Five New Species of Trichoderma From Wetland Soils in China


 Trichoderma isolates were collected from wetland soils in different areas of China. Combined analyses of morphological characteristics and phylogenetic analyses by partial translation elongation factor 1 alpha (TEF1-α) and RNA polymerase II subunit b (RPB2) revealed five new Trichoderma species, namely, Trichoderma macrofasciculatum, T. shangrilaense, T. nordicum, T. vadicola, and T. hailarense. T. macrofasciculatum and T. shangrilaense belonging to the Polysporum Clade were isolated from wetland soils collected from Sichuan and Yunnan Provinces. The conidiation of T. macrofasciculatum typically appeared in white pustules in concentric rings on PDA or MEA, and its conidia had two or more guttules. Conidiation of T. shangrilaense formed white pustules with irregular shape and size, and its conidia were mostly obovoid and smooth. Trichoderma vadicola, T. nordicum, and T. hailarense belonging to the Viride Clade were collected from Shandong Province, Beijing Municipality, and Inner Mongolia Autonomous Region, respectively. The phialides of T. nordicum lageniform were curved on PDA, and its conidia were globose to obovoidal and large. The aerial mycelium of T. vadicola formed strands and floccose mat. The colonies of T. hailarense cannot form conidia on PDA, and the conidia of T. hailarense on other media were obovoid and delicately roughened.


Introduction
Trichoderma Pers. species are economically useful fungi in agriculture, industry, and medicine. They are widely used as biofungicides (Mukherjee et al. 2012), plant growth regulators (Harman 2011), and sources of enzymes for industrial utility. Some Trichoderma species have great potential applications to remediate soil and water pollution (Tripathi et al. 2013). Trichoderma species are cosmopolitan and prevalent components of different ecosystems in a wide range of climatic zones ).
They are mainly found in natural soils and decaying wood and plant material; in addition, they can be isolated from agricultural habitats, living plants as endophytes, mushroom-related substrata, human as opportunistic pathogens, water-related environments, air, and settled dust (Kredics et al. 2014). Through sequence comparison and phylogenetic analysis, more than 288 Trichoderma species have been found and described to date, and new species are constantly being discovered (Bissett et  Trichoderma resources are abundant in China, but only 107 species of Trichoderma have been found and con rmed so far. The research on Trichoderma mostly focused on application but lacked resource mining and taxonomy. To this end, we jointly launched a survey of Trichoderma resource from different ecological environments, such as farmland, forest, grassland and tidal wetland. Our research team is responsible for the excavation and character analysis of Trichoderma resources in wetland soil. In our work, 759 soil samples were collected from 23 provinces of China in 2015-2016, and over 3000 Trichoderma strains were isolated on the basis of morphological and cultural characters. Combined with sequence analyses of TEF1-α and RPB2, 35 species were identi ed, including 17 species known from China (T. guizhouense, T. hamatum, T. citrinoviride, T. asperellum, T. longibrachiatum, T. priscilae, T. pleuroticola, T. pleurotum, T. virens, T. brevicompactum, T. koningiopsis, T. harzianum, T. koningii, T. pseudokoningii, T. viride, T. longibrachiatum, and T. atroviride), 13 Chinese new records (T. crissum, T. afroharzianum, T. atrobrunneum, T. simmonsii, T. paratroviride, T. polysporum, T. oblongisporum, T. mediterraneum, T. trixiae, T. paraviridescens, T. viridescens, T. barbatum, and T. ivoriense), and 5 new species.
The present study performed the phylogenetic analysis of the ve new species of Trichoderma to establish their new status. Loci such as the ribosomal DNA internal transcribed spacer (ITS) region and partial genes encoding for the translation elongation factor 1-alpha (TEF1-α), endochitinase (chi18-5),  Bissett et al. (2015). Thus, only TEF1-α and RPB2 sequences were used for the phylogenetic reconstruction of the ve new species in the present study. The ITS sequences of the new species were submitted to Genbank but not used in phylogenetic analyses.

Materials And Methods
Isolates and specimens.
Specimens were collected from Sichuan, Yunnan, Beijing, Shandong, and Inner Mongolia. Trichoderma strains were isolated from soils on THSM (K 2 HPO 4 0.90 g; MgSO 4 ·7H 2 O 0.20 g; NH 4 NO 3 1.0 g; KCl 0.15 g; glucose 3.0 g; Rose Bengal 0.15 g; Agar 15.0 g; distilled water 1.0 L. Following autoclavation, chloromycetin (0.25 g), streptomycin (0.03 g), and pentachloronitrobenzene (0.2 g) were added) (Martin 1950  the random addition of sequences and tree bisection reconnection as the MP search method. All molecular characters were weighted equally and gaps were treated as missing data. Bootstrap proportions were calculated from 1000 replicates, each with 10 replicates of random addition of taxa.
Phylogenetic analyses showed that the Polysporum and Viride Clades containing the new species were well supported. Notes: Phylogenetically Trichoderma macrofasciculatum WT 37805 is related to T. polysporum represented by C.P.K. 3131 in the "Polysporum" clade ( Fig. 1), but the sequence similarities of TEF1-α and RPB2 between these species were only 93% and 96%, with 94 and 41 bp differences among 1311 and 1152 bp. Trichoderma macrofasciculatum cannot grow at 35 ℃ as T. polysporum, and the former formes large and white pustules in concentric rings at 25 ℃, elongations were rarely observed and conidia had few guttules, which are distinct from T. polysporum (Lu et al. 2004).
Trichoderma shangrilaense G.Z. Zhang, sp. nov. Fig. 7 MycoBank: MB 821300 Etymology: "shangrilaense" was originally found at Shangrila in Yunnan Province of China. Notes: Trichoderma shangrilaense is related to T. parapiluliferum (CBS 120921) in the Polysporum Clade (Fig. 1). The sequence similarity of TEF1-α between these two species is only 96%, but the sequence similarity of RPB2 between these two species was to 99%. The sequence similarity of TEF1-α with the extype culture G.J.S. 91-60 (GenBank Accession No. AY937444) was only 92%. Optimum temperature for growth of T. shangrilaense was 20 ℃, no growth occurred at 30 ℃ as in T. parapiluliferum, and conidiation structures consist of at or cushion-shaped pustules formed near the colony margin on MEA, SNA, and CMD. Trichoderma parapiluliferum, conidiophore main axis with conspicuous spiral sterile apical elongations, conidia ellipsoidal to oblong (Lu et al. 2004). Trichoderma shangrilaense, conidiophore main axis fertile to apex, conidia obovoid to ellipsoid, easily distinguished from that of T. parapiluliferum.

Discussion
In this paper, ve new species of Trichoderma were described from wetland soils. An ML tree was reconstructed based on individual TEF1-α and RPB2 and the combined TEF1-α + RPB2 dataset among 24 Trichoderma taxa to explore the taxonomic positions of the new species. Our phylogenetic analyses showed that the ve new Trichoderma species belong to the Polysporum Clade or the Virde Clade and form a ve branch in the ML or MP tree.
T. macrofasciculatum and T. shangrilaense belong to the Polysporum Clade (Figs. 1-3). This clade (formerly Trichoderma section Pachybasium) was rst de ned by Bissett (1991), including 20 species. However, molecular phylogeny has shown that it is paraphyletic Here, we added two new species, T. macrofasciculatum and T. shangrilaense, which are close to T. polysporum (MLBP = 100%) or T. parapiluliferum (MLBP = 100%). Morphologically, species in this clade are heterogeneous, comprising teleomorphs with upright, stipitate, or small pulvinate stromata. The teleomorphs of T. macrofasciculatum and T. shangrilaense have not been founded at present, but their asexual characteristics, such as conidiation in white pustules, resemble other species in this clade. T. nordicum, T. vadicola, and T. hailarense belong to the Virde Clade (formerly section Trichoderma) (Figs. 1-3). This clade contains one of the most di cult groups of species (Jaklitsch and Voglmayr 2015).
Morphologically, all asexual morphs have green conidia that are often warted. Up to now, this large clade has 69 species to be con rmed and described, among which 52 species have been well located in the six subclades (Hamatum/Asperellum, Koningii, Neorufum, Rogersonii, Viride, and Viridescens), and 17 species have not been located in the unnamed branches. Here, we added three new species, T. nordicum, T. vadicola, and T. hailarense, which are all located in the unnamed branches and close to T. paratroviride, T. caerulescens, T. gamsii, and T. neokoningii (MLBP = 92%, 97%, 84%). Phenotypically, the three new species have green conidia, only T. hailarense has coarsely warted conidia, and two other species are smooth-walled. Figure 1 Phylogenetic tree based on the maximum likelihood (InL=-8884.57) analysis of the TEF1-α dataset.

Declarations
Maximum likelihood bootstrap values (left) and MPBP (right) above 50% are indicated for branches. The tree is rooted with Protocrea illinoensis and Protocrea farinose. New species proposed here are indicated in boldface. Asterisks denote branches in con ict with the MP strict consensus tree.