa.

Bayesian inference (BI) analysis was conducted by MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using a Markov chain Monte Carlo (MCMC) algorithm. MrModeltest 2.3 (Nylander 2004) was used to determine the appropriate nucleotide substitution models and select the best-fit model by Akaike information criterion for the investigated dataset. Four MCMC chains (one cold and three heated) were run for 1,000,000 generations, with the trees sampled every 100 generations. The first 25 % of trees were excluded as the burn-in phase of the analyses, and the remaining trees were used for estimating Bayesian inference posterior probability (BIPP) values. Trees were viewed in TreeView 1.6.6 (Page 1996).

Taxonomy

Trichoderma asterineum W.T. Qin & W.Y. Zhuang, sp. nov. (Fig. 1) MycoBank: MB 813807

Fig. 1
figure 1

Trichoderma asterineum (holotype HMAS 271353). a–m Sexual state. a–d Dry stromata on natural substrates. e Mature stroma after rehydration. f Mature stroma in 3 % KOH after rehydration. g Surface of dry stroma showing ostioles. h Longitudinal section of a stroma. i Perithecium in section. j Cortical and subcortical tissues in section. k Subperithecial tissue in section. l Stroma base in section. m Asci with ascospores. n–x Asexual state. n–p Cultures after 25 days at 25 °C (n CMD, o PDA, p SNA). q Aggregated young stromata (CMD, 26 days). r–u Conidiophores and phialides (CMD, 25 days). v–w Chlamydospores (CMD, 25 days). x Conidia (CMD, 25 days). Scale bars: a, c–f, h = 200 μm. b = 400 μm. g = 100 μm. i = 30 μm. j–l = 20 μm. m, r, u, v–x = 5 μm. n–p = 10 mm. q = 1 mm. s, t = 10 μm

Full size image

Etymology: The specific epithet refers to the stellate cracks surrounding ostiolar dots of the fungus.

Typification: China. Henan, Jiaozuo, Yuntaishan, 35°24′53″N, 113°22′22″E, alt. 800 m, on twig lying on the ground, 24 Sep 2013, H.D. Zheng, Z.Q. Zeng & Z.X. Zhu 8892 (holotype HMAS 271353, isotype HKAS 95057, ex-type culture HMAS 244996).

Stromata solitary, scattered or aggregated in a group of 2–3, pale yellow, pulvinate or lenticular, often narrowly attached, with rounded or widely free margin, ca. 1.5–3.5 mm diam., 0.7–1.0 mm thick (n = 8). Surface convex, comprising a thick chalky or calcareous, white, sometimes pale yellow covering layer, cracked into polygonal plates. Ostiolar dots ochre to brown, densely disposed, numerous, rounded, slightly projecting, convex, minute but distinct, surrounded by stellate cracks. Rehydrated stromata turning orange in 3 % KOH.

In section, cortical tissue of textura angularis mixed with textura intricata, cells hyaline, thin-walled, (3–)3.5–7.5(–8) × (2.5–)3.5–5(–5.5) μm (n = 30), hyphae hyaline, thin-walled, (2–)3–5(–6) μm (n = 30) wide; subcortical tissue of textura angularis mixed with textura intricata, cells hyaline, thin-walled, (4–)4.5–7.5(–8) × (2.5–)3–5 μm (n = 30), hyphae hyaline, thin-walled, 3.5–5(–8) μm (n = 30) wide; subperithecial tissue of textura angularis mixed with textura epidermoidea, cells hyaline, thin-walled, (6.5–)8–18(–32) × (5–)7.5–11(–21) μm (n = 30); tissue at the base of textura angularis, cells hyaline to light brown, thin-walled, (4–)7–17(–18) × (4–)6–10(–13) μm (n = 30). Perithecia subglobose or flask-shaped, crowded, numerous, (171–)197–224(–237) × (118–)124–171(–176) μm (n = 30); peridium light yellow in lactic acid, turning pale tan in 3 % KOH, 10–13(–16) μm thick at flanks, (8–)11–16(–18) μm thick at the base (n = 30). Ostioles projecting up to 28 μm, (47–)53–68(–74) μm high, 32–40(–45) μm wide at the apex (n = 30). Asci cylindrical, (70–)72–77(–79) × 4–5(–5.5) μm, with a stipe 5–10(–11) μm long (n = 40). Ascospores hyaline, verruculose or spinulose, dimorphic, distal cells subglobose to slightly ovoid, 3–4(–4.5) × 3–3.5(–4) μm, l/w 1.0–1.3(–1.4); proximal cells globose, subglobose to nearly wedge-shaped, (3–)4–5 × 2.5–3 μm, l/w 1.3–1.7(–1.9) (n = 50).

Colony on CMD 4–6 mm in radius after 72 h at 25 °C, 28–33 mm after 25 days. Colony hyaline, dense, with lobed or irregular outline; aerial hyphae inconspicuous. Conidiation noted after 10–15 days, first effuse in minute shrubs, later in numerous minute granules and pustules with granulose or plumose surface, turning pale green from the centre, additional new pustules produced consecutively in distal areas. Conidiophores mostly asymmetrically arranged, densely disposed. Phialides often asymmetric, straight, narrowly lageniform or subulate, less commonly curved or sinuous, divergent in whorls of 2–3(–5), also solitary or paired along conidiophores, (7–)9–16(–22) × 2–3(–4) μm, l/w (2.1–)2.6–6.4(–9.1), (1–)1.5–2.5(–3) μm wide at the base (n = 50). Conidia green, mostly ellipsoidal, less commonly subglobose or oblong, smooth, (2.5–)3–4.5(–5.5) × 2–3, l/w 1.0–1.6(–2.1) (n = 50). Chlamydospores noted after 2 weeks, terminal or intercalary, smooth, globose, ellipsoidal or fusoid, (5–)6–10(–16) × (5–)6–8(–9), l/w 1.0–1.3(–2.2) (n = 50). No distinct odour, no diffusing pigment observed.

Colony on PDA 22–26 mm in radius after 25 days at 25 °C. Colonies green, dense, with little mycelium on agar surface. Conidiation noted after 16 days. No distinct odour, no diffusing pigment observed.

Colony on SNA 16–20 mm in radius after 25 days at 25 °C. Colony subhyaline, circular, conidiation noted after 20 days, mostly around the plug, first yellowish green, finally green. No distinct odour, no diffusing pigment observed.

Notes: Trichoderma asterineum is most similar to T. leguminosarum. Both species share scattered pale yellow stromata with ochre to brown ostiolar dots surrounded by stellate cracks that are unknown in any other species of the genus, slow growth in cultures at 25 °C, and green dense colony with a lobed or irregular outline on CMD (Jaklitsch and Voglmayr 2015). They can be separated from each other by the colour of rehydrated stromata in 3 % KOH, colonies on PDA, growth rates and some other characteristics detailed in Table 2. In our phylogenetic analyses, T. asterineum formed a well-supported terminal branch associated with T. leguminosarum (Fig. 5). As to sequence divergence, their rpb2 sequences are similar, while 21-bp differences among 856 bp for acl1 (unpublished data) and 21-bp differences among 1193 bp for tef1 were detected, which indicates that the two fungi are not conspecific.

Table 2 Major morphological comparison between Trichoderma asterineum and T. leguminosarum
Full size table

Trichoderma henanense W.T. Qin & W.Y. Zhuang, sp. nov. (Fig. 2) MycoBank: MB 813808

Fig. 2
figure 2

Trichoderma henanense (holotype HMAS 252891). a–p Sexual state. a–g Dry stromata on natural substrates (a–f mature, g immature). h Mature stroma in 3 % KOH after rehydration. i Longitudinal section of a stroma. j Perithecium in section. k Cortical and subcortical tissues in section. l Subperithecial tissue in section. m Stroma base in section. n–p Asci with ascospores. q–y Asexual state. q–s Cultures after 13 days at 25 °C (q CMD, r PDA, s SNA). t–v Conidiophores and phialides (SNA, 13 days). w Chlamydospores (SNA, 13 days). x Autolytic excretion (SNA, 13 days). y Conidia (SNA, 13 days). Scale bars: a = 1 mm. b = 0.8 mm. c–h = 400 μm. i = 200 μm. j, x = 50 μm. k–m = 20 μm. n–p, y = 5 μm. q–s = 20 mm. t–w = 10 μm

Full size image

Etymology: The specific epithet refers to the locality of the fungus.

Typification: China. Henan, Jiaozuo, Yuntaishan, 35°24′53″N, 113°22′22″E, alt. 800 m, on twig lying on the ground, 24 Sep 2013, H.D. Zheng, Z.Q. Zeng & Z.X. Zhu 8889 (holotype HMAS 252889, isotype HKAS 95055, ex-type culture HMAS 244997).

Stromata solitary, scattered or gregarious in small groups, pulvinate, discoid or subturbinate, centrally attached and with margin free, outline rounded, angular or irregular, dirty yellow to brownish yellow, 1.5–3.5 mm diam., 0.9–1.0 mm thick (n = 20). Surface usually farinose or granular due to spore deposits, rarely smooth. Ostiolar dots brown to dark brown, distinct, densely distributed. Rehydrated stromata turning brownish red in 3 % KOH.

In section, cortical tissue of textura angularis, 8–16 μm thick, cells light yellow, thin-walled, (4.5–)6–8.5(–10.5) × (4–)4.5–8(–10) μm (n = 30), turning brownish yellow in 3 % KOH; subcortical tissue of textura intricata, hyphae hyaline, thin-walled, (2–)2.5–3 μm (n = 30) wide; subperithecial tissue of textura epidermoidea, cells hyaline, thin-walled, 8–19(–24) × 7.5–14(–19) μm (n = 30) wide; tissue at the base of textura angularis mixed with textura intricata, cells hyaline, thin-walled, (5–)6–13(–16) × (4–)5–10(–12) μm (n = 30), hyphae hyaline, thin-walled, (3.5–)4–5(–6) μm (n = 30) wide. Perithecia flask-shaped or subglobose, numerous, (174–)179–200(–216) × (100–)105–158(–197) μm (n = 30); peridium hyaline or light yellow in lactic acid, turning pale tan or slightly darkened in 3 % KOH, 8–13 μm thick at flanks (n = 30), 11–18(–24) μm thick at the base. Ostioles non-papillate, (40–)42–50(–53) μm high, (18–)21–29(–32) μm wide at the apex (n = 30). Asci cylindrical, (54–)57–64 (–67) × 4–4.5 μm, with a stipe 5–8(–9) μm long (n = 40). Ascospores hyaline, spinulose or verruculose, cells monomorphic, globose, subglobose or rarely ellipsoidal, sometimes part-ascospores in the ascus base dimorphic, oblong or cuneate; distal cells 2–2.5(–3) × 2–2.5 μm, l/w 1.0–1.2(–1.5); proximal cells (2–)2.5–3(–4) × 2–2.5(–3) μm, l/w (1.0–)1.1–1.5(–1.6) (n = 50).

Colony on CMD 29–31 mm in radius after 72 h at 25 °C, mycelium covering the plate after 7 days. Colony hyaline, circular, dense, conspicuously zonate; aerial hyphae dense and short, longer towards the distal margin. Odour slightly fruity, no diffusing pigment observed.

Colony on PDA 19–21 mm in radius after 72 h at 25 °C, mycelium covering the plate after 14 days. Colony whitish, dense and compact, with thin, diffuse margin. Surface downy, farinose to floccose, macroscopically homogeneous with irregularly zonate near the plug. Odour slightly fruity, no diffusing pigment observed.

Colony on SNA 30–32 mm in radius after 72 h at 25 °C, mycelium covering the plate after 7 days. Colony hyaline, radially fan-shaped. Aerial hyphae inconspicuous, appearing empty. Conidiophores sparsely disposed, noted after 10 days, with 1–2 whorls arising from the main axis. Phialides mostly asymmetrically arranged, narrowly subulate or slender, (12–)13–23(–26) × (2–)2.5–3(–3.5) μm, l/w (4.5–)5–9(–9.5), 2–3 μm wide at the base (n = 50). Conidia hyaline, subglobose or ellipsoidal, smooth, (2.5–)3–5(–6) × 2–3(–3.5) μm, l/w (1.0–)1.1–2.0(–2.5) (n = 50). Chlamydospores distinctly abundant, noted after 8–10 days, terminal and intercalary, globose or ellipsoidal, (3–)4–8(–10) × (3–)4–6(–7) μm, l/w 1.0–1.4(–1.6) (n = 50). Autolytic excretions abundant, no distinct odour, no diffusing pigment observed.

Notes: Trichoderma henanense is most similar to T. odoratum but they represent different species. Both of them form yellowish stromata with brownish ostiolar dots, monomorphic ascospores, white colonies and hyaline conidia. However, T. odoratum differs in projecting ostioles, larger perithecia, longer asci, smaller growth rate, larger conidia and the presence of a mushroom-like odour in cultures. Detailed morphological distinctions between the two species are shown in Table 3.

Table 3 Morphological comparisons of Trichoderma pseudobritdaniae with T. britdaniae and T. henanense with T. odoratum
Full size table

Phylogenetically, T. henanense formed a branch with T. odoratum; however, the sequence similarity of rpb2 and tef1 between them was only 93.14 % and 94.67 %, respectively.

Trichoderma odoratum W.T. Qin & W.Y. Zhuang, sp. nov. (Fig. 3) MycoBank: MB 813809

Fig. 3
figure 3

Trichoderma odoratum (holotype HMAS 271354). a–n Sexual state. a–e Dry mature stromata on natural substrates. f Mature stroma after rehydration. g Mature stroma in 3 % KOH after rehydration. h Longitudinal section of a stroma. i Perithecium in section. j Cortical and subcortical tissues in section. k Subperithecial tissue in section. l Stroma base in section. m, n Asci with ascospores. o–z Asexual state. o–q Cultures after 17 days at 25 °C (o CMD, p PDA, q SNA). r Scattered young stromata (PDA, 25 days). s Conidiation tufts (PDA, 17 days). t–x Conidiophores and phialides (PDA, 17 days). y Chlamydospores (CMD, 20 days). z Conidia (PDA, 17 days). Scale bars: a–g = 400 μm. h = 200 μm. i = 50 μm. j–l, v, x = 20 μm. m, n = 5 μm. o–q = 20 mm. r = 0.8 mm. s = 1 mm. t, u, w, y, z = 10 μm

Full size image

Etymology: The specific epithet refers to the distinctive odour produced by the fungus.

Typification: China. Hubei, Shennongjia, Muchengshaoka, 31°27′41″N, 110°30′27″E, alt. 860 m, on twig lying on the ground, 22 Sep 2014, W.T. Qin, K. Chen, H.D. Zheng & Z.Q. Zeng 10035 (holotype HMAS 271354, isotype HKAS 95058, ex-type culture HMAS 244998).

Stromata solitary, scattered or aggregated in small fascicles, yellow or greyish yellow, pulvinate or discoid with rounded margin, 1.5–3 mm diam., 0.5–0.8 mm thick (n = 15). Surface usually smooth, occasionally farinose or granular due to spore deposits. Ostiolar dots dark brown or reddish brown, distinct, densely distributed. Rehydrated stromata turning brownish red in 3 % KOH.

In section, cortical tissue of textura angularis, 13–21 μm thick, cells yellow, thin-walled, (4–)5.3–7.9(–12) × (3.7–)4–5.5(–7.9) μm (n = 30), turning dark orange red to brownish red in 3 % KOH; subcortical tissue of textura intricata, hyphae hyaline, thin-walled, (1.3–)2–3(–3.7) μm (n = 30) wide; subperithecial tissue of textura epidermoidea, cells hyaline, thin-walled, (6–)8–15(–16) × (6–)7.4–11 μm (n = 30); tissue at the base of textura angularis mixed with textura intricata, cells hyaline, thin-walled, (5.3–)7.4–10.5(–11.3) × 5.3–7.9 μm (n = 30), hyphae hyaline, thin-walled, (2.8–)3–5.3 μm (n = 30) wide. Perithecia subglobose or flask-shaped, 184–224(–227) × (111–)116–145 μm (n = 30); peridium yellow in lactic acid, turning dark orange red to brownish red in 3 % KOH, 5.8–8(–11) μm thick at flanks, 6.6–10.5(–13.2) μm thick at the base (n = 30). Ostioles projecting by (8–)16–21 μm, 53–66(–74) μm high, (29–)32–40(–42) μm wide at the apex (n = 30). Asci cylindrical, (63–)65–74 (–77) × 3.4–3.8(–4) μm, with a stipe (11–)12–20 μm long (n = 40). Ascospores hyaline, spinulose or verruculose, cells monomorphic, globose, subglobose or ellipsoidal, sometimes part-ascospores in the ascus base dimorphic, oblong or cuneate; distal cells 2.4–3.1 × 2.2–2.8 μm, l/w 1.0–1.2(–1.3); proximal cells 2.4–3.8(–4.1) × 2.1–2.7 μm, l/w (1.0–)1.1–1.5(–1.6) (n = 50).

Colony on CMD 19–22 mm in radius after 72 h at 25 °C, mycelium covering the plate after 10 days. Colony hyaline, thin, circular, dense, with indistinct light/dense and darker/looser concentric zones; denser zones slightly wider. Aerial hyphae apparent toward the downy or floccose distal margin, becoming fertile. No conidiation noted within 20 days at 25 °C. Chlamydospores noted after 12–15 days, terminal or intercalary, smooth, globose or ellipsoidal, (4.8–)5–9 (–11.5) × (4.5–)5–8 μm, l/w 1.0–1.4(–1.8) (n = 50). Autolytic excretions rare, coilings numerous, odour mushroom-like, no diffusing pigment noted.

Colony on PDA 5–7 mm in radius after 72 h at 25 °C, 37–39 mm after 20 days. Colony white, not zonate, hyphae densely disposed with a well-defined or slightly wavy margin. Conidiation noted after 7 days, starting on and around the plug in short minute shrubs, spreading, growing to tufts or pustules, white, effuse, farinose, floccose or cottony. Trichoderma-like to verticillium-like conidiophores formed widely spaced on aerial hyphae. Phialides solitary or commonly divergent in whorls of 2–3, lageniform to subulate, commonly slender, (10.5–)11–18(–21) × (2–)2.5–3(–4) μm, l/w (3.3–)3.9–6(–6.6), 1.8–2.6(–3.2) wide at the base (n = 50). Conidia hyaline, variable in shape, mostly oblong to cylindrical, also ellipsoidal, sometimes subglobose or oval, smooth, (2.5–)2.8–7.2(–8.7) × 2.5–3(–3.5) μm, l/w (1.0–)1.2–2.9(–3.5) (n = 50). Chlamydospores abundant, noted after 8 days, terminal or intercalary, smooth, globose or ellipsoidal, (6.5–)7.2–11.5(–14.4) × (5.5–)6.5–8.2(–9.5) μm, l/w 1.0–1.4(–1.9) (n = 50). Odour mushroom-like, no diffusing pigment observed.

Colony on SNA 19–22 mm in radius after 72 h at 25 °C, mycelium covering the plate after 10 days. Colony hyaline, thin, hardly visible, smooth, radially fan-shaped, not zonate, hyphae loosely disposed. No conidiation noted within 20 days at 25 °C. Chlamydospores noted after 10 days. Autolytic activity moderate, excretions minute, coilings numerous, odour mushroom-like, no diffusing pigment noted.

Notes: Trichoderma odoratum is most similar to T. henanense. See notes under T. henanense for comparisons. Notably, yellow to orange aggregated pseudoparenchymatous stromata of T. odoratum were formed after 8 days on PDA or after 17 days on SNA at 25 °C. Until now, very few species of Trichoderma have been reported producing stromata on artificial media (Table 4).

Table 4 Species of Trichoderma producing stromata on artificial media
Full size table

Trichoderma pseudobritdaniae W.T. Qin & W.Y. Zhuang, sp. nov. (Fig. 4) MycoBank: MB 813810

Fig. 4
figure 4

Trichoderma pseudobritdaniae (holotype HMAS 271355). a–m Sexual state. a–e Dry stromata on natural substrates (a–d mature, e immature). f Mature stroma after rehydration. g Mature stroma in 3 % KOH after rehydration. h Longitudinal section of a stroma. i Perithecium in section. j Cortical and subcortical tissues in section. k Subperithecial tissue in section. l Stroma base in section. m Ascus with ascospores. n–u Asexual state. n–p Cultures after 11 days at 25 °C (n CMD, o PDA, p SNA). q–t Conidiophores and phialides (PDA, 10 days). u Conidia (PDA, 10 days). Scale bars: a, c, d, f, g = 1 mm. b = 2 mm. e = 400 μm. h = 200 μm. i = 50 μm. j–l = 20 μm. m, s, u = 10 μm. n, p = 20 mm. o = 10 mm. q, r, t = 20 μm

Full size image

Etymology: The specific epithet refers to its morphological similarity to Trichoderma britdaniae.

Typification: China. Henan, Lingbao, Yanzishan, 34°28′50″N, 111°05′9″E, alt. 1000 m, on twig lying on the ground, 16 Sep 2013, H.D. Zheng, Z.Q. Zeng & W.T. Qin 8663 (holotype HMAS 271355, isotype HKAS 95056, ex-type culture HMAS 244999).

Stromata solitary, scattered or aggregated in small groups, brownish yellow or greyish yellow, pulvinate, centrally attached and with margin free, outline circular, oblong or irregular, 2–3.5(–5) mm diam., 0.7–1.0 mm thick (n = 25). Surface usually smooth, occasionally farinose or granular. Ostiolar dots dark brown or grey black, distinct, densely distributed. Rehydrated stromata turning brownish red in 3 % KOH.

In section, cortical tissue of textura angularis, 15–24 μm thick, cells yellow, walls up to 1.3 μm thick, (5.5–)6.5–10.5(–12.5) × 5–7.5(–8) μm (n = 30), turning dark orange in 3 % KOH; subcortical tissue of textura intricata, hyphae hyaline to light yellow, thin-walled, (2.5–)3–4.5(–5) μm (n = 30) wide; subperithecial tissue of textura epidermoidea, cells hyaline to light yellow, thin-walled, (6.5–)8.5–18(–24) × (5.3–)6–10.5(–13.2) μm (n = 30); tissue at the base of textura angularis mixed with textura globulosa, cells hyaline to light yellow, walls up to 1.3 μm thick, (6.5–)7.5–18(–21) × (6.5–)7.5–15.5(–16) μm (n = 30). Perithecia flask-shaped, (195–)224–263(–283) × (111–)119–142(–171) μm (n = 30); peridium pale yellow brown in lactic acid, turning dark orange in 3 % KOH, 5.3–13.2 μm thick at flanks, 7.9–13.2 μm thick at the base (n = 30). Ostioles non-papillate, (53–)55–66 μm high, 26–37(–40) μm wide at the apex (n = 30). Asci cylindrical, (62–)68–82(–89) × 4.2–5(–5.5) μm, with a stipe (10–)12–29(–31) μm long (n = 40). Ascospores hyaline, spinulose or verruculose, cells monomorphic, globose, subglobose or broad-ellipsoidal, sometimes the basal part-ascospores dimorphic, occasionally oblong to cuneate; distal cells 2.8–3.5 × (2.2–)2.5–3.2 μm, l/w 1.0–1.5; proximal cells (2.4–)2.9–3.7(–4.2) × 2.1–3.2 μm, l/w 1.0–1.5(–1.9) (n = 50).

Colony on CMD 15–18 mm in radius after 72 h at 25 °C, 62–65 mm after 12 days. Colony hyaline, sparse, with wavy or irregular outline; aerial hyphae inconspicuous. Conidiation noted on or around the plug after 5 days. No distinct odour, no diffusing pigment observed.

Colony on PDA 3–5 mm in radius after 72 h at 25 °C, 28–34 mm after 12 days. Colony pale creamy, with commonly lobed or irregular outline; aerial hyphae inconspicuous. Conidiation noted on or around the plug after 4–5 days. Conidiophores densely disposed, trichoderma- to verticillium-like. Phialides solitary or divergent in whorls of 2–3(–4), straight, lageniform or subulate, (7–)8.5–18(–20) × (2.5–)2.8–4(–4.5) μm, l/w (2.1–)2.7–4.7(–7.2), 1.3–3.2(–3.5) μm wide at the base (n = 30). Conidia hyaline, ellipsoidal or oblong, also subglobose or oval, sometimes cylindrical, smooth, (2.5–)3–7(–7.5) × (2.5–)3–4(–4.5), l/w 1.0–1.9(–2.1) (n = 50). Chlamydospores rare and sparsely disposed, noted after 10 days, terminal and intercalary, globose or ellipsoidal, (4–)5–12 × 4–10 μm, l/w 1.0–1.3 (n = 15). No distinct odour, no diffusing pigment observed.

Colony on SNA 13–15 mm in radius after 72 h at 25 °C, 60–64 mm after 12 days. Colony hyaline, sparse, with rounded or irregular outline. Aerial hyphae inconspicuous, appearing empty. Conidiation noted on or around the plug after 3 days. No distinct odour, no diffusing pigment observed.

Notes: As a sister of T. britdaniae, T. pseudobritdaniae also forms small asci and monomorphic ascospores, whereas stromata of T. britdaniae look like basidiomata of a corticiaceous fungus, are large, undulate, up to ca. 10 cm long and 3 mm thick when fresh, scattered or forming confluent clusters extending up to 80 cm long, which are much larger than those of T. pseudobritdaniae. Trichoderma britdaniae forms short orange-brown hyphal protrusions near the stroma base, which is absent in T. pseudobritdaniae (Jaklitsch and Voglmayr 2012). Table 3 provides the detailed morphological distinctions between them. As sequences were compared, they shared 92.42 % similarity for rpb2 and 89.02 % similarity for tef1, and should be treated as different species.

Results

The PHT (p = 0.01) of rpb2 and tef1 sequences indicated that the individual partitions were generally congruent (Cunningham 1997), and, thus, the genes can be combined for analyses. Phylogenetic positions of the four new species were determined by analyses of the combined sequences of rpb2 and tef1. In the MP analysis, the combined dataset contained 42 taxa and 2411 characters, of which 1337 were constant, 200 were variable and parsimony-uninformative, and 874 were parsimony-informative. In the BI analysis, GTR + I + G was selected as the best-fit model.

All the investigated Trichoderma species clustered together, receiving high statistical supports (MPBP/BIPP = 100 %/100 %). The ten green-ascospored species representing the five clades as defined by Jaklitsch (2009) and Jaklitsch and Voglmayr (2015) formed a relatively high-supported group (MPBP/BIPP = 65 %/100 %). The 21 hyaline-ascospored species were distributed in ten recognisable clades. Among them, Brevicompactum, Deliquescens, Hypocreanum, Polysporum, Psychrophilum and Viride clades were closely related (MPBP/BIPP = 80 %/100 %). The rest of the species were attributed to the Longibrachiatum clade (MPBP/BIPP = 99 %/100 %), Semiorbis clade (MPBP/BIPP = 97 %/100 %), Stromaticum clade (MPBP/BIPP = 100 %/100 %), a clade containing T. asterineum and T. leguminosarum (MPBP/BIPP = 100 %/100 %), and several terminal branches which did not belong to any of the named clades (Fig. 5). Trichoderma britdaniae and T. pseudobritdaniae (MPBP/BIPP = 93 %/100 %), and T. henanense and T. odoratum (MPBP/BIPP = 100 %/100 %) appeared as two sister pairs (Fig. 5). These four species showed a close relationship to the Longibrachiatum clade.

Fig. 5
figure 5

Maximum parsimony phylogram reconstructed from the combined sequences of rpb2 and tef1 (tree length = 5600, CI = 0.3414, HI = 0.6586, RC = 0.1759 and RI = 0.5151), showing the phylogenetic position of the four new Trichoderma species and a new clade (in bold). MPBP above 50 % (left) and BIPP above 90 % (right) are given, respectively. TreeBASE S18017

Full size image

Discussion

The application of molecular tools for fungal taxonomy prompted the researches on morphology-based taxonomy of Trichoderma. Till now, more than 260 species have been recognised in the genus (Samuels et al. 1998; Chaverri and Samuels 2003; Samuels et al. 2006; Jaklitsch 2009, 2011; Jaklitsch and Voglmayr 2014, 2015; Zhu and Zhuang 2014b, c, 2015a, b; Bissett et al. 2015; http://www.isth.info/tools/molkey/index.php). As shown in the previous studies, ITS is not suitable for a genus-wide species reconstruction, and the tef1 introns can only be used in individual clades or certain groups due to their high sequence variability (Samuels et al. 2006). Endochitinase 42, chitinase 18-5, calmodulin- and actin-encoding genes were also tested for a limited number of species, which did not provide a reasonable resolution (Chaverri et al. 2003b; Druzhinina and Kubicek 2005). rpb2 sequences appeared powerful due to their suitable interspecific variations and, therefore, they were accepted as the main marker for the exploration of phylogeny of the group (Zhu et al. 2014; Jaklitsch and Voglmayr 2015). The tef1 exons are less variable among species compared with its introns, but capable of providing additional useful information (Chaverri and Samuels 2003; Jaklitsch et al. 2006; Overton et al. 2006a, b). In this study, the combined sequences of rpb2 and tef1 regions were chosen to clarify the phylogenetic positions of the new Trichoderma species (Fig. 5). The tree topology is basically congruent with the previous reports (Jaklitsch 2009, 2011; Jaklitsch and Voglmayr 2015).

Trichoderma leguminosarum was first discovered as a separate terminal branch and did not belong in any known clades (Jaklitsch and Voglmayr 2015). In the present work, T. asterineum and T. leguminosarum clustered together with high statistical supports (Fig. 5, MPBP/BIPP = 100 %/100 %) and share significant morphological characteristics that are unknown in any other hyaline-ascospored species of the genus. Considering the distinctive morphological similarity and close phylogenetic relationship of these two fungi, we name them the Asterineum clade.

Trichoderma henanense, T. odoratum and T. pseudobritdaniae are associated with but outside the Longibrachiatum clade (Fig. 5). Their densely disposed ostiolar dots and monomorphic ascospores resemble members of the Longibrachiatum clade, but their yellowish stromata, hyaline or yellowish apical fascicle of periphyses in lactic acid and whitish colonies recall species in the Polysporum clade. Trichoderma henanense and T. odoratum are sisters, as well as T. britdaniae and T. pseudobritdaniae (Fig. 5). Although taxa of the individual sisters share certain morphological features, the two species in the former sister differ in size of perithecia, asci and conidia, growth rate in cultures and sequence data; and those of the latter sister are hardly confused in stromatal size, perithecia gross morphology and sequence data.

Trichoderma species inhabit commonly in soil and on woody substrates. The genus is widely distributed in world temperate and tropical regions. China is rich in species diversity of the group. We believe that more new taxa will be discovered. For future research, integrated studies of morphology and phylogeny are needed, which will comprehensively deepen our knowledge of species diversity, phylogeny and potential use of Trichoderma.