﻿Three new species of Trichoderma (Hypocreales, Hypocreaceae) from soils in China

﻿Abstract Trichoderma spp. are diverse fungi with wide distribution. In this study, we report on three new species of Trichoderma, namely T.nigricans, T.densissimum and T.paradensissimum, collected from soils in China. Their phylogenetic position of these novel species was determined by analyzing the concatenated sequences of the second largest nuclear RNA polymerase subunit encoding gene (rpb2) and the translation elongation factor 1– alpha encoding gene (tef1). The results of the phylogenetic analysis showed that each new species formed a distinct clade: T.nigricans is a new member of the Atroviride Clade, and T.densissimum and T.paradensissimum belong to the Harzianum Clade. A detailed description of the morphology and cultural characteristics of the newly discovered Trichoderma species is provided, and these characteristics were compared with those of closely related species to better understand the taxonomic relationships within the Trichoderma.


Introduction
The genus Trichoderma (Ascomycota, Sordariomycetes, Hypocreales) is widely studied and applied because of their economical and ecological significance. In agriculture, they are avirulent plant symbionts used for plant protection and growth promotion (Harman et al. 2004), and as a biological agent to control of fungal diseases (Lorito et al. 2010;Zin and Badaluddin 2020). In addition, Trichoderma species have been applied for the production of enzymes and bioactive compounds of industrial utility (Ahamed and Vermette 2008;Sun et al. 2016;Stracquadanio et al. 2020). Trichoderma species possessing stress tolerance to different environmental factors hold significant promise for addressing environmental issues such as severe contamination (Kredics et al. 2001;Tripathi et al. 2013). Meanwhile, a few of Trichoderma species cause disease in cultivated mushrooms or are reported as causes of serious infections in humans (Kuhls et al. 1999;Savoie and Mata 2003). Members of Trichoderma are widely distributed in varied ecosystems, and are frequently found on soil, decaying wood, compost, or other organic matter and as endophytes in plant tissues (Samuels 2006;Zheng et al. 2021).
Traditionally, Trichoderma species were identified based on their morphology and growth characteristics (Rifai 1969;Bissett 1984Bissett , 1991a. However, as the Trichoderma species richness has increased, it has been difficult to distinguish them because species in this genus are highly similar in morphology (Bissett et al. 2003;Overton et al. 2006). With the development of molecular biology, more reliable identification is provided as DNA barcoding was introduced to recognize Trichoderma (Druzhinina et al. 2006). The most commonly used DNA barcode loci are the internal transcribed spacer (ITS), translation elongation factor 1-alpha encoding gene (tef1) and the second largest nuclear RNA polymerase subunit encoding gene (rpb2) (Druzhinina et al. 2006;Atanasova et al. 2013;Chaverri et al. 2015;Cai and Druzhinina 2021). The combination of multi-gene (rpb2 and tef1) phylogenetic analysis and phenotypic characteristics is usually applied in the species identification of Trichoderma (Chaverri and Samuels 2004;Zhu and Zhuang 2015a, b;Zheng et al. 2021;Cao et al. 2022). Recently, Cai and Druzhinina (2021) have developed an authoritative protocol that provides a standard for the molecular identification of Trichoderma. It is based on rpb2 ≥ 99% and tef1≥ 97%, one species can be identified. If the unique sequences do not meet the rpb2 ≥ 99% or tef1≥ 97%, it can be considered a new species. This protocol is advocated for the identification of Trichoderma species by the International Subcommission on Taxonomy of Trichoderma (https://trichoderma.info/; accessed on 18 Oct 2022).
Fungal diversity is enormous in China (Sun et al. 2012;Lu 2019). Since the first record of Trichoderma from China in 1895, many new Trichoderma species have been ceaselessly discovered, with most of them isolated from soils, litter, mushrooms and endophytes (Zhang et al. 2005;Yu et al. 2007;Zhang et al. 2007;Li et al. 2013;Zhu and Zhuang 2015a, b;Chen and Zhuang 2016;Qin and Zhuang 2016;Chen and Zhuang 2017;Qiao et al. 2018;Gu et al. 2020;Zhang et al. 2020;Zheng et al. 2021;Cao et al. 2022). In a previous study conducted by Dou et al. (2019), a total of 485 Trichoderma strains were obtained from soils in three provinces of China: Shanxi, Shaanxi, Shandong. The online multilocus identification system (MIST) was employed in a previous study conducted by Dou et al. (2020) to re-identify Trichoderma. The present study therefore had to identify new taxa, the sequences of which do not meet the known Trichoderma species, based on the multi loci phylogenetic analysis and morphological features observation.

Isolation of strains
In accordance with a prior study by Dou et al. (2019), a total of 485 Trichoderma strains were extracted from soil samples gathered from three provinces in China. Of these strains, 334 were sourced from Shandong, 107 from Shanxi, and 44 from Shaanxi The isolation of these strains was aided by the use of a selective medium (Dou et al. 2019).
All strains of Trichoderma were kept in 4 °C Refrigerator and -80 °C Ultra Low Temperature Refrigerator in the Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou, China. In addition, the holotype and ex-type culture were deposited in the China General Microbiological Culture Collection Center (CGMCC; https:// www.cgmcc.net/english/; accessed on 16 Sep 2022).

Morphological characterizations
The morphological observation of the colonies was based on strains grown on potato dextrose agar (PDA; 10g potato extract, 20g dextrose, 13g agar, 1 L distilled water), cornmeal dextrose agar (CMD; 40g cornmeal, 20g dextrose, 15g agar, 1 L distilled water), malt extract agar (MEA; 20g malt extract, 15g 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, 15 g agar, 1 L distilled water) medium for 7 d in an incubator at 25 °C with alternating 12 h/12h light/dark cycle. Growth-rate trials were performed on 9 cm Petri dishes with CMD, PDA, MEA and SNA at 25 °C, 30 °C, and 35 °C. The Petri dishes were incubated in darkness for up to 1 week or until the colony covered the agar surface. Colony radii were measured daily, and trials were replicated three times.
Microscopic preparations were made by mounted on lactic acid, and at least 30 measurements per structure were documented and examined under a Nikon Eclipse 80i microscope (Nikon Corp.). Length (L) and width (W) of the phialides, conidia and chlamydospores were measured, respectively, and the ratio of length to width was calculated. Measurement values are expressed as (a-)b-c(-d), where (a) represents the lowest extreme value, b-c contains the minimum value of 90% of the calculated values, and (d) denotes the highest extreme value. The letter "n" indicates the total number of measurements taken (Aignon et al. 2021;Li et al. 2021).

DNA extraction, polymerase chain reaction (PCR) and sequencing
The mycelia of pure cultures were scraped directly from plates after 2-3 d growth on PDA at 25 °C and used to extract DNA, and the genomic DNA was extracted as described by Jiang et al. (2016). For the amplifications of rpb2 and tef1 gene fragments, two different primer pairs were used EF1/EF2 for tef1 (O'Donnell et al. 1998) and fRPB2-7cR/ fRPB2-5F for rpb2 (Liu et al. 1999). The polymerase chain reaction (PCR) amplifications were performed in a total reaction volume of 20 μL, including 10 μL of Easy Flash PCR MasterMix (Easy-Do, China), 0.8 μL of each primer (10 μM), 0.4 μL genomic DNA (~0.2 μg). PCR reactions were run in a LifePro Thermal Cycler (Technology Co., Ltd. Hangzhou, China) following the PCR thermal cycle programs described by Zhu and Zhuang (2015b). PCR products were purified with the PCR product purification kit and sequencing was carried out in both directions with the same primers on an ABI 3730 XL DNA sequencer (Applied Biosystems, Foster City, CA, USA) by Sunya Biotechnology Co., Hangzhou, China. Sequences generated in this study are deposited in GenBank and the accession numbers are provided in Table 1.

Phylogenetic analyses
The phylogeny was constructed with the concatenated sequences of rpb2 and tef1. The species closely related to our strain were determined by NCBI BLAST searches with rpb2 and tef1 sequences (Altschul et al. 1990; https://blast.ncbi.nlm.nih.gov/Blast. cgi/; accessed on 16 Jun 2022), and the closely related sequences were retrieved from NCBI database for subsequent phylogenetic analysis. The GenBank accession numbers of sequences retrieved are provided in Table 1. The sequences were aligned with MAFFT (Katoh and Standley 2013), and then the alignments were manually adjusted with MEGA7 (Kumar et al. 2018) and the fragments that were suitable for molecular identification were trimmed according to Cai and Druzhinina (2021). The trimmed sequences were concatenated using SequenceMatrix v.1.8 (Vaidya et al. 2011). The following phylogenetic analysis was performed in PhyloSuite platform ). The best-fit partition model was selected using ModelFinder (Kalyaanamoorthy et al. 2017) according to BIC criterion. Maximum likelihood (ML) phylogenies were inferred using IQ-TREE (Lam-Tung et al. 2015) under Edge-linked partition model for 5000 ultrafast (Minh et al. 2013) bootstraps, as well as the Shimodaira-Hasegawa-like approximate likelihood-ratio test (Guindon et al. 2010). Bayesian Inference phylogenies were inferred using MrBayes 3.2.6 (Ronquist et al. 2012) under partition model. The phylogenetic tree was visualized in FigTree v1.4.3. (http:/tree.bio. ed.ac.uk/software/figtree/; accessed on 04 Oct 2016) with maximum likelihood bootstrap proportions (MLBP) greater than 70% and Bayesian inference posterior probabilities (BIPP) greater than 0.9, as shown at the nodes.

Sequence analysis
The comparison of rpb2 and tef1 sequences between the query strain and the reference strain revealed that the similarity did not meet the rpb2 ≥ 99% and tef1 ≥ 97% criteria as outlined in Table 2. Additionally, the query strain exhibited unique tef1 and rpb2 Table 1. Strain numbers and corresponding GenBank accession numbers of sequences used for phylogenetic analyses. sequences that do not conform to the sp∃!(rpb2 99 ≅tef1 97 ) standard for known Trichoderma species, according to Cai and Druzhinina (2021). These findings suggest that these strains could potentially be classified as new species, and therefore, phylogenetic analyses were conducted on their rpb2 and tef1 sequences.

Multi-locus phylogeny
Multi-loci phylogenetic analyses were performed on sequences obtained from 43 strains, consisting of 30 strains from the Harzianum Clade, 10 strains from the Atroviride Clade, and 3 strains from the Viride Clade. The combined rpb2 and tef1 regions were further analyzed by the methods of ML and BI, with Protocrea farinosa CBS 121551 and P. pallida CBS 299.78 as the outgroup. The tree topology derived from the ML analysis ( Fig. 1) was consistent with that obtained in a BI analysis. However, details regarding the BI analysis were not provided in the text. All strains formed a monophyletic group with higher statistical support, designated as T. nigricans (MLBP/BIBP = 100/1.00), T. densissimum (MLBP/BIBP = 100/1.00) and T. paradensissimum (MLBP/BIBP = 99/1.00). Of the three new species, T. nigricans belonged to the Atroviride Clade, whereas T. densissimum and T. paradensissimum were located in the Harzianum Clade (Fig. 1). Trichoderma nigricans was closely related with T. atroviride, and associated with T. obovatum, T. uncinatum, and T. paratroviride. This clade had high statistics support (MLBP/BIBP = 94/0.99). Trichoderma densissimum was closely related with T. paradensissimum, and associated with T. pholiotae, T. guizhouense, T. asiaticum and T. simile, with high support value (MLBP/ BIBP = 95/1.00).  Etymology. The Latin specific epithet "nigricans" refers to the "blackish green" color of the mass of conidia. Diagnosis. Phylogenetically, T. nigricans was found to form a distinct clade and was closely related to T. atroviride, T. paratroviride, T. obovatum, and T. uncinatum (Fig. 1). In terms of growth characteristics, T. nigricans was observed to have a larger colony radius on CMD after 72 h, and its mycelium covered the plate at both 25 °C and 30 °C. On PDA, T. nigricans grew faster than T. atroviride, T. paratroviride, T. obovatum, and T. uncinatum  Colony radius on MEA after 72 h: 58-60 mm at 25 °C, 53-55 mm at 30 °C, 11-12 mm at 35 °C. Colony also similar to CMD, but conidiation is yellow green, more abundant around the inoculation plug, uniform distribution all around. No diffusing pigment noted, odor indistinct.

Diagnosis.
T. paradensissimum is characterized by the green to yellow and white pustules formed inconspicuously zonate on PDA or MEA at 25 °C of a 12-h photoperiod after 7 d.
Description. Optimum temperature for growth is 30 °C on CMD, PDA and SNA and 25 °C on MEA. Chlamydospores were common on all media.
Colony radius on CMD after 72 h: 40-42 mm at 25 °C, 63-64 mm at 30 °C, 38-40 mm at 35 °C. Colony well-defined, white, aerial myceli loose and radial. White minute pustules were noted after 2 d around the inoculation plug, white at first, turning yellow green after 3-4 d, then dark green after 5-6 d. Around the point of inoculation, conidiation from dark green to pale green, inconspicuously zonate. Distinctive odor absent. The production of pigment was related to light, media and temperature: around the point of inoculation, it was yellowish at 35 °C in the dark.
Colony radius on PDA after 72 h: 59-65 mm at 25 °C, 64-67 mm at 30 °C, 20-24 mm at 35 °C. Colonies similar to that on MEA. Pustules were noted after 4-5 d. After 7 d, the green to yellow and white pustules were formed as inconspicuously zonate. Distinctive odor absent. The production of pigment was related to light and temperature; it was yellowish at 35 °C in the dark.
Sexual morph. Unknown. Substrate. Soil. Distribution. China, Shanxi Province.  Notes. Similar species can be distinguished according to the pigment: T. paradensissimum can produce yellowish pigment on PDA and CMD at 35 °C in the dark, whereas T. guizhouense typically at 35 °C reverse forming a dull orange to brown pigment. However, T. densissimum, T. asiaticum, T. simile and T. zelobreve cannot produce diffusing pigment on PDA. Trichoderma pholiotae and T. paradensissimum can both produce yellow pigment on PDA, but T. pholiotae has a slightly fruity odor on both PDA and CMD, while T. paradensissimum does not have a distinctive odor (Cao et al. 2022).

Discussion
All three new species were isolated from soils. Based on morphology and phylogenetic analyses, the taxonomic positions of three new species were explored. Of these species, T. nigricans was grouped into the Atroviride Clade, while T. densissimum and T. paradensissimum were associated with the Harzianum Clade.
The genus Trichoderma contains at least eight infrageneric clades, of which the Harzianum clade is one of the largest (Cai and Druzhinina 2021). The Harzianum clade consists of more than 95 accepted species, which are morphologically heterogeneous and phylogenetically complicated (Cao et al. 2022). Two of the newly described species, T. densissimum and T. paradensissimum, belong to the Harzianum Clade, which are closely related to T. pholiotae, associated with T. guizhouense, T. asiaticum, and T. simile. The chlamydospores of the Harzianum Clade members are usually either rarely numerous or not observed, and this is consistent with observations for T. guizhouense, T. asiaticum, T. breve, T. bannaense, and T. atrobrunneum, among others. In T. simile, the chlamydospores are either elliptic or round in shape (Li et al. 2013;Chaverri et al. 2015;Jang et al. 2018;Gu et al. 2020). In contrast, the chlamydospores of T. densissimum and T. paradensissimum are numerous, globose to subglobose, and relatively large, especially in T. densissimum. Our phylogenetic analyses revealed that T. densissimum and T. paradensissimum are closely related due to the minimal genetic variation observed in their ITS and tef1 sequences. Moreover, both species exhibit similar growth characteristics and possess numerous chlamydospores. However, their genetic variation in the sequences of rpb2 (similarity < 99%) differentiate them as distinct species. In addition, T. densissimum exhibits green conidiation with 3-4 distinct concentric zones and no diffusing pigment, while T. paradensissimum exhibits inconspicuously zonate green to yellow conidiation with white pustules and yellowish pigment.
Trichoderma atroviride and T. paratroviride were classified to the Viride Clade (Jaklitsch and Voglmayr 2015). However, with the addition of T. obovatum and T. uncinatum, they were assigned to the Atroviride Clade by (Zheng et al. 2021). In this study, the new species T. nigricans was also identified as a member of the Atroviride Clade. The results of the phylogenetic analysis indicated a close relationship between T. nigricans and T. atroviride. Morphologically, T. nigricans shares many similarities with T. atroviride, including the production of a strong coconut odor in PDA cultures and the presence of abundant chlamydospores. Trichoderma nigricans exhibits a faster growth rate on PDA in comparison to T. atroviride, with the former's mycelium covering a larger area of the plate and its colony radius measuring between 42.8-60.5 mm after 72 h at 25 °C. Colony radius is T. nigricans 16 mm vs. T. atroviride (0~)0.3~3.2(~8.3) mm at 35 °C (Samuels et al. 2002).
Numerous biological control agents have been derived from species in the Atroviride and Harzianum clade to effectively control soil-borne diseases (Chaverri et al. 2015), such as T. atroviride, T. guizhouense, T. afroharzianum, and T. atrobrunneum (Longa et al. 2010;Rees et al. 2022;Zhang et al. 2022;Zhao et al. 2022). The discovery of T. nigricans, T. densissimum, and T. paradensissimum in this study highlights the diversity of Trichoderma in China and provides valuable information for the development of Trichoderma-based biocontrol agents. Further research is necessary to explore the diversity of Trichoderma in China and to investigate their potential as biocontrol agents against plant diseases.