Species Diversity of Trichoderma Associated With Soil In The Zoige Alpine Wetland of Southwest China

Background: The Zoige alpine wetland is one of the most important wetlands in China because of its complex natural environment, abundant ecological resources, and unique climatic conditions. The ecology of soil fungi is poorly understood, and recent comprehensive reports on Trichoderma are not available for any region, including the Zoige alpine wetland ecological region in China. Our results may be used as a reference for a greater understanding of soil microorganism at various ecological regions, ecological rehabilitation and reconstruction and as microbial resources. Results: One hundred soil samples were collected from different soil types and soil layers in Zoige alpine wetland ecological regions in 2013. Using the traditional suspension plating method, a total of 80 Trichoderma strains were isolated. After a preliminary classication of morphological characteristics and the genes glyceraldehyde-3-phosphate dehydrogenase (gpd), 57 strains were representatively selected and eventually identied as seven species via phylogenetic analyses of multilocus sequences based on the genes transcription elongation factor 1 alpha (tef1), encoding RNA polymerase II subunit B (rpb2) and ATP citrate lyase (acl1). Among them, Trichoderma harzianum was the dominant species and had the highest isolation frequency (23%) in this zone, while Trichoderma polysporum and Trichoderma pyramidale were rare species, with isolation frequencies of less than 1%. Conclusions: Our detailed morphological observation and molecular phylogenetic analyses support the recognition of Trichoderma zoigense was described for the rst time as a new species. 30-day-old length and width of conidia (n = 50), length of phialides (n = 50), width of phialides at the base (n = 50), and width of phialides at the widest point (n = 50). Nomarski differential interference contrast (DIC) was used for observations and measurements, and were with showed all had different growth rates but were differentiated from each other on CMD. These were roughly divided into four groups based on their optimum growth temperature. soil in the Zoige alpine ecological region of Southwest China, morphological characteristics and multilocus phylogenetic analyses were performed to identify 80 strains as T. harzianum (48 strains, 60%), T. alni (15 strains, 18.75%), T. zoigense (a new species, 8 strains, 10%), T. rossicum (4 strains, 5%), T. atrobrunneum (3 strains, 3.75%), T. polysporum (1 strain, 1.25%) and T. pyramidale (1 strain, 1.25%). This is the rst comprehensive report on the population structure of Trichoderma in the Zoige alpine wetland. A specialized analysis of Trichoderma from 100 soil samples shows high richness of the Trichoderma species in this region and indicates the presence of latent resources that require further study, such as new species. surface reverse-diffusing to pale brown hyphae are numerous to punctate and long, forming radial strands, with white mycelial patches appearing in aged Autolytic excretions are rare, with no coilings observed. Conidiation noted 3–4 d at 25 ℃ , a yellow or greenish color appears after 7 d, conidiation is effuse and in intense tufts, erect conidiophores occur around the plug and on aerial hyphae, and they are mainly concentrated along the colony center, show a white color that turns green, and then nally degenerate, with conidia often adhering in chains. Conidiophores are short and simple with asymmetric branches. Branches produce phialides directly. Phialides are generally solitary along main axes and side branches and sometimes paired in the terminal position of the main axes, sometimes in whorls of 2–3. Phialides are × 2–5 µm ( x̄ 7.5 ± 1.5 × 3 ± n and 1.5–2.5 µm ( x̄ 2 ± 0.2) wide at the base, lageniform or ampulliform, mostly uncinate or slightly curved, less straight, and often distinctly widened in the middle. Conidia are 3–4.5 × 2.3–4 µm ( x̄ = 3.5 ± 0.3 × 3 ± 0.3, n = 50) and initially hyaline, green guttules,

for any region, including the Zoige alpine wetland ecological region in China. In fact, only Feng et al. (2009) has analyzed the fungal community structure in the soil of this region via a combination of BIOLOG analysis and traditional culture methods. Because morphological and molecular tools are ideal for assessments of the species diversity in all geographical regions, the work described here was designed to investigate the species diversity of the genus Trichoderma in the uniquely ecological environment of the Zoige alpine wetland, with an emphasis on four major soil types (peat soil, meadow soil, subalpine meadow soil and aeolian sandy soil). Our results may be used as a reference for a greater understanding of soil microorganism at various ecological regions, ecological rehabilitation and reconstruction and as microbial resources.

Study region
The Zoige alpine wetland (32°10′~34°10′N, 101°45′~103°55′E) is located in the northwest part of Sichuan Province in China and the eastern edge of the Qinghai-Tibet Plateau and has an average altitude of 3400 m above sea level and an area of 19600 km 2 . It is a relatively pristine natural area with an annual temperature of 0.6-1.0 ℃ and annual precipitation level of 580-860 mm. The cold, humid weather slows the decomposition of the soil organic matter and facilitates its accumulation in the soil (Sun 1998;Ding et al. 2004;Feng 2009). Peat soil, meadow soil, subalpine meadow soil and aeolian sandy soil are extensively developed and the most common soil types in this area, because of its unique ecological conditions.

Isolates and specimens
A total of 100 soil samples were collected in June 2013 across a range of soil types (peat soil, meadow soil, subalpine meadow soil and aeolian sandy soil) and soil layers (depth 0-10, 10-20, 20-30, 30-50, and 50-100 cm) in the Zoige alpine wetland ecological regions. Global positioning system technology (GPS Map 76; Garmin Ltd, USA) was used to determine the sampling locations. After removal of vegetation debris, approximately 300 g of each soil sample was immediately placed in a sterile plastic bag in a cooler, transported to the laboratory within 48 h and then stored at 4 ℃.
Soil fungi were isolated using the suspension plating method (Mueller et al. 2011). Brie y, suspensions (1 mL) of various dilutions (10 − 1 , 10 − 2 and 10 − 3 ) were placed on 90 mm diameter petri plates and Martin medium was then added and mixed evenly with the suspension. The plates were kept in the dark at 25 ℃ for 5 d, and the colonies of fungi were observed and counted. Three replicates were performed for each concentration. According to the colony characteristics, the puri ed fungal colonies were transferred onto potato dextrose agar (PDA) and kept in tube slants and glycerol for further taxonomic identi cation. The specimens were deposited in the Fungal Herbarium of Sichuan Agricultural University, with accession numbers of T1-T80. And the holotype of new species and new record species were deposited in China General Microbiological Culture Collection Center (CGMCC), with accession numbers of CGMCC3.20145 and CGMCC3.20167.
Fungal colony characteristics were observed on the CMD, PDA, MEA and SNA media and grown under 12 h of white light and 12 h of darkness at 20 ℃ and 25 ℃. Colony textures and the presence or absence of exudates were recorded using a stereomicroscope (OLYMPUS SZX16, Japan). Colony morphologies were observed weekly with a digital camera (Nikon D3100, Japan). Micromorphological characteristics were observed after 3-7 d or 14 d of cultivation, and microscopic observations were performed in 3% KOH. Chlamydospores were measured from 7-30-day-old cultures on CMD or SNA plates under a compound microscope using a 100× objective. The following characteristics of each isolate were measured: length and width of conidia (n = 50), length of phialides (n = 50), width of phialides at the base (n = 50), and width of phialides at the widest point (n = 50). Nomarski differential interference contrast (DIC) was used for observations and measurements, and data were gathered using a Carl Zeiss microscope (Axio Imager Z2, Germany). Colors were determined with Methuen's Handbook of Colour (Kornerup & Wanscher 1981).
To identify the optimal growth temperature and differentiate growth rates of the species, 3 representative strains or all strains (≤ 3 in total) for each species were selected to determine the growth rate on CMD at ve temperature levels (15 ℃, 20 ℃, 25 ℃, 30 ℃ and 35 ℃) as described previously with minor modi cations (Jaklitsch 2009). The strains were pre-grown on PDA for 48 h or 72 h at 25 ℃. For new cultures, 5-mm agar blocks were cut from the margin of the colonies and transferred to fresh medium from the edge of the 9-cm petri dish. The maximum colony radius was measured every day until the plates were entirely covered with mycelium. The growth rate was calculated by linear regression of t versus r (t = time of incubation and r = radius measured from the edge of the agar plug). Every treatment was repeated twice, with three replicates each time.

Phylogenetic analyses
For approximate identi cation, all sequences of the 57 strains listed in Table 2 were compared with the NCBI sequence database using the BLAST algorithm. The two markers (ITS and gpd) sequenced in the present study were analyzed separately. Their closest matches were aligned by ClustalX (Thompson et al. 1997), and a distance tree was built with the neighbor-joining (NJ) algorithm in MEGA v. 6.0 with 1000 bootstrap replicates (Tanaka et al. 2009;Tamura et al. 2011). Combined rpb2, tef1 and acl1 gene sequences were analyzed based on a multilocus dataset. A phylogenetic analysis was performed for the sequences of a total of 101 strains obtained from the present study or other references in previous studies and complemented with GenBank sequences (Jaklitsch 2009; Jaklitsch & Voglmayr 2015) ( Table 2).
The strains from this study are indicated in bold letters. (T = ex-type). T42, T44, T48 were deposited in China General Microbiological Culture Collection Center (CGMCC), and the rest in the Fungal Herbarium of Sichuan Agricultural University.
Maximum parsimony (MP) analyses of the combined DNA matrix was performed with PAUP* v. 4.0 b10 (Swofford 2002) using 1000 replicates of a heuristic search with the random addition of sequences. All molecular characteristics were unordered and given equal weight, and all gaps were treated as missing data.

Relationship with ecological factors
The isolation frequency was calculated at the species level using the following formula: where F = the isolation frequency (%), n = the number of species isolated from soil samples, and N = the number of total soil samples. The relationships between the isolation frequency and soil types and soil layers were subsequently analyzed.

Trichoderma species collection
A total of 80 strains were obtained from 100 soil samples collected from Zoige alpine wetland ecological regions in China. Details of the strains isolated from soil samples are given in Table 1. All strains were subsequently used for morphological identi cation, while fty-seven were used for phylogenetic analysis.

Phylogenetic analysis
The ITS region used preliminarily as a species identi cation criterion was applied to TrichOKey at www.ISTH.info ). However, the ITS region has a low number of variable sites and long insertions in certain species, thus, it is not suitable for a phylogenetic reconstruction of this group ). In our study, most fragments of the genes tef1, rpb2 and acl1 were successfully ampli ed. We also designed a pair of new primers based on the full-length tef1 gene, 5'-GAGAAGTTCGAGAAGGTGAGC-3' and 5'-ATGTCACGGACGGCGAAAC-3', with which a 1.4-kb fragment was ampli ed for most isolates.
All samples analyzed in our study were divided into 4 primary clades based on the gpd gene region, including 49 strains from the Trichoderma harzianum complex, 3 Trichoderma rossicum strains, 1 Trichoderma polysporum strain and one unknown species (4 Trichoderma sp. strains) (Fig. 1). Maximum parsimony analysis was conducted among 101 strains, with Protocrea farinosa (CPK 2472) and P. pallida (CBS 299.78) used as outgroup ( Table 2). The dataset for the rpb2, tef1 and acl1 genes contained 3403 characteristics, among which 1152 were parsimonyinformative, 988 were variable and parsimony-uninformative, and 1263 were constant. The most parsimonious trees are shown in Fig. 2

Growth rates
As shown in Fig. 3, the genus Trichoderma from Zoige alpine wetland ecological regions was able to grow in a range from 15-35 ℃, and the suitable growth temperature for most species ranged from 20 ℃ to 30 ℃. All seven species identi ed had normal viability at relatively low temperature (15 ℃), and they rarely grew well over 35 ℃ except for T. zoigense. For T. atrobrunneum, T. harzianum and T. pyramidale, the optimum growth temperature on CMD was 25-30 ℃. Trichoderma alni and T. rossicum preferred a cool growth environment, with an optimum temperature of 25 ℃, whereas T. zoigense was more partial to a hot environment, with an optimum temperature of 30 ℃, and it even grew well up to 35 ℃. T. polysporum was the only slow-growing species that grew with less than 6.0 mm/d between 15 ℃-30 ℃ and did not survive at 35 ℃. The above results showed that all species had different growth rates but were not completely differentiated from each other on CMD. These species were roughly divided into four groups based on their optimum growth temperature.

Relationship with ecological factors
Our results revealed a substantial disparity in the number and distribution of Trichoderma species among Zoige alpine wetland ecological regions (Tables 3, 4). Table 3 showed that T. harzianum was found in all four soil types, but most isolates of this species were obtained from peat soil. T. rossicum, T. alni and T. zoigense were also present in meadow soil and subalpine meadow soil, whereas T. atrobrunneum was found in aeolian sandy soil and peat soil. T. polysporum was found only in peat soil. T. rossicum  Table 4, T. harzianum was widely distributed in the ve soil layers at depths of 0-100 cm. T. rossicum, T. alni and T. zoigense were isolated mainly from the soil layers at depths of 0-50 cm. Both T. atrobrunneum and T. pyramidale were only isolated from depths of 0-10 cm, and T. polysporum was found only in the soil layers at depths of 50-100 cm.
Regarding isolation frequency, T. harzianum was the most common of the seven species with a 23% isolation frequency, and it was therefore the dominant species in the zone, while the rare species T. polysporum and T. pyramidale had the lowest isolation frequencies at 1%.

Discussion
To characterize the biodiversity and establish the species composition of Trichoderma associated with soil in the Zoige alpine wetland ecological region of Southwest China, morphological characteristics and multilocus phylogenetic analyses were performed to identify 80 strains as T. harzianum (48 strains, 60%), T. alni (15 strains, 18.75%), T. zoigense (a new species, 8 strains, 10%), T. rossicum (4 strains, 5%), T. atrobrunneum (3 strains, 3.75%), T. polysporum (1 strain, 1.25%) and T. pyramidale (1 strain, 1.25%). This is the rst comprehensive report on the population structure of Trichoderma in the Zoige alpine wetland. A specialized analysis of Trichoderma from 100 soil samples shows high richness of the Trichoderma species in this region and indicates the presence of latent resources that require further study, such as new species.
Although many studies have focused on the identi cation of Trichoderma, identifying Trichoderma species based on only morphological characteristics remains di cult. Ampli cation of four universal fungal genes, gpd, acl1, rpb2 and tef1, showed that the gpd gene could be used to divide approximately the 57 representative strains into 4 clades, which were exactly aligned with the previous 4 morphological groups. The gpd gene was suitable for categorizing large groups but was not useful for the accurate identi cation of speciation within the Trichoderma complex (Druzhinina et al. 2010). In fact, any single gene among acl1, rpb2 and tef1 can play an important role in the identi cation of Trichoderma species but cannot accurately distinguish Trichoderma at the species level. Notably, although the primer pair EF1-728F and TEF1LLErev for tef1 was useful, it did not always successfully amplify all tested DNA materials. Admittedly, there are many factors affecting PCR ampli cation, not all of which can be attributed to primers, among which the quality of DNA may also be one of the factors. Phylogenetic studies of many species have proven that the most accurate method of species identi cation is to combine phylogenetic analysis with morphological phenotypic characteristics. In this study, when the genes acl1, rpb2 and tef1 were used in multilocus phylogenetic analysis, the phylogenetic relationships among taxa were consistent with those identi ed in previous studies in which the phylogenetic tree was built based on the genes rpb2 and tef1 either singly or in combination ( We found that the Longibrachiatum clade contained a new species, T. zoigense, which was phylogenetically distinct from any other species of Trichoderma (Fig. 2) and provided a low level of support for relationships with T. citrinoviride (C.P.K. 2005) and T. saturnisporum (ATCC 18903) (Fig. 2, MPBP = 62%). Compared to their morphological characteristics of the above two species, T. zoigense was di cult to distinguish from T. citrinoviride and T. saturnisporum by colony and spores. However, T. zoigense was able to produce yellow pigment dispersion and a fragrance in all tested media and easily produces chlamydospores (Samuels et al. 1998 The results of our studies demonstrated signi cant differences in the abundance and distribution of Trichoderma species isolated in the Zoige alpine wetland natural region. T. harzianum showed the highest abundance among the species isolated from ve soil layers and four soil types, implying that this species had good adaptability and can survive under most environmental conditions. Only T. polysporum was isolated at a soil depth of 50-100 cm, indicating that it prefers to live in a low-temperature environment (Domsch et al. 2007). In general, it is assumed that some Trichoderma species have stricter requirements for the growth environment and, thus, a narrower range for survival ).  Etymology: zoigense (Latin), the speci c epithet in reference to the place where the type was found.

Conclusion
Description: Cultures and anamorph: optimal growth at 25 ℃ on all four media. On CMD after 72 h, growth is 25-28 mm at 20 ℃ and 28-31 mm at 25 ℃. Colony is dense and has a wavy to crenate margin. Surface becomes distinctly zonate and white to grayish-green but celadon to atrovirens later, and it is granular in the center and distinctly radially downy outside and shows whitish surface hyphae and reverse-diffusing croci to pale brown pigment. Aerial hyphae are numerous to punctate and long, forming radial strands, with white mycelial patches appearing in aged cultures. Autolytic excretions are rare, with no coilings observed. Conidiation noted after 3-4 d at 25 ℃, a yellow or greenish color appears after 7 d, conidiation is effuse and in intense tufts, erect conidiophores occur around the plug and on aerial hyphae, and they are mainly concentrated along the colony center, show a white color that turns green, and then nally degenerate, with conidia often adhering in chains. Conidiophores are short and simple with asymmetric branches. Branches produce phialides directly. Phialides are generally solitary along main axes and side branches and sometimes paired in the terminal position of the main axes, sometimes in whorls of 2-3. Phialides are 4.5-10.5 × 2-5 µm (x̄ = 7.5 ± 1.5 × 3 ± 0.5, n = 50) and 1.5-2.5 µm (x̄ = 2 ± 0.2) wide at the base, lageniform or ampulliform, mostly uncinate or slightly curved, less straight, and often distinctly widened in the middle. Conidia are 3-4.5 × 2.3-4 µm (x̄ = 3.5 ± 0.3 × 3 ± 0.3, n = 50) and initially hyaline, and they turn green and are oblong or ellipsoidal, almost with constricted sides, and smooth, eguttulate or with minute guttules, with indistinct scars.
On PDA, after 72 h, growth is 35-41 mm at 20 ℃ and 50-55 mm at 25 ℃; and mycelium covers the plate after 5 d at 25 ℃. Colonies are dense with wavy to crenate margins; and mycelia are conspicuously differentiated in width of the primary and secondary hyphae. Surface becomes distinctly zonate, yellowish-green to prasinous in color and celadon to atrovirens later, and it is farinose to granular in the center, distinctly radially downy outside, with whitish of surface hyphae and reverse-diffusing brilliant yellow to fruit-green pigment. Aerial hyphae are numerous, long and ascend several millimeters, forming radial strands, with white mycelial patches appearing in aged cultures. Autolytic excretions are rare; and no coilings are observed. Odor is indistinct or fragrant. Chlamydospores examined after 7 d at 4.5-9 × 4.5-7.5 µm (x̄ = 6 ± 1.1 × 6 ± 0.7, n = 50), and they are terminal and intercalary, globose or ellipsoidal, and smooth. Conidiation is noted after 3-4 d and yellow or greenish after 7 d. Conidiophores are short and simple with asymmetric branches. Phialides are similar to CMD. Conidia are greenish, ellipsoidal, and smooth.
On SNA, after 72 h, growth is 13-15 mm at 20 C and, 16-21 mm at 25 ℃; and mycelium covers the plate after 12-13 d at 25 ℃. Colony is similar to that on CMD, with a little wave margin, although mycelia are looser and slower on the agar surface. Aerial hyphae are relatively inconspicuous and long along the colony margin. Autolytic activity and coiling are absent or inconspicuous. No diffusing pigment or distinct odor are produced. Conidiation noted after 3-4 d at 25 ℃, and many amorphous, loose white or aqua cottony tufts occur, mostly median from the plug outwards, and they are con uent to masses up and white but then turn green. From the inside after 4-5 d, conidiation becoming dense within the tufts, which are loose at their white margins with long, straight or slightly sinuous sterile ends in the periphery. Tufts consisting of a loose reticulum with branches often in right angles give rise to several main axes. Main axes are regular and tree-like, with few or many paired or unpaired side branches. Branches are exuous and phialides are solitary along the main axes and side branches, and they are sometimes paired in terminal position of the main axes, sometimes in whorls of 2-3 that are often cruciform or in pseudo-whorls up to 4. Phialides and conidia are similar to that on CMD. often constricted below the tip to form a narrow neck of 4.5-8 × 2-3.5 µm (x̄ = 6 ± 0.8 × 2.5 ± 0.3, n = 50) and 1-2.5 µm (x̄ = 2 ± 0.3) wide at the base. Conidia are subglobose to ovoid, 3-4.5 × 2.5-3.3 µm (x̄ = 3.5 ± 0.3 × 3 ± 0.2, n = 50), laurel-green to bright green, smooth, and ellipsoidal. Description: Cultures and anamorph: optimal growth at 25 ℃ on all media, with little growth at 35 ℃. On CMD, after 72 h, growth is 29-32 mm at 20 ℃ and 48-53 mm at 25 ℃; and mycelium covers the plate after 5-6 d at 25 ℃. Colonies show distinct zonation. Mycelium is loose and thin; hyphae are narrow, sinuous and often form strands on the margin. Aerial hyphae are slight, form a thin white to green downy, uffy or occose mat. Brown pigment is shown, but no distinct odor noted. Conidiophores are hyaline and thick with side branches on several levels at the base of the elongations that are mostly paired and in right angles with phialides in whorls of 3-5. Phialides are 5-9.5 × 2.5-3 µm (x̄ = 7 ± 1.1 × 3 ± 0.3, n = 50) and 1-2.5 µm (x̄ = 1.5 ± 0.3) wide at the base and often short, wide, and ampulliform. Conidia are 2.5-4 × 2.5-3.5 µm (x̄ = 3.5 ± 0.3 × 3 ± 0.2, n = 50), green, smooth, and ellipsoidal.
On SNA, after 72 h, growth is 33-35 mm at 20 ℃ and 38-40 mm at 25 ℃; and mycelium covers the plate after 7-8 d at 25 ℃. Colonies show distinct zonation. Mycelium is thin, yellow to green; hyphae are wide, sinuous, with indistinct strands on the margin. Margin is thin and ill de ned. Aerial hyphae are slight and form a thin white downy, uffy or occose mat in distal parts of the colony. No diffusing pigment or distinct odor noted. Conidiation similar to CMD. Description: Cultures and anamorph: optimal growth at 25 ℃ on all media. On CMD, growth of 10-11 mm/d at 20 ℃ and 15-17 mm/d at 25 ℃; and mycelium covers the plate after 6-7 d at 20 ℃. Colony is dense with a wavy margin, and the surface becomes distinctly zonate. Aerial hyphae are numerous, long, and villiform in the plate. No diffusing pigment or odor. Autolytic activity is variable, and coilings are scarce or inconspicuous. Conidiation noted after 3-4 d at 20 ℃. Conidiation is effuse and in intense tufts that are hemispherical or irregular, and they show wide wheel grain banding that is gray green to deep green. Conidiophores radiate from the reticulum and are broad, straight, sinuous or helically twisted, show distally slightly pointed elongations, taper from the main axes to top branches, and present primary branches arranged in pairs or in whorls of 2-3, with secondary branches to solitary. Phialides are 4.5-14 × 2.5-4 µm (x̄ = 7 ± 1.5 × 3.5 ± 0.3, n = 50) and 2-3.5 µm (x̄ = 3 ± 0.4) wide at the base, ampulliform, and in whorls of 3-6. Conidia are 3.5-5.5 × 2.5-4 µm (x̄ = 4.5 ± 0.5 × 3 ± 0.2, n = 50), short cylindrical, and a gray color when single and pea green to yellow green in a group.
On SNA, growth is 8-13 mm/d at 20 ℃ and 8-12 mm/d at 25 ℃; and mycelium covers the plate after 6-7 d at 25 ℃. Colony is hyaline, thin and dense; and mycelium degenerate rapidly. Aerial hyphae are inconspicuous, autolytic activity is scant, and coilings are distinct. Conidiation noted after approximately 4 d and starts in white uffy tufts spreading from the center to form concentric zones, and they compact to pustules with a white to greenish color. Figure 1 Neighbor-joining tree based on partial gpd gene sequences from 57 Trichoderma isolates. Parsimony bootstrap values of more than 50% are shown at nodes