Soil samples were collected from the rocky desertification region in Shilin County, Kunming City, Yunnan Province (24.6° N, 103.4° E; altitude 1940 m a.s.l.). This area is characterized by exposed bedrock interspersed with gravel, sand, and soil. Despite the degradation, crops such as maize and soybeans are still cultivated, indicating that the region has not reached a fully desertified state. A total of 90 soil samples were collected from three sampling sites, with each located approximately 20 km apart. At each site, 30 samples were collected using a random sampling method, maintaining a minimum spacing of 5 m between sampling points. Samples were taken from a depth of 5–10 cm after the removal of surface plant debris and gravel. All samples were labeled with unique identifiers and detailed collection information. Subsequently, the samples were transferred to the laboratory and stored at 4°C until further analysis.

The soil fungal isolation steps were as follows: 10 g of soil were mixed with 90 mL of sterile water with an appropriate amount of sterile glass beads and then shaken thoroughly at 220 r/min⁻¹ for 1 h. After allowing the suspension to stand for 2 min, the supernatant was collected and subjected to serial dilutions (10⁻¹ to 10⁻⁴). Aliquots of 100 μL from the 10⁻² to 10⁻⁴ dilutions were plated in triplicate onto Rose Bengal Agar (RBA; Guangdong Huankai Microbial Science and Technology Co., Ltd., China) supplemented with antibiotics (streptomycin, 40 mg/L; ampicillin, 30 mg/L) to suppress bacterial growth. The inoculated plates were incubated in a temperature-controlled chamber at 25°C for 5–7 days and monitored daily for colony growth.

After mycelia growth, well-developed colonies were subcultured onto potato dextrose agar plates (PDA: 200 g potato, 20 g dextrose, 18 g agar, and 1,000 mL distilled water) for further purification and identification. The resulting pure cultures were deposited in the Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University (YMF), Kunming, China.

Growth rates were measured on 9-cm-diameter Petri dishes containing three different media: PDA, cornmeal agar (CMA: 20 g cornmeal, 18 g agar, and 1,000 mL distilled water), and synthetic nutrient-poor agar (SNA: 1 g KH₂PO₄, 1 g KNO₃, 0.5 g MgSO₄, 0.5 g KCl, 0.2 g glucose, 0.2 g sucrose, 18 g agar, and 1,000 mL distilled water), at 25, 30, and 35°C under alternating 12-h light and 12-h dark cycles. After 3 days of incubation, the colony diameter was recorded, and the time required for complete colony coverage was documented. Furthermore, the morphological characters of colonies, such as colony appearance, color, and conidia production, were recorded at the same time. For microscopic morphology, including hyphae, conidiophores, phialides, conidia, and other structures, images were taken using an Olympus BX51 microscope (Tokyo, Japan) connected to a DP controller digital camera. At least 30 datasets were measured for each structure. Colonies were photographed after 7 days, and conidia were photographed after 14 days of production.

Genomic DNA was extracted following the method described by Ye et al. (2024). Briefly, 0.5 g of mycelia was transferred into a 2.0-mL microcentrifuge tube, to which steel beads and 700–800 μL of urea extraction buffer [7 mol/L of urea, 50 mmol/L of Tris–HCl, 62.5 mmol/L of NaCl, 10 g/L of sodium dodecyl sulfate (SDS)] were also added, followed by 5 min of disruption at 50 Hz oscillation. The mixture was centrifuged at 12,000 r/min for 5 min, after which the supernatant was transferred to a 1.5-mL centrifuge tube and an equal volume of DNA extraction (phenol/chloroform/isoamyl alcohol, 25:24:1) was added. The mixture was centrifuged at 12,000 r/min for 5 min, and the supernatant was transferred to a new 1.5-mL centrifuge tube, to which an equal volume of isopropanol and 1/10 volume of 3 mol/L of NaAc were added, followed by incubation at −20°C for 20 min. The mixture was then centrifuged at 12,000 r/min for 5 min, and the upper aqueous phase was discarded. The DNA pellets were washed twice with 70% ethanol, dried at 40°C, and then resuspended in 50 μL of sterile water for PCR analysis (Liu et al., 2005).

The ITS, rpb2, and tef1-α fragments were amplified using three pairs of primers: ITS4 and ITS5 for ITS (White et al., 1990), frpb2-5f and frpb2-7cr for rpb2 (Liu et al., 1999), and EF1-728F (Carbone and Kohn, 1999) and TEF1LLErev (Jaklitsch et al., 2005) for tef1-α. PCR amplifications were conducted in a 25-μL reaction system containing 12.5 μL of 2 × Master Mix (Accurate Biology), 9.5 μL of double-distilled water, and 1 μL each of forward primer, reverse primer, and DNA template. The PCR reactions were carried out using an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) following the thermal cycling program described in Table 1. The PCR products were purified using a PCR product purification kit (Biocolor Bioscience and Technology Co., Shanghai, China) and subsequently sequenced in both directions using amplification primers on an ABI 3730 XL DNA sequencer (Applied Biosystems, Foster City, California). The obtained sequences were deposited in the GenBank database at the National Center for Biotechnology Information (NCBI), and the corresponding accession numbers are provided in Table 2.

Preliminary BLASTn searches were conducted using the ITS, rpb2, and tef1-α sequences of the newly isolated strains against the NCBI database to identify closely related species. Both the reference sequences and the newly generated sequences in this study are listed in Table 2. Phylogenetic reconstruction was performed based on the concatenated sequences of the ITS, rpb2, and tef1-α loci. Sequence alignment was conducted using Clustal X 1.83 (Thompson et al., 1997) with default parameters, followed by trimming to appropriate lengths using MEGA11 (Tamura et al., 2021). Sequence assembly and alignment were carried out in BioEdit version 7.0 (Hall, 1999), with manual concatenation of the aligned sequences from the three loci. Missing nucleotide positions were filled with question marks “?” to facilitate subsequent analyses and to optimize the quality of sequence assembly. A sequence matrix (FASTA file) containing three gene loci was generated using BioEdit version 7.0, with a total of 3,024 characters (669 from ITS, 1,041 from rpb2, and 1,314 from tef1-α). The alignment data used in the phylogenetic analyses were deposited in TreeBASE.

Phylogenetic reconstruction of the newly identified species was conducted through both maximum likelihood (ML) and Bayesian inference (BI) approaches. For the ML analysis, the concatenated sequence matrix in FASTA format, assembled using BioEdit version 7.0 (Hall, 1999), was analyzed in IQ-TREE software (Nguyen et al., 2015). The optimal nucleotide substitution model was selected through ModelFinder, executed with the command iqtree -s example.fas -m MF -nt AUTO, which identified the TNe + I + G4 model as the best-fit evolutionary model based on the Bayesian Information Criterion (BIC). Bootstrap support values were estimated from 1,000 replicates following the outgroup designation. Bayesian trees were constructed using MrBayes v3.1.2 (Huelsenbeck and Ronquist, 2001), with the best model chosen through MrModeltest 2.3. The Markov chain Monte Carlo (MCMC) analysis was initiated with four parallel chains (one cold and three heated) per run, which proceeded for five million generations with sampling intervals of 500 generations until the average standard deviation of split frequencies fell below 0.01. The initial 25% of sampled generations were discarded as burn-in, with the remaining samples utilized to compute posterior probabilities for Bayesian phylogenetic reconstruction. Phylogenetic trees were visualized using FigTree version 1.4, with the nodal support values indicated by both maximum likelihood bootstrap proportions (MLBPs≥75%) and Bayesian posterior probabilities (BIPPs≥0.85).

A total of 57 strains of Trichoderma were isolated and purified from rocky desertification soils based on the initial colony morphology. Among these strains, 47 were identified as known species, and 10 were designated as putative new species based on the BLASTn search results of the ITS sequence.

Phylogenetic analyses inferred from the ITS sequence were conducted to identify known species. The detailed species and their isolation frequencies are provided in Table 3. The highest isolation frequency was observed in T. harzianum, reaching 26.31%. The isolation frequencies of the remaining species were as follows: 12.28% each for Trichoderma koningiopsis and Trichoderma sulphureum, 8.77% for Trichoderma gamsii, 7.02% for T. hamatum, 5.26% for T. virens, and 3.51% each for T. atroviride, T. cerinum, and T. spirale.

A concatenated dataset comprising ITS, rpb2 and tef1-α sequences (total length: 3,024 characters) was analyzed to determine the phylogenetic placement of the novel species. Phylogenetic trees were reconstructed through both ML and BI methods and exhibited consistent topological structures (Figure 1). Based on combined morphological characteristics and phylogenetic evidence, 10 isolates were identified as 4 new Trichoderma species, which are distributed across three different clades. The four new species were proposed as T. calcicola, T. exigua, T. karsti, and T. xerophilum, and each was supported by robust phylogenetic evidence and distinct morphological characteristics.

Two isolates were assigned to the Longibrachiatum clade, forming a new subclade corresponding to a novel species, designated as T. karsti (MLBP/BIPP = 100/1.00). In the Viride clade, two isolates formed a new subclade, defined as a novel species, designated as T. calcicola (MLBP/BIPP = 100/1.00). In the Harzianum clade, six isolates formed two new subclades, which were identified as novel species and named T. xerophilum (MLBP/BIPP = 100/1.00) and T. exigua (MLBP/BIPP = 100/1.00).

Trichoderma karsti Z. F. Yu & X. W. Dai, sp. nov. Figure 2.

MycoBank No: 860053.

Etymology: Latin, karsti, refers to the holotype being isolated from karst soil.

Description: Sexual morph: Unknown. Asexual morph: Conidiophores consisting of a recognizable main axis with branches arranged either in pairs or singly, arising at an angle slightly less than 90° with respect to the main axis. The distance between two neighboring branches ranges from 3.5 to 13.2 μm. Phialides are commonly singly, opposite, ampulliform or narrowly vase-shaped, and the tip is long and curved, oriented an indefinite direction. They measure (6.2–)7.5–9.5(−11.3) × 2.3–4.1 μm, with a length-to-width (l/w) ratio of 1.9–4.6(−5.1), and, at base, they are 1.6–2.4(−3.5) μm wide and widest around the middle. Conidia are oval, elliptic, pale yellow-green, and smooth-walled, measuring 3.8–5.2 × 2.8–3.2 μm, with an l/w ratio of 1.1–1.4. Chlamydospores were observed growing at the tip of hyphae, round, measuring 8.2–10.8 × 7.1–9.7 μm, with a l/w ratio of 1.0–1.1.

Culture characteristics: optimum temperature for growth 30°C.

After 72 h, the colony radius on PDA was 64 mm at 25°C, 72 mm at 30°C, and 59 mm at 35°C, covering the plate after 3 days at 30°C. The colony is white, circular, and turns green after 3 days. Aerial hyphae are abundant, forming a dense mat. Pure yellow pigments are noted. A slight odor was noted.

Colony radius on CMA after 72 h: 12 mm at 25°C, 29 mm at 30°C, and 15 mm at 35°C. The colony lucency is circular, darkening to deep green as the incubation time extended. There was no diffusing pigment noted, and the odor was indistinct.

Colony radius on SNA after 72 h: 13 mm at 25°C, 34 mm at 30°C, and 30 mm at 35°C. The colony is white and turns green after 5 days. Sulphur yellow pigment was noted, and a slight odor was noted. Chlamydospores noted in all media.

Materials examined: China, Yunnan Province, Shilin Country, from soil of rocky desertification, August 2024, Z. F. Yu, (holotype YMF 1.09950). lbid. (cultures: YMF 1.09951).

Notes: From a systematic perspective, T. karsti is closely related to T. thermophilum and associated with T. rugosum. T. thermophilum and T. rugosum, which exhibit a sexual morph, T. karsti has only been observed in its asexual state (Qin and Zhuang, 2016; Zhang and Zhuang, 2018). The phialides of T. thermophilum and T. rugosum are relatively regular in morphology, while those of T. karsti are more curved and asymmetrical. In addition, the conidia of T. karsti are larger than those of T. thermophilum (3.8–5.2 × 2.8–3.2 vs. 2.7–6 × 2.3–3) and T. rugosum (3.8–5.2 × 2.8–3.2 vs. 3–4 × 2.2–3). The colonies of all three species are yellow on PDA and CMA, while they appear transparent or translucent on SNA. T. karsti has a mild odor, whereas no distinct odor was detected in the other two species.

Trichoderma xerophilum Z. F. Yu & X. W. Dai, sp. nov. Figure 3.

MycoBank NO: 860054.

Etymology: Latin, xerophilum, refers to the arid karst soil.

Description: sexual morph: unknown. Asexual morph: conidiophores comprising a recognizable main axis, primary branches that are mostly paired, occasionally 3 verticillate or solitary, and arise at an angle of approximately 90° from the main axis. Each branch terminates in a paired and a whorl of 3 together with a terminal phialide. Phialides ampulliform to narrowly vase-shaped, straight or slightly curved, mostly paired or whorls of 3 on terminal branches of the conidiophore, occasionally solitary, (5.5–)6.0–8.7(−11.5) × 2.1–3.2 μm, l/w ratio 2.3–4.9, 1.4–3.2 μm wide at base, widest around the middle. Conidia are oval, elliptic to subspheroidal, green, smooth, (3.1–)3.3–3.9(−4.2) × (2.5–)2.7–3.4 (−3.6) μm, with a l/w ratio of 1.0–1.2. The chlamydospores observed at the tips of hyphae exhibited two distinct morphological types: elliptical, measuring 10.0–12.9 × 7.8–9.2 μm with a length-to-width ratio of 1.2–1.6, and subglobose, measuring 8.5–11.2 × 6.6–9.4 μm with a length-to-width ratio of 1.0–1.2.

Culture characteristics: Optimum temperature for growth is 30°C.

Colony radius on PDA after 72 h: 55 mm at 25°C, 63 mm at 30°C, and 40 mm at 35°C, covering the plate after 3 days at 30°C. The colony is translucent, circular, and radial, with a white to pale grayish green. Aerial hyphae are abundant, forming a dense mat. Pure yellow pigments noted, slight odor noted.

Colony radius on CMA after 72 h: 25 mm at 25°C, 32 mm at 30°C, and 29 mm at 35°C. The colony lucency is circular, 1–2 zonate, darkening to green as incubation time extended. No diffusing pigment was noted, and odor was indistinct.

Colony radius on SNA after 72 h: 44 mm at 25°C, 50 mm at 30°C, and 30 mm at 35°C. The colony lucency is circular, three or more zonate, and the color changes to yellowish green after 3 days. Pure yellow pigments were noted, and a slight odor was noted. Chlamydospores were noted in all media.

Materials examined: China, Yunnan Province, Shilin Country, from soil of rocky desertification, August 2024, Z. F. Yu, (holotype YMF 1.09953). lbid. (cultures: YMF 1.09952, YMF 1.09958, YMF 1.09959).

Notes: Based on phylogenetic analyses, the four strains of T. xerophilum formed a single clade, sistering to the clade formed by T. afroharzianum, T. atrobrunneum, T. pyramidale, and T. pseudopyramidale. The phialides of T. afroharzianum (5.2–10.2 × 2.0–3.5 μm) and T. atrobrunneum (5.5–8.0 × 2.2–3.7 μm) are lageniform to ampulliform, whereas T. xerophilum exhibits longer phialides (6.0–8.7 × 2.4–3.2 μm) with a more pronounced length-to-width ratio, making it distinctive within the group. T. atrobrunneum does not mention the formation of chlamydospores, while T. afroharzianum rarely produces them (Chaverri et al., 2015). In contrast, T. xerophilum forms chlamydospores at the tips of hyphae, exhibiting two distinct morphological types. Furthermore, the phialides of T. pyramidale exhibit greater morphological diversity, ranging from lageniform to ampulliform and occasionally inequilateral or sigmoid, compared to the more uniform phialides of T. xerophilum (5.5–11.5(−17.5) × 2.8–3.7(−4.5) vs. 6.0–8.7(−11.5) × 2.1–3.2), thereby reflecting the morphological differences between the two species (Chaverri et al., 2015). Similarly, the phialides of T. pseudopyramidale (5.3–8.6(−9.1) × 2.2–2.9(−3.2) μm) are predominantly ampulliform to lageniform and usually formed in whorls, showing a somewhat narrower width than those of T. xerophilum (6.0–8.7(−11.5) × 2.1–3.2 μm) (del Carmen et al., 2021). These morphological characteristics distinctly set T. xerophilum apart from other closely related species.

Trichoderma calcicola Z. F. Yu & X. W. Dai, sp. nov. Figure 4.

MycoBank NO: 860055.

Etymology: Latin, calcicola, referring to the limestone-rich karst soil from which the strain was isolated.

Description: Sexual morph: Unknown. Asexual morph: Conidiophores and branches form a pyramidal structure, the distance between two neighboring branches (6.7–)8.0–25.0(−30.0) μm. Branches paired asymmetrically or solitary, occasionally in a whorl of 3 at an angle less than or near 90° concerning the main axis, branches terminating in a single, paired, or a whorl of three phialides. Phialides are spindle-shaped and lageniform, (6.4–)7.5–11.4(−12.0) × (2.6–)2.9–3.8(−4.0) μm, l/w ratio 1.4–3.5. Conidia thin-walled, ellipsoidal, rarely globose, green, smooth, (3.5–)3.8–4.5(−4.8) × (2.6–)2.8–3.2(−3.5) μm, l/w ratio 1.2–1.4. Chlamydospores were noted at the tip of hyphae, round, and measure 7.1–10.23 × 6.2–8.4 μm, with a l/w ratio of 1.1–1.2.

Culture characteristics: Optimum temperature for growth is 25°C.

Colony radius on PDA after 72 h: 55 mm at 25°C, 50 mm at 30°C, and 24 mm at 35°C, covering the plate after 3 days at 25°C. The colonies are white, circular, and fuzzy; aerial hyphae are abundant. No diffusing pigment noted, slight odor noted.

Colony radius on CMA after 72 h: 35 mm at 25°C, 32 mm at 30°C, and 24 mm at 35°C. The colony lucency is circular, the central air mycelia of the colony exiguity, and the margin dense. No diffusing pigment noted, slight odor noted.

Colony radius on SNA after 72 h: 40 mm at 25°C, 33 mm at 30°C, and 30 mm at 35°C. The colonies are white, circular, and fuzzy, aerial hyphae hairy to floccose, dense. Slight odor noted. Chlamydospores were observed in all media.

Materials examined: China, Yunnan Province, Shilin Country, from soil of rocky desertification, August 2024, Z. F. Yu, (holotype YMF 1.09956). lbid. (cultures: YMF 1.09957).

Notes: T. calcicola and T. hailarense are phylogenetically related but exhibit distinct differences in morphological and culture characteristics (Zhang et al., 2022). Regarding phialides, T. hailarense features longer lageniform phialides (8.0–15.5 μm × 2.5–3.6 μm), while T. calcicola possesses spindle- to lageniform-shaped phialides (6.4–12.0 μm × 2.6–4.0 μm). For conidia, T. hailarense yields delicately roughened, obovoid conidia (4.2–4.9 μm × 3.4–4.1 μm), whereas T. calcicola produces smooth, ellipsoidal conidia (3.5–4.8 μm × 2.6–3.5 μm). T. hailarense exhibits faster growth at 30°C, whereas T. calcicola shows better adaptation to growth conditions at 25°C.

Trichoderma exigua Z. F. Yu & X. W. Dai, sp. nov. Figure 5.

MycoBank NO: 860056.

Etymology: Latin, exigua, refer exiguous conidiation.

Description: Sexual morph: Unknown. Asexual morph: Conidiophores more or less symmetrical, main axis recognizable, branches arising at an angle of less than 90° concerning the main axis. Most branches are paired or form a whorl of 3, occasionally solitary. Phialides concentrated on the apex of conidiophores, arranged in 2–5 whorls, less solitary, ampulliform or narrowly vase-shaped, straight or curved, with an indefinite direction. (5.5–)6.5–9.7(−12.0) × (2.0–) 2.6–3.5(−4.2) μm, l/w ratio1.6–3.5. Conidia oval, elliptic, green, smooth, (2.7–)2.9–3.5(−4.1) × (2.4–)2.5–2.8(−3.0) μm, l/w ratio 1.1–1.3. Chlamydospores not found.

Culture characteristics: Optimum temperature for growth is 30°C.

Colony radius on PDA after 72 h: 51 mm at 25°C, 60 mm at 30°C, and 42 mm at 35°C, covering the plate after 3 days at 30°C. The colony is white, circular, and turns primrose after 3 days. Aerial hyphae are abundant, forming a dense mat. No diffusing pigment noted, slight odor noted.

Colony radius on CMA after 72 h: 30 mm at 25°C, 35 mm at 30°C, and 27 mm at 35°C. The colony is lucency, with the air mycelium having more edges and less center; no diffusing pigment was noted, and odor was indistinct.

Colony radius on SNA after 72 h: 40 mm at 25°C, 51 mm at 30°C, and 33 mm at 35°C. The colony is white, circular. Three days later, the center of the colony turns yellow-green. No diffusing pigment was noted, and odor was indistinct.

Materials examined: China, Yunnan Province, Shilin Country, from soil of rocky desertification, August 2024, Z. F. Yu, (holotype YMF 1.10219). lbid. (cultures: YMF 1.10220).

Notes: T. exigua and T. guizhouense are phylogenetically related but exhibit distinct differences in morphological and culture characteristics (Li et al., 2013). However, T. exigua possesses distinctly longer lageniform phialides than the ampulliform to lageniform phialides of T. guizhouense (6.5–9.7 × 2.6–3.5 vs. 4.5–10 × 2–3); the phialides of the former are organized in 2–5 whorls, while those of the latter are often in a whorl of 3. Moreover, conidia of T. exigua are smooth, oval to elliptic, and larger (2.4–3.0 vs. 2–3), while conidia of T. guizhouense are globose.

Finally, regarding culture characteristics, T. guizhouense exhibits rapid growth, with a colony radius of 57–58 mm on PDA at 25°C after 72 h, whereas T. exigua shows slower growth. Both species lack diffusing pigments, although T. guizhouense may produce a brown diffusing pigment in some strains, and both species emit a slight odor.