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

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 trichodermalike 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.


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 andZhuang (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.

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 cyclesequenced 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.

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).

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).
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.
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.
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 × 3.2-4.0μm) 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.
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.
Habitat and distribution.On the surface of rotten wood in humid forests of south central and east China.

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.

Table 1 .
Materials including strain numbers and GenBank accessions of sequences used for phylogenetic analyses.