Two new green-spored species of Trichoderma (Sordariomycetes, Ascomycota) and their phylogenetic positions

Zhao-Xiang Zhu; Hao-Xiang Xu; Wen-Ying Zhuang; Yu Li

doi:10.3897/mycokeys.26.14919

Abstract

Two new species of Trichoderma are described based on the collections producing ascomata or asexual morphs on woody substrates, and named as Trichoderma fujianense and T. zonatum. Trichoderma fujianense produces gliocladium to verticillium-like conidiophores, slender to lageniform phialides, green and ellipsoidal to cylindrical conidia. Trichoderma zonatum is characterized by pulvinate, pale yellow to light brown stromata with densely disposed dark green to black ostioles, monomorphic ascospores, simple trichoderma-like conidiophores, green, (sub)globose to pyriform conidia. Their phylogenetic positions were investigated inferred from sequence analyses of the combined RNA polymerase II subunit b and translation elongation factor 1-α genes. The results indicate that T. fujianense, along with T. aureoviride and T. candidum, represents an independent lineage with high statistical support. Trichoderma zonatum belongs to the Chlorosporum clade and is associated with but clearly separated from T. rosulatum and T. costaricense. Morphological distinctions and sequence divergences between the new species and their close relatives were discussed.

Key words

Hypocreales , morphology, phylogeny, taxonomy

Introduction

Trichoderma Pers. (Ascomycota, Sordariomycetes, Hypocreales, teleomorph Hypocrea Fr.) species are frequently found on dead wood and bark, on other fungi, in soil and living within healthy plant roots, stems and leaves (Mukherjee et al. 2013). Species of the genus belong to one of the most useful groups of microbes to have had an immense impact on human welfare. Some species are widely used as effective biocontrol agents for several soil-borne plant pathogens (Harman et al. 2004, Hasan et al. 2012, Liu et al. 2012), producers of enzymes, antibiotics and heterologous proteins for food, feed, textile and biofuel industries (cellulases, hemicellulases) (Samuels 1996, Almeida et al. 2007, Cheng et al. 2012, Lopes et al. 2012, Mukherjee et al. 2013). Many members are treated as agents for improving seed germination and nutrient use efficiency, breaking of seed dormancy, as well as source of transgenes and herbicides, and are long known to improve plant growth through the production of phytohormones and certain secondary metabolites (Harman et al. 2004, Shoresh et al. 2010), whereas others are causal agents of opportunistic infections of humans and animals (Samuels 1996, Kuhls et al. 1999, Kredics et al. 2003), and due to association of certain species with economically significant production losses in commercial mushroom farms (Samuels et al. 2002, Park et al. 2006, Kim et al. 2012a, 2012b).

The genus Trichoderma was established in 1794 including four species (Samuels 1996). In recent years, the number of Trichoderma species increases dramatically. Bissett et al. (2015) presented a list of 254 names of species and two names of varieties in Trichoderma with name or names against which they are to be protected, following the ICN (Melbourne Code, Art. 14.13). More recently, In a large-scale survey of Trichoderma from rotten wood and soil in China, Qin and Zhuang (2016a, b, c, d, e, 2017) published 27 new species based on the collections producing ascomata or asexual morphs on woody substrates; Chen and Zhuang (2016) described two new species based on soil samples from the Hubei and Tibet regions of China; Montoya et al. (2016) found three new taxa in the attine ant environment; Sun et al. (2016) described a new fungicolous Trichoderma species which was isolated from surface of the stroma of Hypoxylon anthochroum. Until now, 287 Trichoderma species have been described.

During our investigation of the diversity of Trichoderma species in China, two species were found to represent undescribed new taxa, on the basis of both morphological and cultural characters and DNA sequence analyses of partial nuc translation elongation factor 1-α encoding gene (TEF1-α) and the gene for nuc RNA polymerase II second largest subunit (RPB2). Differences between the new species and their close relatives are discussed, and a phylogenetic analysis is provided.

Materials and methods

Specimens and strains

Specimens were collected from Henan and Fujian provinces, China, and deposited in the Mycological Herbarium of Jilin Agricultural University (HMJAU). Strains were obtained either by single ascospore isolation from fresh stromata of sexual morphs or by direct isolation from asexual morphs on the substrates. Cultures are deposited in the China General Microbiological Culture Collection Center (CGMCC).

Morphological study

Dried stromata were rehydrated and longitudinal sections through ascomata were made with a freezing microtome (Leica CM1950) at a thickness of 5–10 μm. Agar media employed were cornmeal dextrose agar (CMD, Difco, Sparks, MD, USA, with dextrose 20 g/L), potato dextrose agar (PDA, Solarbio, Beijing, CHINA) and synthetic low nutrient agar (SNA, Nirenberg 1976, pH adjusted to 5.5). Colonies were incubated in 9 cm diam Petri dishes at 25 °C with alternating light/darkness (12/12 h) at 20 °C, 25 °C, 30 °C and 35 °C and measured daily until the dishes were covered with mycelium. The characteristics of asexual and sexual states were described following the methods of Jaklitsch (2009) and Zhu and Zhuang (2015). Photographs were taken using a Leica DFC450C digital camera (Tokyo, Japan) connected to a Zeiss Axioskop 2 Plus microscope (Göttingen, Germany) for anatomical structures and to a Zeiss Stemi 2000C stereomicroscope for gross morphology.

DNA extraction, amplification and sequencing

Genomic DNA was extracted from mycelium harvested from colonies on PDA after 1–2 wk with a NuClean Plant Genomic DNA Extraction Kit (CoWin Biosciences, Beijing, China) according to the manufacturer’s protocol. Fragments of the nuc rDNA internal transcribed spacers (ITS1-5.8S-ITS2 = ITS), TEF1-α and RPB2 were amplified with the primer pairs ITS4 and ITS5 (White et al. 1990), EF1-728F (Carbone and Kohn 1999) and TEF1LLErev (Jaklitsch et al. 2005), fRPB2-5f and fRPB2-7cr (Liu et al. 1999), respectively. PCR products were purified with the PCR Product Purification Kit (Biocolor BioScience and Technology Co., Shanghai, China) and cycle-sequenced on an ABI 3730 XL DNA Sequencer (Applied Biosciences, Foster City, Calofornia) with the same primer in fragments amplification for ITS and primers reported by Jaklitsch (2009) for TEF1-α, and RPB2 at Beijing Tianyihuiyuan Bioscience and Technology, China. The strains and the NCBI GenBank accession numbers of DNA sequences used in this work are listed in Table 1.

Table 1.

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Materials including strain numbers and GenBank accessions of sequences used for phylogenetic analyses.

Name	Strain	GenBank accession number
Name	Strain	RPB2	TEF1-α
Trichoderma aerugineum Jaklitsch	CBS 120541	FJ860516	FJ860608
T. aureoviride Rifai	C.P.K. 2848	FJ860523	FJ860615
T. ceramicum P. Chaverri & Samuels	CBS 114576	FJ860531	FJ860628
T. chlorosporum P. Chaverri & Samuels	G.J.S. 88-33	AY391903	AY391966
T. chromospermum P. Chaverri & Samuels	G.J.S. 94-68	AY391913	AY391974
T. costaricense (P. Chaverri & Samuels) P. Chaverri, Jaklitsch & Voglmayr	P.C. 21	AY391921	AY391980
T. cremeoides Jaklitsch & Voglmayr	S112	KJ665253	KJ665456
T. cremeum P. Chaverri & Samuels	G.J.S. 91-125	AF545511	AF534598
T. cuneisporum P. Chaverri & Samuels	G.J.S. 91-93	AF545512	AF534600
T. danicum (Jaklitsch) Jaklitsch & Voglmayr	CBS 121273	FJ860534	FJ860634
T. estonicum P. Chaverri & Samuels	G.J.S. 96-129	AF545514	AF534604
T. fujianense Z.X. Zhu, W.Y. Zhuang &Y. Li	HMJAU 34830	MF374808*	MF374811
T. gelatinosum P. Chaverri & Samuels	G.J.S. 88-17	AF545516	AF534579
T. gliocladium Jaklitsch & Voglmayr	S81	KJ665271	KJ665502
T. helicum Bissett, C.P. Kubicek & Szakács	DAOM 230021	DQ087239	KJ871125
T. longipile Bissett	CBS 120953	FJ860542	FJ860643
T. nigrovirens P. Chaverri & Samuels	G.J.S. 99-64	AF545518	AF534582
T. parestonicum Jaklitsch	CBS 120636	FJ860565	FJ860667
T. phyllostachydis P. Chaverri & Samuels	G.J.S. 92-123	AF545513	AF534576
T. pseudocandidum Minnis, Samuels & P. Chaverri	P.C. 59	AY391899	AY391962
T. rosulatum Z.X. Zhu & W.Y. Zhuang	HMAS 252548	KF730005	KF729984
T. sinuosum P. Chaverri & Samuels	G.J.S. 90-88	AY391932	AY391990
T. spinulosum (Fuckel) Jaklitsch & Voglmayr	CBS 121280	FJ860589	FJ860699
T. stipitatum Z.X. Zhu & W.Y. Zhuang	HMAS 266613	KF730012	KF729991
T. strictipile Bissett	C.P.K. 1601	FJ860594	FJ860704
T. surrotundum P. Chaverri & Samuels	G.J.S. 88-73	AF545540	AF534594
T. thailandicum P. Chaverri & Samuels	G.J.S. 97-61	AY391957	AY392005
T. thelephoricola P. Chaverri & Samuels	CBS 120925	FJ860600	FJ860711
T. virescentiflavum (Speg.) Jaklitsch & Voglmayr	P.C. 278	AY391959	AY392007
T. zonatum Z.X. Zhu, W.Y. Zhuang &Y. Li	HMJAU 34820	MF374806	MF374809
T. zonatum Z.X. Zhu, W.Y. Zhuang &Y. Li	HMJAU 34825	MF374807	MF374810
Nectria eustromatica Jaklitsch & Voglmayr	CBS 121896	HM534886	HM534875
N. berolinensis (Sacc.) Cooke	CBS 127382	HM534883	HM534872

Phylogenetic analyses

Sequences were assembled, aligned and manually adjusted when needed with BioEdit 7.0.5.3 (Hall 1999). NEXUS files were generated with Clustal X 1.83 (Thompson et al. 1997). To identify the phylogenetic positions of Trichoderma fujianense and T. zonatum, RPB2 and TEF1-α sequences were combined for the analyses. Thirty-three sequences representing 30 Trichoderma taxa were selected for analyses, with Nectria eustromatica and N. berolinensis selected as outgroup taxa. Alignments are deposited in TreeBASE accession number 21272.

Maximum parsimony (MP) analysis was performed with PAUP 4.0b10 (Swofford 2002) using 1000 replicates of heuristic search with random addition of sequences and subsequent tbr (tree bisection and reconnection) branch swapping. Analyses were performed with all characters treated as unordered and unweighted, gaps treated as missing data. Topological confidence of resulted trees was tested by maximum parsimony bootstrap proportion (MPBP) with 1000 replications, each with 10 replicates of random addition of taxa.

Bayesian Inference (BI) analysis was conducted via MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using a Markov Chain Monte Carlo (MCMC) algorithm. Nucleotide substitution models were determined by MrModeltest 2.3 (Nylander 2004). gtr+i+g was estimated as the best-fit model for combined sequences. Four MCMC chains were run from random trees for 2 000 000 generations and sampled every 100 generations. The first 5000 trees were discarded as the burn-in phase of the analyses, and Bayesian inference posterior probability (BIPP) was determined from the remaining trees. Trees were visualized in TreeView 1.6.6 (Page 1996).

Results

Phylogenetic analyses

The partition homogeneity test (P = 0.01) of RPB2 and TEF1-α sequences indicated that the individual partitions were generally congruent (Cunningham 1997). Phylogenetic positions of the new species were determined by analyses of the combined RPB2 and TEF1-α dataset containing 33 taxa and 2396 characters, of which 1304 characters were constant, 366 variable characters were parsimony-uninformative and 726 were parsimony-informative. Five most-parsimonious trees with the same topology were generated (Figure 1) (tree length = 3178, CI = 0.4685, HI = 0.4572, RI = 0.5493 and RC = 0.2982).

Thirty-three sequences representing 30 green-spored Trichoderma species and two outgroup taxa Nectria berolinensis and N. eustromatica were used to construct the phylogenetic tree (Figure 1). All the green-spored species formed a monophyletic group (100 % MPBP/BIPP), which is basically consistent with the previous study by Jaklitsch (2009) and Zhu and Zhuang (2015).

In our phylogenetic tree, the five major clades, Chlorosporum, Spinulosum, Virescentiflavum, Ceramicum and Strictipile were basically well supported. The first four clades received 100%/100%, 98%/100%, 100%/100% and 100%/- (MPBP/BIPP) support in the tree, respectively, but the Strictipile clade was less strongly supported at 61% (MPBP) in the tree.

In the Chlorosporum clade (Figure 1), two samples of Trichoderma zonatum (HMJAU 34820 and 34825), sharing identical sequences, constituted a well-supported independent lineage (MPBP/BIPP = 100 %/100 %). Although T. zonatum is morphologically similar to T. chromospermum, they are not phylogenetically close and have relatively low sequence similarities in RPB2, TEF1-α and ITS.

Figure 1.

Maximum parsimony phylogram reconstructed from the combined squences of RPB2 and TEF1-α. MPBP above 50% (left) and BIPP above 90% (right) are indicated at the nodes.

Trichoderma aureoviride, T. candidum and T. fujianense form an independent lineage with high statistical support (MPBP/BIPP = 100%/100%), which still remains unnamed (Chaverri and Samuels 2003, Jaklitsch 2009).

Taxonomy

Trichoderma fujianense Z.X. Zhu, W.Y. Zhuang & Y. Li, sp. nov.

MycoBank No: 821807

Figure 2

Diagnosis

Characterized by slender to lageniform, long phialides (14–23 × 2–3.5 μm), gliocladium to verticillium-like conidiophores, ellipsoidal to cylindrical conidia (4.5–5.5 × 2.5–3.5 μm).

Figure 2.

The new species Trichoderma fujianense, holotype (HMJAU 34830). a–s Asexual state a–c Cultures after 13 d at 25 °C (aPDA, bCMD, cSNA). d–q Conidiophores and phialides (SNA, 20 d) r, s Conidia (SNA, 20 d). Scales bars: 20 mm (a–c); 20 μm (d–h); 10 μm (i–s).

Type

CHINA. Fujian: Quanzhou City, Qingyuan mountain. 24°56'51"N, 118°36'31"E, 150 m alt., on bark, 6 Aug 2015, Z.X. Zhu 230 (HMJAU 34830, holotype), Ex-type culture CGMCC 3.18757.

Description

Colony radius on CMD after 72 h 2.5–5 mm at 20 °C, 13–15 mm at 25 °C, 3.5–5 mm at 30 °C, no growth at 35 °C, mycelium covering the plate after 2 wk at 25°C. Colony circular, dense, ﬁnely zonate, becoming hairy to ﬂoccose by conidiophores, ﬁrst whitish, turning light green. Aerial hyphae virtually absent. Autolytic excretions, pigment and coilings absent. Conidiation starting after 4 d in densely disposed gliocladium-like conidiophores, short-effuse, turning green after 1 wk.

Colony radius on PDA after 72 h 7.5–8.5 mm at 20 °C, 8.5–10 mm at 25 °C, 0.5–1 mm at 30 °C, no growth at 35 °C, mycelium covering the plate after 2 wk at 25 °C. Colony circular, compact with distinctly zonate, with commonly lobed or coarsely wavy margin, centre dense, green, margin relatively looser, whitish. Conidiation noted around the plug after 3–4 d, effuse, spreading from the centre over the entire colony surface. No distinct odor, no diffusing pigment observed.

Colony radius on SNA after 72 h 1.5–3 mm at 20 °C, 4–5 mm at 25 °C, 1–2 mm at 30 °C, no growth at 35 °C, mycelium covering the plate after 24 d at 25 °C. Colony hyaline, thin, irregular, not zonate, surface mycelium scant. Aerial hyphae inconspicuous, short. Conidiophores sparsely disposed, noted after 7 d, gliocladium to verticillium-like, with 1–3(–4) whorls arising from the main axis. Phialides arising in more or less narrow angles from cylindrical metulae, phialides slender to lageniform, somewhat curved, (10–)14–23(–28) × 2–3.5(–4) μm, l/w 4.8–7.2(–9.2), (1.5–)1.8–2.7(–3.2) μm wide at the base (n = 100). Conidia green, ellipsoidal to cylindrical, smooth, (4–)4.5–5.5(–6) × 2.5–3.5(–4) μm, l/w (1.2–)1.3–2.0 (n = 100). No distinct odor, no diffusing pigment observed.

Habitat and distribution

On the surface of rotten wood in humid forests of east China.

Etymology

The epithet “fujian”, indicating occurrence of the fungus in Fujian province.

Teleomorph

Not known.

Remarks

Morphologically, the new species is most similar to Trichoderma costaricense in conidiophore character and phialide shape and size; while the latter fungus produces abundant chlamydospores on CMD, has relatively larger conidia (5.2–6.0 × 3.2–4.0 μm) and faster growth on PDA and SNA, and grows well and sporulates at 35 °C (Chaverri and Samuels 2003). Furthermore, sequence similarity of ITS and RPB2 between these species was only 90.1% and 92.1%, with 60 bp and 68 bp differences among 606 bp and 864 bp, respectively. Among the species with green ascospores, T. gelatinosum, T. nigrovirens, T. chromospermum and T. thelephoricola also generated gliocladium to verticillium-like conidiophores, but they are not phylogenetically closely related.

The phylogenetic positions of the new taxa (Figure 1) demonstrated that Trichoderma fujianense is found to be closely related to T. aureoviride and T. candidum, and three of them form an independent lineage with high statistical support. However, T. aureoviride is distinctive by shorter conidia (3.8–4.0 × 3.0–3.3 μm, l/w 1.2–1.3); T. candidum differs by shorter phialides (7.3–)9.0–13.5(–16.5) μm, globose to subglobose and smaller conidia (3.2–3.5 × 3.0–3.2 μm, l/w (1.0–)1.1(–1.3) (Chaverri and Samuels 2003).

Trichoderma zonatum Z.X. Zhu, W.Y. Zhuang & Y. Li, sp. nov.

MycoBank No: 821806

Figure 3

Diagnosis

Characterized by pulvinate, pale yellow to light brown stromata with densely disposed dark green to black ostioles, long asci (93–112 × 5.8–6.6 μm), monomorphic and subglobose ascospores (4.2–5 × 4–4.7 μm), simple trichoderma-like conidiophores, green, (sub)globose to pyriform conidia (2.8–3.8 × 2.3–2.8 μm).

Figure 3.

The new species Trichoderma zonatum, holotype (HMJAU 34820). a–o Sexual state. a–g Dry stromata on nature substrate h Mature stroma after rehydration i Perithecium in section j Cortical and subcortical tissue in section k Subperithecial tissue in section l Stroma base in section. m–o Ascus with part-ascospores. p–aa Asexual state. p–r Cultures after 7 d at 25 °C (pPDA, qCMD, rSNA). s–y Conidiophores and phialides (SNA, 7 d). z, aa Conidia (SNA, 7 d). Scales bars: 2 mm (a); 1 mm (b, g); 500 μm (c–f, h); 20 μm (i–l, s, x, y); 5 μm (m–o, z, aa); 20 mm (p–r); 10 μm (t–w).

Type

CHINA. Henan: Xinyang City, Jigong mountain. 31°49'2"N, 114°04'16"E, 1500 m alt., on bark, 16 Jul 2015, B. Zhang 220 (HMJAU 34820, holotype), Ex-type culture CGMCC 3.18758.

Description

Stromata generally solitary, scattered, gregarious, or aggregated in small groups, broadly attached, pulvinate to somewhat flattened, outline circular or with lobed margin, (0.5–)1.0–2.5(–3) mm diam (n = 20), (0.3–)0.5–0.8 mm high (n = 20). Surface flat, smooth, with slight perithecial protuberances, pale yellow to light brown, not changing colour in KOH, ostiolar openings obvious due to the green ascospores.

In section stroma cortical tissue of textura angularis, 13–28 μm thick, not changing colour in 3% KOH, cells yellow, thin-walled, 6–12(–17) × 5–10(–13) μm (n = 40); subcortical tissue of textura angularis, cells hyaline, thin-walled, 4–10 × 5–8 μm (n = 40); subperithecial tissue of textura epidermoidea, cells hyaline, thin-walled, 10–22 × 8–17 μm (n = 40); tissue at the base of textura intricata, hyphae hyaline, thin-walled, (2.5–)3.5–6(–8) μm (n = 40) wide. Perithecia subglobose or flask-shaped, crowded, 178–216 ×120–165 μm (n = 40); peridium yellow in lactic acid, not changing colour in 3% KOH, (8–)10–14(–17) μm thick at the sides, (12–)14–21(–27) μm at the base (n = 40). Ostioles conical or cylindrical, 51–70 μm high, 31–54 μm wide at the apex (n = 40). Asci cylindrical, 93–112 × 5.8–6.6(–7) μm, with a stipe (13–)18–23 μm long (n = 60). Part-ascospores green, turning brown in KOH, distinctly verrucose, cells monomorphic, subglobose, also slightly ovoid, 4.2–5 × 4–4.7 μm (n = 100), l/w 1.0–1.1.

On CMD colony radius after 72 h 30–43 mm at 20 °C, 32–46 mm at 25 °C, 17–34 mm at 30 °C, no growth at 35 °C. Colony hyaline, circular, loose, forming obvious zonate, covering the plate after 5–7 d at 25 °C. Aerial hyphae radially arranged. Conidiation at 25 °C noted after 3 d, ﬁrst effuse, soon followed by formation of granules or pustules, particularly along the margin, spreading from the centre across the entire plate. No distinct odor, no diffusing pigment observed.

On PDA after 72 h 38–48 mm at 20 °C, 55–62 mm at 25 °C, 28–30 mm at 30 °C, no growth at 35 °C; mycelium covering the plate after 8 d at 25°C. Colony circular, conspicuously dense, becoming zonate with broad, slightly downy zones and narrow, well-defined, convex, white to green farinose zones. Aerial hyphae numerous, mostly short, becoming fertile from the centre. Conidiation at 25 °C starting after 2 d, green after 4 d, first simple, mostly on short aerial hyphae concentrated in the centre and in denser zones, later abundant in pustules. Autolytic activity lacking or inconspicuous, no coilings seen. No diffusing pigment, no distinct odour noted.

On SNA after 72 h 12–14 mm at 20 °C, 18–20 mm at 25 °C, 15–17 mm at 30 °C, no growth at 35 °C; mycelium covering the plate after 8–9 d at 25°C. Colony hyaline, thin, loose, irregularly lobed, not zonate. Aerial hyphae inconspicuous. Autolytic activity moderate. Conidiophores visible after 4 d, trichoderma-like, with 2–3(–4) whorls arising from the main axis. Phialides solitary or divergent in whorls of 2–3, mostly asymmetrically arranged, lageniform, (5–)7–11(–14) × 2–3(–4) μm, l/w 1.7–2.8(–4) (n = 60). Conidia green, (sub)globose to pyriform, smooth, (2.5–)2.8–3.8 × 2.3–2.8 μm, l/w (1.0–)1.1–1.3(–1.5) (n = 70). No chlamydospores formed. No distinct odor, no diffusing pigment observed.

Habitat and distribution

On the surface of rotten wood in humid forests of south central and east China.

Etymology

The speciﬁc epithet refers to the zonate colony on PDA.

Other specimens examined

CHINA. Fujian: Quanzhou City, Qingyuan mountain. 24°55'53"N, 118°36'31"E, 200 m alt., on bark, 6 Aug 2015, Z.X. Zhu 225, HMJAU 34825, Ex-type culture CGMCC 3.18759.

Remarks

Phylogenetic analyses based on RPB2 and TEF1-α indicated that Trichoderma zonatum belongs to the Chlorosporum clade, previously consisting of eight species, T. sinuosum, T. cremeum, T. surrotundum, T. chlorosporum, T. thelephoricola, T. rosulatum, T. cremeoides and T. costaricense. Phylogenetically, T. zonatum is most related to T. rosulatum and T. costaricense, but T. rosulatum is clearly distinguishable by dimorphic ascospores, gliocladium-like conidiophores, production of abundant chlamydospores and rosulate colony on CMD (Zhu and Zhuang 2015); T. costaricense produces dimorphic and larger ascospores (5.5–6.0 × 5.2–5.7 μm), verticillium-like conidiophores and ellipsoidal to cylindrical conidia (Chaverri and Samuels 2003, Zhu and Zhuang 2015).

Species of the Chlorosporum clade usually produce pale yellow or pale green, semi-translucent stromata, globose to subglobose ascospores and gliocladium-like or verticillium-like conidiophores (Chaverri and Samuels 2003, Zhu and Zhuang 2015). Trichoderma zonatum is characterized by pulvinate, pale yellow to light brown stromata with densely disposed dark green to black ostioles, monomorphic ascospores, simple trichoderma-like conidiophores, green, (sub)globose to pyriform conidia. Morphologically, stromata of T. zonatum are not typical of the Chlorosporum clade and differ from all other species by relatively larger and non-transparent. It is most similar to T. chromospermum in gross stromata morphology, while the latter fungus clearly differs by much shorter asci [(78–)85–90(–102) μm], gliocladium-like conidiophores and ellipsoidal to cylindrical conidia (Chaverri and Samuels 2003, Zhu and Zhuang 2015).

Discussion

Phylogenetic analyses of Trichoderma species with green spores based on sequences of RPB2 and TEF1-α were performed by Chaverri and Samuels (2003). In the more recent study by Zhu and Zhuang (2015) a phylogenetic tree with 45 species having green-spored was inferred from RPB2 and TEF1-α sequences. In our study analyses of the combined sequences of the same genes of 30 related Trichoderma species were carried out to ascertain the phylogenetic positions of our new species. The tree topology is basically consistent with previous researches (Chaverri and Samuels 2003, Jaklitsch 2009, Zhu and Zhuang 2015). The study of Chaverri and Samuels (2003) suggested that phenotypic characters, alone are usually not useful in understanding phylogenetic relationships in Trichoderma, because teleomorph characters, for example, the tissue structure of the stroma, the size and character of the perithecia, asci and ascospores, are generally highly conserved and anamorph characters tend to be morphologically divergent within monophyletic groups, clades or species complex. Based on the results of the present study, we conclude that similarity in teleomorphic characters is not indicative of close phylogenetic relationships, holomorphs must be studied in order to effectively determine both life cycles and species concepts.

Acknowledgments

The authors thank Dr. Bo Zhang for collecting specimens jointly for this study. This project was supported by Science and Technology Developing Plan of Jilin Province (20160520054JH), China Postdoctoral Science Foundation and University S & T Innovation Platform of Jilin Province for Economic Fungi (#2014B-1).

References

Almeida FB, Cerqueira FM, Silva RN, Ulhoa CJ, Lima AL (2007) Mycoparasitism studies of Trichoderma harzianum strains against Rhizoctonia solani: evaluation of coiling and hydrolytic enzyme production. Biotechnology letters 29: 1189–1193. https://doi.org/10.1007/s10529-007-9372-z

Bissett J, Gams W, Jaklitsch WM, Samuels GJ (2015) Accepted Trichoderma names in the year 2015. IMA Fungus 6: 263–295. https://doi.org/10.5598/imafungus.2015.06.02.02

Carbone I, Kohn LM (1999) A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91: 553–556. https://doi.org/10.2307/3761358

Chaverri P, Samuels GJ (2003) Hypocrea/Trichoderma (Ascomycota, Hypocreales, Hypocreaceae): species with green ascospores. Studies in Mycology 48: 1–116.

Chen K, Zhuang WY (2016) Trichoderma shennongjianum and Trichoderma tibetense, two new soil-inhabiting species in the Strictipile clade. Mycoscience 57: 311–319. https://doi.org/10.1007/s00284-017-1282-2.

Cheng CH, Yang CA, Peng KC (2012) Antagonism of Trichoderma harzianum ETS 323 on Botrytis cinerea mycelium in culture conditions. Phytopathology 102: 1054–1063. https://doi.org/10.1094/Phyto-11-11-0315

Cunningham CW (1997) Can three incongruence tests predict when data should be combined? Molecular Biology and Evolution 14: 733–740. https://doi.org/10.1093/oxfordjournals.molbev.a025813

Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.

Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species–opportunistic, avirulent plant symbionts. Nature Reviews in Microbiology 2: 43–56. https://doi.org/10.1038/nrmicro797

Hasan MM, Rahman SM, Kim GH, Abdallah E, Oh DH (2012) Antagonistic potentiality of Trichoderma harzianum towards seed-borne fungal pathogens of winter wheat cv. Protiva in vitro and in vivo. Journal of Microbiology and Biotechnology 22: 585–591. https://doi.org/10.4014/jmb.1107.07063

Jaklitsch WM (2009) European species of Hypocrea I. The green-spored species. Studies in Mycology 63: 1–91. https://doi.org/10.3114/sim.2009.63.01

Jaklitsch WM, Komon M, Kubicek CP, Druzhinina IS (2005) Hypocrea voglmayrii sp. nov. from the Austrian Alps represents a new phylogenetic clade in Hypocrea/Trichoderma. Mycologia 97: 1365–1378. https://doi.org/10.3852/mycologia.97.6.1365

Kim CS, Shirouzu T, Nakagiri A, Sotome K, Nagasawa E, Maekawa N (2012a) Trichoderma mienum sp nov. isolated from mushroom farms in Japan. Antonie van Leeuwenhoek 102: 629–641. https://doi.org/10.1080/15572536.2006.11832743

Kim CS, Yu SH, Nakagiri A, Shirouzu T, Sotome K, Kim SC, Maekawa N (2012b) Re-evaluation of Hypocrea pseudogelatinosa and H. pseudostraminea isolated from shiitake mushroom (Lentinula edodes) cultivation in Korea and Japan. The Plant Pathology Journal 28: 341–356. https://doi.org/10.5423/PPJ.OA.05.2012.0068

Kredics L, Antal Z, Dóczi I, Manczinger L, Kevei F, Nagy E (2003) Clinical importance of the genus Trichoderma. Acta Microbiologica et Immunologica Hungarica 50: 105–117. https://doi.org/10.1556/AMicr.50.2003.2-3.1

Kuhls K, Lieckfeldt E, Börner T, Guého E (1999) Molecular re-identification of human pathogenic Trichoderma isolates as Trichoderma longibrachiatum and Trichoderma citrinoviride. Medical Mycology 37: 25–33. https://doi.org/10.1080/02681219980000041

Liu M, Liu J, Wang WM (2012) Isolation and functional analysis of Thmfs1, the first major facilitator superfamily transporter from the biocontrol fungus Trichoderma harzianum. Biotechnology Letters 34: 1857–1862. https://doi.org/10.1007/s10529-012-0972-x

Liu YJ, Whelen S, Hall BD (1999) Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Molecular Biology and Evolution 16: 1799–1808. https://doi.org/10.1093/oxfordjournals.molbev.a026092

Lopes FAC, Steindorff AS, Geraldine AM, Brandao RS, Monteiro VN, Lobo M, Coelho ASG, Ulhoa CJ (2012) Biochemical and metabolic profiles of Trichoderma strains isolated from common bean crops in the Brazilian Cerrado and potential antagonism against Sclerotinia sclerotiorum. Fungal Biology 116: 815–824. https://doi.org/10.1016/j.funbio.2012.04.015

Montoya QV, Meirelles LA, Chaverri P, Rodrigues A (2016) Unraveling Trichoderma species in the attine ant environment: description of three new taxa. Antonie Van Leeuwenhoek 109: 633–51. https://doi.org/10.1007/s10482-016-0666-9

Mukherjee PK, Horwitz BA, Singh US, Mukherjee M, Schmoll M (2013) Trichoderma: Biology and Applications. CABI, 327 pp. https://doi.org/10.1079/9781780642475.0000

Nylander JAA (2004) MrModeltest 2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University.

Page RDM (1996) TreeView: an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12: 357–358.

Park MS, Bae KS, Yu SH (2006) Two new species of Trichoderma associated with green mold of oyster mushroom cultivation in Korea. Mycobiology 34: 111–113. https://doi.org/10.4489/MYCO.2006.34.3.111

Qin WT, Zhuang WY (2016a) Four new species of Trichoderma with hyaline ascospores from central China. Mycological Progress 15: 811–825. https://doi.org/10.1007/s11557-016-1211-y

Qin WT, Zhuang WY (2016b) Four new species of Trichoderma with hyaline ascospores in the Brevicompactum and Longibrachiatum clades. Mycosystema 35: 1317–1336. https://doi.org/10.13346/j.mycosystema.160158

Qin WT, Zhuang WY (2016c) Seven wood-inhabiting new species of the genus Trichoderma (Fungi, Ascomycota) in Viride clade. Scientific Reports 6: 27074. https://doi.org/10.1038/srep27074

Qin WT, Zhuang WY (2016d) Three Trichoderma species new to China. Mygosystema 35: 1008–1017. https://doi.org/10.13346/j.mycosystema.150230

Qin WT, Zhuang WY (2016e) Two new hyaline-ascospored species of Trichoderma and their phylogenetic positions. Mycologia 108: 205–214. https://doi.org/10.3852/15-144

Qin WT, Zhuang WY (2017) Seven new species of Trichoderma (Hypocreales) in the Harzianum and Strictipile clades. Phytotaxa 305: 121–139. https://doi.org/10.11646/phytotaxa.305.3.1

Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. https://doi.org/10.1093/bioinformatics/btg180

Samuels GJ (1996) Trichoderma: a review of biology and systematic of the genus. Mycological Research 100: 923–935. https://doi.org/10.1016/S0953-7562(96)80043-8

Samuels GJ, Dodd SL, Gams W, Castlebury LA, Petrini O (2002) Trichoderma species associated with the green mold epidemic of commercially grown Agaricus bisporus. Mycologia 94: 146–170. https://doi.org/10.1080/15572536.2003.11833257

Shoresh M, Harman GE, Mastouri F (2010) Induced systemic resistance and plant responses to fungal biocontrol agents. Annual Review of Phytopathology 48: 21–43. https://doi.org/10.1146/annurev-phyto-073009-114450

Sun JZ, Pei YF, Li E, Li W, Hyde KD, Yin WB, Liu XZ (2016) A new species of Trichoderma hypoxylon harbours abundant secondary metabolites. Scientific Reports 6: 37369. https://doi.org/10.1038/srep37369

Swofford DL (2002) PAUP 4.0b10: Phylogenetic analysis using parsimony. Sinauer Associates, Sunderland, MA, U.S.A.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876–4882. https://doi.org/10.1093/nar/25.24.4876

White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR Protocols: A Guide to Methods and Applications.Academic Press, New York, 315–322. https://doi.org/10.1016/b978-0-12-372180-8.50042-1

Zhu ZX, Zhuang WY (2015) Trichoderma (Hypocrea) species with green ascospores from China. Persoonia 34: 113–129. https://doi.org/10.3767/003158515X686732