New Species of Trichoderma Isolated as Endophytes and Saprobes from Southwest China

During the investigation of endophytic fungi diversity in aquatic plants and the fungal diversity in soil in southwest China, we obtained 208 isolates belonging to Trichoderma, including 28 isolates as endophytes from aquatic plants and 180 isolates as saprobes from soil, respectively. Finally, 23 new species of Trichoderma are recognized by further studies. Their phylogenetic positions are determined by sequence analyses of the combined partial sequences of translation elongation factor 1-alpha (tef1) and gene encoding of the second largest nuclear RNA polymerase subunit (rpb2). The results revealed that the 23 new species are distributed in nine known clades. The morphology and culture characteristics are observed, described and illustrated in detail. Distinctions between the new species and their close relatives were compared and discussed. These include: Trichoderma achlamydosporum, T. amoenum, T. anaharzianum, T. anisohamatum, T. aquatica, T. asiaticum, T. asymmetricum, T. inaequilaterale, T. inconspicuum, T. insigne, T. obovatum, T. paraviride, T. pluripenicillatum, T. propepolypori, T. pseudoasiaticum, T. pseudoasperelloides, T. scorpioideum, T. simile, T. subazureum, T. subuliforme, T. supraverticillatum, T. tibetica, and T. uncinatum.


Introduction
Trichoderma Pers, the anamorphic state of Hypocrea Fr. (Ascomycota, Sordariomycetes, Hypocreales), is an ecologically and economically important genus. According to the International Code of Nomenclature for algae, fungi, and plants [1], pleomorphic individuals no longer have more than one name, Trichoderma is now the legal name and Hypocrea is considered synonym [2]. Members of Trichoderma are widely distributed, having been found in various ecosystems, such as soil, decaying wood, plant leaves, bark and root systems, and also living as endophytes in plant tissues [3][4][5][6][7][8][9]. Many Trichoderma species have been used or encountered in many human activities, including as biocontrol agents of plant diseases, promoters of plant growth, enhancers of soil fertility in agriculture, producers of enzymes and antibiotics, processors of food, and handlers of pulp in industry [3,10,11]. In addition, they have great potential in soil and water pollution remediation, e.g., T. viride Pers. and T. atroviride P. Karst. are good bioremediators for some heavy metal ions [12,13]. Some Trichoderma species can also be used to manufacture gold or silver nanoparticles in nanotechnology [14,15]. However, the members of some species of the genus Trichoderma are the causal agents of diseases in commercially produced mushrooms, resulting in serious losses in mushroom production [16,17].
The genus Trichoderma, typified with T. viride Pers., was proposed as a genus by Persoon in 1794 [18], and originally included three other species based on the different colors of conidia. It has been proven that only T. viride remained in the genus. In 1871, Harz proposed the first accurate definition of Trichoderma, and emphasized the importance of microscopic characteristics, especially phialide. Subsequently, the first taxonomy system of Trichoderma species was put forward by Rifai in 1969, divided Trichoderma into nine species aggregates, mainly according to the morphological characteristics of conidiophores and phialides [19]. Since then, the number of Trichoderma species has increased dramatically. Currently, there are more than 340 species recognized in the genus (Index Fungorum, May 2021).
Most Trichoderma species were isolated as saprobes from soil. In comparison, there are also some Trichoderma species found as endophytes or isolated from aquatic ecosystems. Of these Trichoderma species, which isolated as endophytes, most belong to the Harzianum, Koningiopsis, and Hamatum clades. Previous studies investigated some Trichoderma species living as endophytes and found that Trichoderma species as innoxious endosymbionts were abundant in woody plants stems (vascular cambium and phloem), such as Cola spp., Herrania spp., Hevea spp., Theobroma spp. [20][21][22][23][24][25]. Trichoderma species in aquatic environment also showed biodiversity, especially in marine habitats. For example, Paz et al. [26] reported 10 Trichoderma species associated with Mediterranean sponges and Gal-Hemed et al. [27] isolated 29 Trichoderma strains from Mediterranean Psammocinia sp. sponges. However, endophytic Trichoderma species from freshwater ecosystems have not been reported.
Traditionally, taxonomic studies of members of the genus Trichoderma were based on morphology. However, as more and more novel species have been discovered, it has been difficult to distinguish them only by means of morphological observation because species in this genus are highly similar in morphology. For instance, most species in this genus usually grow fast, produce highly branched conidiophores, cylindrical to nearly subglobose phialides and ellipsoidal and globose conidia. Moreover, the morphological characters may change with different environmental conditions [28]. Therefore, the use of DNA sequence analysis became a new method in fungal phylogenetics and systematics. Of these, multi-locus molecular phylogeny enables the rapid and accurate identification of Trichoderma species, and a significant number of Trichoderma species have been reported based on molecular phylogenetic evidence [29][30][31][32][33].
China has enormous fungal diversity, with the Southwestern region in China assessed as one of the world's 34 biodiversity hotspots [34]. In recent years, we investigated the endophytic fungi diversity in aquatic plants in southwestern China, and obtained over 2000 isolates. After preliminary examination and classification by ITS (the internal transcribed spacer region) barcoding, 28 isolates were found to belong to Trichoderma. Furthermore, we also surveyed the fungal diversity in soil in Yunnan Province, and isolated 180 isolates belonging to Trichoderma species. Based on the multi-locus phylogenetic analysis and morphological features for all 208 isolates, 23 new species were recognized within the genus Trichoderma. This study significantly expands the worldwide diversity of Trichoderma and provides descriptions of new taxa.

Sample Collection
Soil samples were mainly collected from Yunnan Province, China, and placed into sterile, self-sealing plastic bags. Aquatic plants were collected from lakes, rivers, ponds, reservoirs and wetlands in provinces of Yunnan, Guizhou, Sichuan, and Tibet Autonomous Region. These plant samples were also placed into sterile self-sealing plastic bags. All the samples were transported to the lab and stored at 4 • C until processing.

Isolation of Fungi
For soil samples, soil fungi were isolated by gradient dilution and the spread plate method [35]. Three dilutions (10 −1 , 10 −2 , and 10 −3 ) were prepared with 10 g soil and 90 mL sterile water, 0.2 mL of each dilution was spread on a 90 mm Rose Bengal Agar (RBA) plates (Guangdong Huankai Microbial Sci and Tech, Guangzhou, Guangdong Province, China), supplemented with two antibiotics (0.5 g l −1 penicillin G and 0.5 g l −1 streptomycin) [36]. Then, these plates were cultured at 25 • C. After 3-5 days, single colonies were isolated into pure culture and grown on potato dextrose agar plates (PDA; 200 g potato, 20 g glucose, 18 g agar, 1 L distilled water). For plant samples, endophytic fungi were isolated by incubating surface-disinfected tissue segments (5 mm diam.) on RBA plates according to the method described by Zheng et al. [37]. These Petri dishes were sealed, incubated at 25 • C, and examined periodically. When fungi grew out from the tissue segment, a few hyphal fragments were picked up and transferred to PDA plates.
The pure cultures and dried cultures were deposited in the Herbarium of the Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University, Kunming, Yunnan, China (YMF), the China Center for type Culture Collection (CCTCC), and the China General Microbiological Culture Collection Center (CGMCC).

Growth Rate Determination and Morphology
Growth rates were determined on 9-cm-diameter Petri dishes of PDA, cornmeal dextrose agar (CMD; 40 g cornmeal, 20 g glucose, 18 g agar, 1 L distilled water) and synthetic low nutrient agar (SNA; 1 g KH 2 PO 4 , 1 g KNO 3 , 0.5 g MgSO 4 , 0.5 g KCl, 0.2 g glucose, 0.2 g sucrose, 18 g agar, 1 L distilled water) at 25, 30, and 35 • C under alternating 12 h light and 12 h darkness [38]. Colony diameters were measured after 3 days and the time when mycelia entirely covered the surface of plate was also recorded. Furthermore, the morphological characters of colonies, such as colony appearance, color, and spore production, were recorded at the same time. For microscopic morphology, photographs were taken with an Olympus BX51 microscope (Tokyo, Japan) connected to a DP controller digital camera. Microscopic characteristics were made from cultures growing on CMD or SNA at 25 • C. Colonies were photographed after 7 days and conidia were photographed after 14 days of incubation.

DNA Extraction, PCR Amplification and Sequencing
DNA was extracted from fresh mycelium harvested from PDA plates after 4 days, as described by Turner et al [39]. Fragments of the internal transcribed spacers (ITS), RNA Polymerase II subunit B (rpb2), and translation elongation factor 1-alpha (tef 1) were amplified with the following primer pairs: ITS4 and ITS5 for ITS [40], EF1-728F [41] and TEF1LLErev [42] for tef 1, and fRPB2-5f and fRPB2-7cr for rpb2 [43], respectively. Each 25 µL PCR reaction volume consisted of 12.5 µL T5 Super PCR Mix (containing Taq polymerase, dNTP and Mg 2+ , Beijing TsingKe Biotech Co., Ltd., Beijing, China), 1 µL of forward primer (10 µM), 1 µL of reverse primer (10 µM), 1µL DNA template, 5 µL of PCR buffer and 4.5 µL sterile water. PCR reactions were run in an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) following the PCR thermal cycle programs described by Chen & Zhuang [44]. PCR products were purified by using the PCR product purification kit (Biocolor BioScience and Technology Co., Shanghai, China), and forward and reverse sequenced on an ABI 3730 XL DNA sequencer (Applied Biosystems, Foster City, CA, USA) with the same primers, using a Thermo Sequenase Kit as described by Kindermann et al. [45]. These sequences were deposited in the GenBank database at the National Center for Biotechnology Information (NCBI) and the accession numbers are listed in Supplementary Table S1.

Phylogenetic Analyses
The phylogeny was reconstructed with sequences of rpb2 and tef 1. Most of the sequences analyzed here were obtained from GenBank based on previous publications [6,44,46], and the remaining sequences were obtained by BLAST searches. Both the reference sequences and new generated sequences in this study are listed in Supplementary Table S1. DNA sequence data of rpb2 and tef 1 were aligned using Clustal X 1.83 [47] with the default parameters, then the consensus sequences were manually adjusted and linked through BioEdit v.7.0 [48]. Manual gap adjustments were done to improve the alignment and ambiguously aligned regions were also excluded. We finally obtained the combined sequence matrix (Fasta file) generated by BioEdit v.7.0, containing 1777 nucleotide positions from two genes (761 from rpb2, 1016 from tef 1), and the matrix was uploaded to TreeBASE (www.treebase.org (accessed on 5 June 2021).; accession number: S28206).
Maximum likelihood (ML) analysis was computed by using RAxML [49] with the PHY files generated with CLUSTAL_X version 1.83 [47], using the GTR-GAMMA model. Maximum likelihood bootstrap proportions (MLBP) were also computed with 1000 replicates. Bayesian inference (BI) analysis was conducted by using MrBayes 3.1.2 [50] with the NEXUS file converted MEGA6 [51]. The Akaike information criterion (AIC) implemented in jModelTest version 2.0 [52] was used to select the best fit models after likelihood score calculations were done. TPM1uf + I + G was estimated as the best-fit model under the output strategy of AIC. Metropolis-coupled Markov chain Monte Carlo (MCMCMC) searches were run for 5,000,000 generations sampling every 500th generation. Two independent analyses with four chains each (one cold and three heated) were run until stationary distribution was achieved. The initial 25% of the generations of MCMC sampling were excluded as burn-in. The refinement of the phylogenetic tree was used for estimating Bayesian inference posterior probability (BIPP) values. The tree was viewed in TreeView 1.6.6 [53] with maximum likelihood bootstrap proportions (MLBP) greater than 70% and Bayesian inference posterior probabilities (BIPP) greater than 90%, as shown at the nodes.

Multi-Locus Phylogeny
To place these Trichoderma isolates, the sequences of rpb2 and tef 1 regions from 83 strains, representing nine clades of Trichoderma, were analyzed by the methods of ML and BI, with Protocrea pallida CBS 299. 78 and Protocrea farinosa CBS 121551 as the outgroup. The individual sequence datasets did not show any conflicts in the tree topologies for the 70% reciprocal bootstrap trees, which allowed the two genes for the multi-locus analysis to be combined. The ML analysis showed similar tree topology and was congruent with that obtained in the BI analysis ( Figure 1). However, the support values with BI analysis are relatively higher than the ML bootstrap support values.
In our phylogenetic analysis, the 40 selected isolates (in bold) were distributed among nine independent clades: three from the Longibrachiatum clade, ten from the Harzianum clade, one from the Virens clade, four from the Spirale clade, three from the Viridescens clade, one from the Viride clade, three from the Atroviride clade, eleven from the Hamatum clade, three from the Koningii clade, and one forming its own clade.
MycoBank MB 834556 Etymology. Latin, prope-, meaning near, -polypori, referring to the phylogeny affinities with Trichoderma polypori. Conidiophores straight or slightly curved, comprised developed main axis and paired branches at relatively regular intervals along the central axis, branches tended towards the conidiophore terminus in steep angles. Phialides ampulliform with sinuous, often constricted below the tip to form a narrow neck, less frequently solitary, often in whorls of 2-5, mostly inequilateral, equilateral only in central whorls, (  Notes: Trichoderma propepolypori is distinctive by the cotton-like aerial hyphae on PDA. Phylogenetically, T. propepolypori is closely related to T. polypori and T. longifialidicum. However, T. propepolypori can be easily distinguished by slower growth rate, shape of phialides, and size of conidia. The phialides of T. propepolypori are sinuously ampulliform phialides, while T. polypori is lageniform and subulate [44]. The conidia of T. propepolypori are much bigger than those of T. polypori, Conidiophores verticillium-like, straight or curved, mostly emerging in aerial hyphae, typically with 1-3 branching levels, branches orientating slightly towards the conidiophore terminus, the main branches often unpaired or irregular, the side branches simple, rebranching 1-2 times, replaced by a solitary phialide or paired phialides. Phialides ampulliform, sometimes with narrow cylindrical or slightly bent neck, solitary or in whorls of 2-4, symmetric or inequilateral, (5. Notes: Trichoderma pseudoasperelloides is phylogenetically close to T. asperelloides Samuels. They are similar in shape of the phialides and conidia. But for T. asperelloides, secondary branches tend to be paired, also commonly unilateral and consist of a single cell near the tip of the conidiophore, which cannot be represented in T. pseudoasperelloides for its fascicular phialides near the tip [65]. Conidiophores straight or curved, comprised a slightly curved main axis and generally verticillate branches, the main axis often terminating in a whorl of 2-3 divergent phialides, the base of branches about 2.1-4.3 µm wide, branches generally toward the top of main axis and sometimes sterile, terminating 1-3 divergent phialides, the distance between two neighbor branches 12.8-18.8 µm. Phialides commonly narrow lageniform, some ellipsoidal, slightly swollen in the middle, the necks of phialides sometimes curved, sometimes arose singly from the main axis or branches, (6.5-)6.8-12.7(-13.3) × 2.0-3.9 µm, l/w ratio (1.8-)2.1-4.5(-4.7). Conidia commonly globose to subglobose, a few ovoidal to ellipsoidal, hyaline, thin-wall, green, smooth, 3.3-4.4 × 2.4-3.8 µm, l/w ratio 1.0-1.7.  Notes: Trichoderma scorpioideum is phylogenetically closely related to two species: T. viridescens and T. sempervirentis Jaklitsch & Voglmayr, the branches of T. scorpioideum and T. viridescens are slightly curved, whereas the branches of T. viridescens is paired and often terminated in 1 or 2 phialides [59], which distinctly differs from verticillate branches of T. scorpioideum. As for the phialides of T. viridescens, it often forms a submoniliform chain of 2-6 cells when cultured on CMD [59], in contrast, the structure is inconspicuous on the phialides of T. scorpioideum. Furthermore, the conidia of T. viridescens sometimes are wider Conidiophores tree-like, formed densely intricate reticulum, main axis unrecognizable, mostly curved, integrated into the reticulum, side branches arising from main axis asymmetrically, perpendicular to the axis, some slightly orientated towards the conidiophore terminally, rebranching 1-3 times. Phialides varied, borne in regular levels around the axis, mostly paired arrangements or in whorls of 2-5, sometimes crowded at the stipe terminus, less commonly singly, straight or curved, ampulliform, less lageniform with long, symmetrical or slightly bent necks, (3.  Notes: Phylogenetically, Trichoderma simile is closely related to T. guizhouense. However, T. simile is distinguished from T. guizhouense by producing chlamydospores. Moreover, there are significant differences in size and shape of phialides and conidia, for instance, the phialides of T. guizhouense, (4.5-10 × 2.0-3.0 µm), are narrower than T. simile, (3.8-)4.3-11.9(-14.3) × (2.3-)2.7-3.9 µm, and T. guizhouense has globose conidia while the conidia of T. simile are oval [64].
Trichoderma Conidiophores straight or slightly curved, typically comprising 1-2 levels branched with phialides arising at the top in whorls of 2-4(-5), less commonly solitarily along stipes, with side branches short at the main axis terminus and much longer subjacent, branches paired or in whorls of 1-3, disposed perpendicular to the axis, some orientating slightly towards the conidiophore terminus. Phialides ampulliform to lageniform, with symmetrical or slightly curved necks, (4.7-)5. Conidiophores verticillium-like, phialides not formed directly around the axis, side branches emerging from the main axis, perpendicular relative to the stipe axis, or orientating slightly towards the conidiophore terminus, often paired or in verticils, also solitary, mostly straight. Secondarybranches appear, no tertiary branches noted. Phialides subuliform, sometimes long lageniform with long neck, symmetrical or slightly curved, in whorls   Notes: Trichoderma subuliforme is phylogenetically closed to T. spirale. In morphology, T. subuliforme has slenderer phialides than T. spirale, (2.9-)3. Conidiophores regularly tree-like, side branches from main axis typically paired, perpendicular to the axis at irregular intervals, some slightly orientating towards the conidiophore terminus. Stage 2 branches present, no stage 3 branches noted. Phialides formed mostly around the stipes at regular levels, in whorls of 3-4, paired or solitary directly on the main axis, ampuliform to spindly, homogeneous, straight, less curved or sinuous, with symmetrical or slightly bent long necks, (5.5-)7.5-11.5(-13.6) × (2. Notes: Trichoderma supraverticillatum forms a sole clade with relatively high statistical support. It can be distinguished by two types of conidia, which are ellipsoidal or obpyriform to obovoid with an apparent protuberance. Morphologically, T. supraverticillatum is similar to T. subuliforme with characteristics of conidiophores and phialides. However, T. subuliforme has oblong or ellipsoid conidia, and smaller than those of T. supraverticillatum,   Conidiophores consist of a discernible, slightly curved main axis and generally paired branches, the main axis usually terminated in three cruciform phialides, the branches often slightly upward and sometimes perpendicular to main axis, the distance between two neighbor branches 10.5-34.3 µm, base slightly swollen, about 2.4-3.6 µm wide, terminating in a whorl of 2-4 divergent phialides. Phialides usually lageniform, sometimes globose, ellipsoidal, or pyramidal, the neck of phialides sometimes hooked or degenerated, phialides around the tip sometimes arose singly from the main axis, (5.2-)5.9-11.8(-12.3) × (2.1-)2.3-3.5 µm, l/w ratio1.9-4.5 (-4.9). Conidia ellipsoidal to ovoid, subglobose rarely noted, green, smooth, 3.7-4.8 × (2.9-)3.1-4.0 µm, l/w ratio 1. Notes: Trichoderma tibetica phylogenetically belongs to the Koningii clade, and is closely related to T. petersenii Samuels, Dodd & Schroers. T. tibetica and T. petersenii showed no significant difference in the morphological features of conidiophores, which both species having a well-defined main axis and generally paired branches, and the phialides of two species being lageniform, sometimes cylindrical, and slightly swollen in the middle [55]. But the phialides of T. tibetica are sometimes globose or pyramidal, and sometimes hooklike on the bent neck, which is rarely noted in T. petersenii. The conidia of T. tibetica are mostly ellipsoidal, sometimes ovoidal or subglobose, which distinguishes them from the generally ellipsoidal conidia of T. petersenii, and the conidia of T. petersenii are commonly smaller than those of T. tibetica (3.5-4.5 × 2.7-3.0 µm). In addition, no conidia formed on CMD for T. tibetica, only vegetative hyphae and pigment were obviously noted, but the conidia of T. petersenii appeared within 7 days on CMD [55]. Conidiophores comprised a hard-discernable, slightly curved main axis, irregular alternate branches, the distance between two neighbor branches of 11.2-26.2 µm, base sometimes swollen, about 2.6-4.2 µm wide, the main axis often terminating in two divergent phialides, every branch often terminating in a whorl of 2-5 phialides. Phialides commonly lageniform, sometimes ampulliform to subglobose, sometimes sterile, sometime the neck of phialides uncinate or constricted sharply, rarely single, (4.3-)5.2-9.3(-10.3) × 2.3-3.9 µm, l/w ratio 1.3-3.6. Conidia globose to subglobose, rarely ovoidal or ellipsoidal, thin-walled, green, smooth, 3.  Notes: Trichoderma uncinatum is close to T. paratroviride in the phylogenetic tree. Morphologically, they share some characters, such as spindly branches generally toward the tip, lageniform to ampulliform phialides sometimes with curved necks, and subglobose conidia [46]. However, the distance between two neighboring branches in T. paratroviride is generally longer, the phialides of T. paratroviride are commonly in whorls of 2-4 vs. in whorl of 2-5 in T. uncinatum, and the phialides are spindlier (6.2-11 × 2.5-3.2 µm) than in T. uncinatum.

Discussion
China is considered an important reservoir of Asian biodiversity; it is estimated that this area harbours an inestimable diversity of fungi. The genus Trichoderma serves as a good example. The known species of the genus in China occupy 40% of the world records. Sixty-two new Trichoderma species have been reported since 2017 [29,30,32,44,66], which is evidence that China has tremendous Trichoderma diversity. Of these 62 species, 20 were discovered in southwest China, including 14 from soils, 4 from rotten woods, and 2 as endophytes from plants. For the past few years, soil has been an important substrate for investigating Trichoderma species, and studies focused on soil-inhabiting species of the genus have been carried out by different researchers around the world [44,46,63,67,68]. In fact, the number of Trichoderma species will continue to increase because many other habitats in China have not yet been investigated in a large scale. The biodiversity of Trichoderma on aboveground habitats may exceed that from the soil.
In recent years, we have been investigating the fungal diversity in southwest China, including in soils, submerged leaves, and aquatic plants, and described many new taxa [32,35,37,[69][70][71][72][73][74][75][76][77][78][79]. In our studies, the endophytic microbiota found in these studies only collected infrequent Trichoderma isolates, which obtained over 2000 isolates but only 28 isolates belong to the genus Trichoderma. Conversely, the soil samples in these study yielded 180 isolates of Trichoderma, with a highly diverse taxonomic range. Finally, 23 new Trichoderma species are described herein, combining a phylogenetic analysis and morphological features.
The 23 new species were assigned to nine clades in Trichoderma based on the two-gene phylogenetic tree, which are the Longibrachiatum, the Haizianum, the Virens, the Spirale, the Koningii, the Atroviride, the Viridescens, the Viride, and the Hamatum, respectively. However, the new species T. supraverticillatum forms phylogenetically lone lineages, and is distantly related to any other clade.
Of these 23 new species, Trichoderma pluripenicillatum and T. aquatica were grouped into the Longibrachiatum clade with strong support values, and their morphology also showed compelling supports in this clade as revised by Samuels et al. [6], which Trichoderma species in this clade are typically growing well and sporulation at 35 • C, and often produce diffusing yellow pigments on PDA. T. pluripenicillatum and T. aquatica produce yellow pigments on PDA at three temperatures, and sporulate abundantly at 30-35 • C. In addition, the conidia of this clade are typically ellipsoidal to oblong and less subglobose; T. aquatica fits the feature. However, T. pluripenicillatum has morphological differences from other species of the Longibrachiatum clade. T. pluripenicillatum produces ampuliform phialides, differing from the generally lageniform phialides of the other members of the Longibrachiatum clade [6].
Six new species, Trichoderma achlamydosporum, T. propepolypori, T. simile, T. anaharzianum, T. asiaticum, and T. pseudoasiaticum, belong to the Harzianum clade, which was reported by Chaverri & Jaklitsch [56] and had been specialized in multifarious morphology and complicated phylogeny. Members of the Harzianum clade usually form diverse pustules in culture, with different conidiophore types, phialide shapes, and varied conidia [57,67]. So far, the Harzianum clade, which includes over 40 species, is the largest clade of the green-spored species groups. Previously, T. polypori was phylogenetically related to T. velutinum Bissett, C.P.
Kubicek & Szakács, but it is closely related to the new species T. propepolypori in this study. T. guizhouense was shown to have a close relationship with T. harzianum in previous studies, but is now clustered with T. simile, which forms a separate clade nearing to T. harzianum and T. anaharzianum. Both T. asiaticum and T. achlamydosporum form a separate subclade in the Harzianum clade, and differ morphologically from other species in the clade.
The new species Trichoderma inaequilaterale was identified as a member of the Virens clade, which also belongs the group of green-spored species in Trichoderma, based on morphology and phylogeny. Consistent with previous studies [57], T. crassum and T. virens form a separate clade in the Virens clade. The general characteristics of species in this clade have a rapid growth rate and gliocladium-like conidiophores.
Two new species, Trichoderma subuliforme and T. subazureum, belong to the Spirale clade, which is newly established by Chen and Zhuang [44] to accommodate three Trichoderma species, T. hunanense, T. longisporum K. Chen & W.Y. Zhuang and T. spirale. However, the phylogenetic position of T. spirale was variable. Early research by Chaverri and Samuels found that T. spirale was closely related to T. polysporum Rifai in the Polysporum clade [57]. Later, T. spirale was assigned to the Strictipile clade and found to be closely related to T. longipile Bissett and T. strictipile Bissett in Jaklitsch's study [67]. Jaklitsch and Voglmayr described T. spirale as a separate terminal branch [46]. Based on recent study by Chen and Zhuang [44], species in the Spirale clade share resemble characteristics: forming downy pustules, producing yellow pigments in culture, and having oblong conidia. T. subuliforme and T. subazureum also share these characteristics with other species of the clade.
The new species T. paraviride belongs to the Viride clade, which is the largest and most diverse group of the genus Trichoderma. Species in this clade can be isolated from diverse sources with a wide geographic distribution [80]. The Viride clade was originally under the name "section Trichoderma" including the type species of the genus, T. viride [81]. Samuels et al. [55] treated them in the T. koningii aggregate based on the combined phenotypic and molecular data, then, Jaklitsch et al. [60] disentangled the complex. Afterwards, Jaklitsch and Voglmayr renamed the clade the Viride clade and provided an updated comprehensive phylogenetic tree [46]. Species in this clade mostly produce trichoderma-, verticilliumor pachybasium-like conidiophores with paired, verticillate phialides and green conidia.
Considering the significance of Trichoderma species in industry, agriculture, and ecology, the diversity and taxonomy of Trichoderma species are being investigated and studied by more and more researchers. The taxonomy Trichoderma had been studied by Samuels et al. [55], Jaklitsch [63], Jaklitsch and Voglmay [46], Chen and Zhuang [44], etc. In addition, extensive molecular studies in recent years have rapidly added new species to the genus. The taxonomy of Trichoderma in China dates back to 1895 [82]. Over more than a century, successive findings have brought the number of known species of the genus in China up to over 100, and species of the genus are located throughout the country. For instance, these 75 wood-inhabiting species have been found in Anhui, Fujian, Guangdong, Guangxi, Guizhou, Hainan, Hebei, Henan, Hubei, Hunan, Jiangsu, Jiangxi, Jilin, Liaoning, Shandong, Sichuan, Taiwan, Yunnan, Zhejiang provinces and Tibet region in China.
Our previous studies on the endophytic diversity of fungi in aquatic plants from southwest China found that there is abundant fungal diversity in aquatic plants. However, the isolated frequency of Trichoderma species is very low. Previous studies on Trichoderma as endophytes in terrestrial plants, particularly in their original wild to semi-wild situation, showed a considerable diversity of species. Notable examples are in Theobroma cacao [9,68,83] and Hevea brasiliensis [22,84]. Therefore, we speculate that the diversity of Trichoderma species in terrestrial environments is more abundant than in aquatic environments, and we will continue to study Trichoderma species in terrestrial plants in southwest China.
In summary, it is not easy to identify Trichoderma species, and impossible to define or recognize a species solely based on morphology, especially when the sexual state is absent. Now, the identification of Trichoderma species mainly depends on morphology, including micromorphological and cultural characters, and phylogeny. More and more DNA fragments are available for Trichoderma species, such as ITS, tpb2, tef 1, and ACT. Of these gene fragments, ACT was introduced to study the genus, and has turned out to be efficient [60]. In the future, the species concepts of Trichoderma may be firmly established with the application of phylogenetic analyses at genomic level. Furthermore, the information on the ecology of the Trichoderma species and their function is still limited because there are many unexplored areas in China and other countries. Although our study only revealed a handful of Trichoderma species in the southwest region of China, our knowledge of Trichoderma will also provide useful information for the sufficient utilization of fungal resources. Further studies are required to understand the potential diversity of Trichoderma in southwest China, especially extensive surveys of unexplored areas.

Data Availability Statement:
The data presented in this study are available in Supplementary Table S1.