Three New Trichoderma Species in Harzianum Clade Associated with the Contaminated Substrates of Edible Fungi

Trichoderma is known worldwide as biocontrol agents of plant diseases, producers of enzymes and antibiotics, and competitive contaminants of edible fungi. In this investigation of contaminated substrates of edible fungi from North China, 39 strains belonging to 10 Trichoderma species isolated from four kinds of edible fungi were obtained, and three novel species belonging to the Harzianum clade were isolated from the contaminated substrates of Auricularia heimuer and Pholiota adipose. They were recognized based on integrated studies of phenotypic features, culture characteristics, and molecular analyses of RNA polymerase II subunit B and translation elongation factor 1-α genes. Trichoderma auriculariae was strongly supported as a separate lineage and differed from T. vermifimicola due to its larger conidia. Trichoderma miyunense was closely related to T. ganodermatigerum but differed due to its smaller conidia and higher optimum mycelial growth temperature. As a separate lineage, T. pholiotae was distinct from T. guizhouense and T. pseudoasiaticum due to its higher optimum mycelial growth temperature and larger conidia. This study extends the understanding of Trichoderma spp. contaminating substrates of edible fungi and updates knowledge of species diversity in the group.


Introduction
Trichoderma Pers. is ubiquitous in various niches and around the world. The genus contains at least eight infrageneric clades, of which the Harzianum clade is one of the largest [1]. According to our investigated statistics, the Harzianum clade consists of more than 95 accepted species, which are morphologically heterogeneous and phylogenetically complicated. They play important roles in agriculture, industry, and other fields and are employed as biocides or biofertilizers for plant growth [2][3][4], act as producers of enzymes and antibiotics, and are endophytic in plants that can resist both physiological stress and pathogen invasion [5,6].
Green mold contamination caused by Trichoderma spp. in the cultivation and various growth stages of edible fungi has been one of the biggest biological constraints in the industry since the 1980s [7], with the economic losses accounting for 10-20% of total production [8]. At present, green mold is one of the most devastating diseases in nearly all production areas of cultivated edible fungi due to its high disease incidence and serious economic loss [9,10]. Mycelia of Trichoderma spp. show stronger competitiveness than those of edible fungi, and thus they can inhibit mycelial growth or decrease the fruiting rate of edible fungi. Lots of green conidia of Trichoderma will gradually cover the contaminated substrates or fruiting bodies, and the contaminated fruiting bodies will eventually shrivel and rot.
In order to better understand the Trichoderma species contaminating substrates of edible fungi and preserve biological control resources, substrates of edible fungi contaminated by green mold in North China were investigated, and three undescribed species belonging to the Harzianum clade were found on contaminated substrates of Auricularia heimuer and Pholiota adipose. Their phylogenetic positions were determined based on sequence analyses of the combined translation elongation factor 1-alpha (tef1-α) and the second largest nuclear RNA polymerase subunit (rpb2) genes. Similarities and differences in morphological characteristics between the new species and their closely related species were investigated and compared in detail.

Isolates and Specimens
Specimens were separately collected from contaminated substrate of edible fungi in North China from 2020 to 2022 (Table S1), and strains were isolated following the method of a previous study [11]. The ex-type strains were deposited in the culture collection of Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences (JZB culture collection).

Morphology and Growth Characterization
For morphological studies, growth rates were determined on three different media: potato dextrose agar (PDA; 200 g potato, 18 g dextrose, 18 g agar, and 1 L distilled water), cornmeal dextrose agar (CMD; 40 g cornmeal, 20 g glucose, 18 g agar, and 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 ·7H 2 O, 0.5 g KCl, 0.2 g glucose, 0.2 g sucrose, 18 g agar, and 1 L distilled water) at 25, 30, and 35 • C in darkness. Mycelial discs (5 mm diameter) were incubated in Petri dishes (90 mm diameter) with three replicates for each isolate. Colony diameters were measured after 3 days. The time when mycelia entirely covered the surface of the plate and the morphological characteristics of colonies, such as colony appearance, color, and spore production, were recorded [12]. For microscopic morphology, photographs were taken with an Axio Imager Z2 microscope (Carl Zeiss, Jena, Germany). Microscopic characteristics and micromorphological data were examined on the cultures grown on SNA and PDA for 7-9 days at 25 • C.

Species Voucher
GenBank Accession Number Numbers in bold indicate newly submitted sequences in this study. T : type strains. ET : ex-type strains.

Phylogenetic Analyses
Sequences for all isolates generated in this study were blasted against the NCBIs Gen-Bank nucleotide datasets (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and MIST (http://mmit.china-cctc.org/index.php) [17] to obtain an initial identification. To identify the phylogenetic positions of Trichoderma species isolated from contaminated substrates of edible fungi, rpb2 and tef1-α sequences of all Trichoderma species belonging to the Harzianum clade were combined for the analyses, with T. atroviride and T. paratroviride selected as outgroup taxa. Their sequences of type or ex-type strains based on previous publications were downloaded from NCBI database and assembled using BioEdit 7.0.5.3 [18]. Alignment was generated and converted to nexus files with Clustal X 1.83 [19].
Maximum parsimony (MP) analysis was performed with PAUP 4.0b10. Starting trees were obtained via random stepwise addition with 1000 replicates and subsequent branch-swapping algorithm using tree bisection-reconnection (TBR) [20]. Analyses were performed with all characters treated as unordered and unweighted, and gaps treated as missing data. MaxTrees was set to 1000, and branches collapsed when maximum branch length was zero. Maximum parsimony bootstrap proportion (MPBP) was calculated to test topological confidence of the resulting trees.
Bayesian inference (BI) trees were calculated using MrBayes v. 3.1.2 [21]. The bestfit nucleotide substitution model GTR+I+G was selected using MrModeltest 2.3 [22]. Four chains were run from random trees for 6,000,000 generations and sampled every 100 generations. The first 25% of trees were discarded as the burn-in phase of the analyses, and Bayesian inference posterior probability (BIPP) was determined from the remaining trees. Trees were visualized in FigTree v1.4.3 [23].

Phylogenetic Analyses
The partition homogeneity test of rpb2 and tef1-α sequences indicated that the individual partitions were generally congruent (p = 0.01). The combined rpb2 and tef1-α dataset was subsequently used for phylogenetic analysis to determine the positions of the new species. In MP analysis, the dataset contained 140 taxa and 2307 characters, of which 1468 characters were constant, 150 variable characters were parsimony uninformative, and 689 were parsimony informative. Five most parsimonious trees with the same topology A total of 140 sequences representing 95 Trichoderma species, including our three new species, were used for constructing the phylogenetic tree, and T. atroviride and T. paratroviride were used as outgroups. Results showed that all the investigated Trichoderma species formed a strongly supported group (MPBP/BIPP = 100%/100%), which was generally congruent with the previous studies [24].

Taxonomy
Trichoderma auriculariae Z. J. Cao and W.T. Qin, sp. nov. MycoBank MB845141 (Figure 2). Etymology: The specific epithet refers to the host from which the fungus was isolated. around the inoculum, effuse in the aerial hyphae, forming a few inconspicuous rings. Small pustules formed around the inoculum, first white, turning green after 3 days, with hairs protruding beyond the surface. No diffusing pigment.
On SNA after 72 h, colony radius 47-49 mm at 25 • C, 51-55 mm at 30 • C, and 5-7 mm at 35 • C. Colony hyaline, mycelium loose. Conidial production noted after 2 days, starting around the inoculum, effuse in the aerial hyphae, forming a few inconspicuous rings. Small pustules formed around the inoculum, first white, turning green after 3 days, with hairs protruding beyond the surface. No diffusing pigment.
Trichoderma miyunense Z. J. Cao and W.T. Qin, sp. nov. MycoBank MB845142 (Figure 3).    Conidiophores pyramidal, with a relatively obvious main axis, multiple branches unpaired, with the longest branches near the base of the main axis. Branches perpendicular to the main axis or at acute angles with the main axis, with septa conspicuous and producing barrel-shaped or cylindrical metulae. Phialides densely disposed at the terminal of branches, often formed in whorls of 2-4, variable in shape and size, ampulliform to lageniform, (5.  (Table S2).
On PDA after 72 h, colony radius 67-68 mm at 25 • C, 70-72 mm at 30 • C, and 8-10 mm at 35 • C. Colonies white in the center, with the zone around the central part of the colony forming a distinct circular and green part. Aerial hyphae distinctly radial, abundant, dense, floccose to cottony. Light diffusing yellow pigment, odor slightly fruity.
On SNA after 72 h, colony radius 49-50 mm at 25 • C, 54-55 mm at 30 • C, and 8-10 mm at 35 • C. Colonies translucent and round-like. Aerial hyphae short, radial distribution. Pustules abundant, irregular in shape, from white to green, with the formation of concentric rings. No diffusing pigment noted. On CMD after 72 h, colony radius 71-72 mm at 25 °C, 73-74 mm at 30 °C, and 13-18 mm at 35 °C. Colonies hyaline, fan-shaped, tending to aggregate toward the distal parts of the colony. Aerial hyphae loose, sparse, radial. Conidiation effuse in aerial hyphae or in loosely disposed pustules. Pustules minute, irregular in shape, relatively abundant in the zonation regions, formed concentric rings around the outer ring, white at first, then gradually green. No diffusing pigment noted, odor slightly fruity.
On PDA after 72 h, colony radius 67-68 mm at 25 °C, 70-72 mm at 30 °C, and 8-10 mm at 35 °C. Colonies white in the center, with the zone around the central part of the colony forming a distinct circular and green part. Aerial hyphae distinctly radial, abundant, dense, floccose to cottony. Light diffusing yellow pigment, odor slightly fruity.
On SNA after 72 h, colony radius 49-50 mm at 25 °C, 54-55 mm at 30 °C, and 8-10 mm at 35 °C. Colonies translucent and round-like. Aerial hyphae short, radial distribution. Pustules abundant, irregular in shape, from white to green, with the formation of concentric rings. No diffusing pigment noted.
Conidiophores typically pyramidal with opposing branches, formed densely intricate reticulum, with one terminal whorl of generally 3-4 phialides and mostly paired side branches, less frequently solitary. Branches mostly perpendicular to the main axis with septa conspicuous. Phialides varied, borne in regular levels around the axis, some regular ampulliform or lageniform and others apex and inequilateral to curved,   [12]. In addition, T. pholiotae and T. pseudoasiaticum could be distinguished by the branching pattern, with T. pholiotae being pyramidal and T. pseudoasiaticum being verticillium-like (Table S3).

Discussion
During exploration of contaminated substrates of edible fungi in North China, 39 strains representing 10 Trichoderma species were isolated from four kinds of edible fungi and examined, and three new species were recognized based on integrated studies of phenotypic and molecular data (Table S1). To explore their taxonomic positions, a phylogenetic tree containing all species of the Harzianum clade was constructed based on analyses of the combined sequences of rpb2 and tef1-α. The three new species were well located in the Harzianum clade with separate terminal branches and were clearly distinguishable from any of the existing species. The results of this study have a number of practical implications to identify and diagnose Trichoderma species contaminating edible fungi. This work provides useful information on the epidemiological and geographical distribution of Trichoderma, which will help in the development of targeted interventions aimed at comprehensive management and control of green mold contamination of edible fungi.
With further study of Trichoderma classification, researchers have reached a consensus that accurate identification of Trichoderma species cannot depend only on the morphological identification as sometimes there is high ambiguity in the morphological features of Trichoderma spp. [32,33]. Trichoderma spp. isolated from the fruiting bodies or substrates of edible fungi is usually anamorph with high morphological similarity with many species, which is not conducive to identification. With DNA-based techniques gradually perfected and widely used, the integrative (polyphasic) taxonomy approach for species delimitation is recommended, including the combination of genealogy and multiparametric phenotypes [34,35], especially for examining the presence of species complexes and cryptic species [31]. Therefore, we hypothesized that T. harzianum, which was originally identified by ITS sequence and morphology in previous studies, probably belonged to the T. harzianum complex. However, the present study showed that the complex still contained many taxa, indicating that the previous identification was not accurate. Furthermore, it is also difficult to identify species of the Harzianum clade according to exclusive tef1-α or rpb2 sequence data [24,25]. Therefore, the combination of tef1-α and rpb2 sequences for phylogenetic analysis is highly recommended to identify species in the Harzianum clade.
Taxonomy of Trichoderma dates back to the late 18th century [36], and some of them cause economic losses in commercial mushroom farms [37]. Over more than a century, successive findings have brought the number of known species of the genus to over 441 [1,23,38]. Trichoderma species are located throughout the world, and more than 30 of them are mushroom inhabiting ( Figure 1, Table 2). They are isolated from the substrate or fruiting bodies of Agaricus bisporus, Lentinula edodes, Pleurotus ostreatus, Ganoderma lingzhi, etc. and are mainly located in the Harzianum, Longibrachiatum, and Viride clades [39]. There may still be many unknown Trichoderma species associated with the growth of edible fungi and their related living environment. The phylogenetical difference between Trichoderma spp. on edible fungi substrates and from other sources deserves further analysis.
Analysis of the biological characteristics of Trichoderma species from contaminated substrates showed that the optimum growth temperature of many Trichoderma species was generally around 30 • C, which was consistent with the phenomenon that contamination of Trichoderma on edible fungi is more likely to occur at high temperatures. Therefore, reasonable control the growth environment temperature of edible fungi may be a reasonable approach to prevent or delay the outbreak of Trichoderma contamination during production. More broadly, research is also needed to analyze the mechanism of occurrence of Trichoderma spp. contamination, such as the correlation between contamination occurrence and the growth environment of edible fungi.
With the increased number of species joining the Harzianum clade, understanding of Trichoderma spp. will become more sophisticated and intelligible, and reasonable species concepts will be firmly established. Accumulated knowledge of Trichoderma, especially the Harzianum clade, will provide useful information for sufficient utilization of resources and for the prevention of contamination of edible fungi. T. koningiopsis Dictyophora rubrovolvata, P. eryngii [52,53] T. lentinulae L. edodes [24] T. longibrachiatum L. edodes, P. ostreatus, A. aegerita [9,43,50] T

Conclusions
In this study, 39 strains belonging to 10 Trichoderma species isolated from four kinds of edible fungi in North China were obtained, and three novel species belonging to the Harzianum clade were isolated from the contaminated substrates of Auricularia heimuer and Pholiota adipose. More than 30 mushroom-inhabiting Trichoderma species throughout the world mainly located in the Harzianum, Longibrachiatum, and Viride clades were indicated. This study enrich the biodiversity of Trichoderma and provide important support for systematic development of the Harzianum clade.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof8111154/s1. Table S1: Strain information and their accession numbers. Table S2: Comparison of the morphological characteristics of Trichoderma auriculariae and its relatives. Table S3: Comparison of the morphological characteristics of Trichoderma miyunense and its relatives. Table S4: Comparison of the morphological characteristics of Trichoderma pholiotae and its relatives. Table S5: The growth rate of three new species in this study incubated at different temperatures and media.