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Research Article
Five new species of Trichoderma from moist soils in China
expand article infoGuang-Zhi Zhang‡, He-Tong Yang‡, Xin-Jian Zhang‡, Fang-Yuan Zhou‡, Xiao-Qing Wu‡, Xue-Ying Xie‡, Xiao-Yan Zhao‡, Hong-Zi Zhou‡
‡ Qilu University of Technology, Jinan, China

Corresponding author: Xin-Jian Zhang ( zhangxj@sdas.org )

Academic editor: Rungtiwa Phookamsak
© 2022 Guang-Zhi Zhang, He-Tong Yang, Xin-Jian Zhang, Fang-Yuan Zhou, Xiao-Qing Wu, Xue-Ying Xie, Xiao-Yan Zhao, Hong-Zi Zhou.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Zhang G-Z, Yang H-T, Zhang X-J, Zhou F-Y, Wu X-Q, Xie X-Y, Zhao X-Y, Zhou H-Z (2022) Five new species of Trichoderma from moist soils in China. MycoKeys 87: 133-157. https://doi.org/10.3897/mycokeys.87.76085
Open Access

Abstract

Trichoderma isolates were collected from moist soils near a water source in different areas of China. ITS sequences were submitted to MIST (Multiloci Identification System for Trichoderma) and meets the Trichoderma [ITS76] standard. Combined analyses of phylogenetic analyses of both phylograms (tef1-α and rpb2) and morphological characteristics, revealed five new species of Trichoderma, namely Trichoderma hailarense, T. macrofasciculatum, T. nordicum, T. shangrilaense and T. vadicola. Phylogenetic analyses showed T. macrofasciculatum and T. shangrilaense belong to the Polysporum clade, T. hailarense, while T. nordicum and T. vadicola belong to the Viride clade. Each new taxon formed a distinct clade in phylogenetic analysis and have unique sequences of tef1-α and rpb2 that meet the Trichoderma new species standard. The conidiation of T. macrofasciculatum typically appeared in white pustules in concentric rings on PDA or MEA and its conidia had one or few distinctly verrucose. Conidiophores of T. shangrilaense are short and rarely branched, phialides usually curved and irregularly disposed. The aerial mycelium of T. hailarense and T. vadicola formed strands to floccose mat, conidiation tardy and scattered in tufts, conidiophores repeatedly rebranching in dendriform structure. The phialides of T. nordicum lageniform are curved on PDA and its conidia are globose to obovoidal and large.

Keywords

Hypocreales, phylogenetic analysis, soil fungi, Sordariomycetes, taxonomy

Introduction

The genus Trichoderma belongs to one of the most useful groups of microbes to have had an impact on human welfare in recent times. They are most widely used as biofungicides and plant growth modifiers and are sources of enzymes of industrial utility, including those used in the biofuels industry (Mukherjee et al. 2013). Some Trichoderma species have great potential applications to remediate soil and water pollution (Tripathi et al. 2013). Trichoderma is a hyperdiverse fungal genus (Jaklitsch and Voglmayr 2015). Formerly the species-level identification of Trichoderma was performed, based on their morphological characteristics (Gams and Bissett 1998) and is becoming more and more difficult because there are only a few relatively invariable morphological characteristics, leading to overlap amongst species (Samuels 2006).

DNA sequence analysis was introduced and provided more reliable identification of Trichoderma species (Druzhinina et al. 2006; Samuels 2006; Samuels et al. 2006). Given their low sequence variability or missing adequate sequence data, ITS, cal1 and chi18-5 are rarely used for new Trichoderma species identifications (Bissett et al. 2015; Cai and Druzhinina 2021). Tef1-α and rpb2 facilitate reliable species identifications through phylogenetic analyses (Bissett et al. 2015; Jaklitsch and Voglmayr 2015; Cai and Druzhinina 2021) and have been used in the phylogenetic analysis and identification of new Trichoderma species in recent years. This has resulted in the exponential expansion of Trichoderma taxonomy, with up to 20 new species recognised per year (Cai and Druzhinina 2021). As of July 2021, a total of 405 species has been reported and recognised (Bustamante et al. 2021; Cai and Druzhinina 2021; Rodríguez et al. 2021; Zheng et al. 2021). The new molecular identification protocol provides a standard for the molecular identification of Trichoderma (Cai and Druzhinina 2021; www.trichoderma.info). According to this protocol, the new species should meet the Trichoderma [ITS76] standard and has unique sequences of rpb2 or tef1 (does not meet the sp∃!(rpb299≅tef197) standard for known species).

Trichoderma species are cosmopolitan and prevalent components of different ecosystems in a wide range of climatic zones (Kubicek et al. 2008). They are mainly found in natural soils and decaying wood and plant material (Kredics et al. 2014). Many new Trichoderma species were first discovered in China, with up to 115 new Trichoderma species being reported since 2016 (Zhu and Zhuang 2015a, b, 2018; Chen and Zhuang 2016, 2017a, b, c, d; Qin and Zhuang 2016a, b, c, 2017; Sun et al. 2016; Zeng and Zhuang 2017, 2019; Zhang and Zhuang 2017, 2018, 2019; Li et al. 2018; Qiao et al. 2018; Zhao et al. 2018; Ding et al. 2020; Gu et al. 2020; Liu et al. 2020; Zheng et al. 2021). Amongst these 115 species, 75 were isolated from soils, 36 were collected from the plant branch or rotten twigs, while the other four species were collected from mushroom, pollen or rotten fruit.

Trichoderma has been segregated into many clades (Bissett 1991; Atanasova et al. 2013). The Polysporum clade (formerly section Pachybasium) was first defined by Bissett (1991), including 20 species. However, molecular phylogeny has shown that it is paraphyletic (Kindermann et al. 1998; Kullnig-Gradinger et al. 2002) and the species composition was subdivided subsequently into five unrelated clades, such as Ceramica, Harzianum, Semiorbis, Strictipilosa or Stromaticum (Chaverri and Samuels 2003; Jaklitsch 2009; Jaklitsch 2011). Trichoderma hamatum and some other species were found to belong to the section Trichoderma. The removal of T. hamatum determined that Bissett’s sectional name could not be used anymore. Lu et al. (2004) refined the clade containing the remaining species around T. polysporum/Hypocrea pachybasioides and it was named the Pachybasium core group by Jaklitsch (2011), which includes 13 species. In subsequent years, several new species were added to this clade, increasing the number of Trichoderma species to 21 species (Jaklitsch and Voglmayr 2015; Zhu and Zhuang 2015; Qin and Zhuang 2016c; Chen and Zhuang 2017b).

The Virde clade is basically in accordance with Bissett’s (1991) concept, but later, some other species have been added constantly. As of 2015, this large clade has 72 species to be confirmed and described, amongst which 55 species have been well located in the six subclades (Hamatum/Asperellum, Koningii, Neorufum, Rogersonii, Viride and Viridescens) and 17 species have not been located in the unnamed branches (Park et al. 2014; Bissett et al. 2015; Jaklitsch and Voglmayr 2015). In subsequent years, 25 new species were added to this clade, increasing the number of Trichoderma species to 97 (Montoya et al. 2016; Qin and Zhuang 2016a; Chen and Zhuang 2017c; Zeng et al. 2017; Zhang and Zhuang 2017; du Plessis et al. 2018; Qiao et al. 2018; Zhang and Zhuang 2018; Crous et al. 2019; Ding et al. 2020; Tomah et al. 2020). Cai and Druzhinina (2021) reconstructed the core topology of the phylogram, based on the Maximum Likelihood (ML) phylogeny of the 361 rpb2-barcoded Trichoderma species and 361 species have been located in the eight main clades (numerically named 1–8). All Trichoderma species in the adjacent Polysporum and Viride clades were remerged into the 5th clade, which also included several Trichoderma species from the Harzianum and lone lineage clades (Jaklitsch and Voglmayr 2015; Sun et al. 2016; Zhang and Zhuang 2017).

The present study performed the phylogenetic analysis of the five new species of Trichoderma to establish their new status. Five new species were collected from moist soils near water in different areas of China. Tef1-α and rpb2 sequences were used for the phylogenetic reconstruction of the five new species in the present study and meet the Trichoderma new species standard (Cai and Druzhinina 2021).

Materials and methods

Isolates and specimens

Specimens were collected from Sichuan, Yunnan, Beijing, Shandong and Inner Mongolia. Trichoderma strains were isolated from soils on Trichoderma Selective Medium (K2HPO4 0.90 g; MgSO4·7H2O 0.20 g; NH4NO3 1.0 g; KCl 0.15 g; glucose 3.0 g; Rose Bengal 0.15 g; Agar 15.0 g; distilled water 1.0 litre. Post autoclaving, chloromycetin (0.25 g), streptomycin (0.03 g) and pentachloronitrobenzene (0.2 g) were added) (Martin 1950). Ex-type living cultures of new species were deposited in the Agricultural Culture Collection of China (ACCC) (Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, China).

Morphological characterisations

Morphological observation of the colonies and conidium-bearing structures was based on isolates grown on PDA (potato dextrose agar, Difco), CMD (Difco cornmeal agar + 2% w/v dextrose), MEA (malt extract agar, Difco) and Nirenberg’s SNA medium (Nirenberg 1976) for 2 weeks in an incubator at 25 °C with alternating 12 h/12 h fluorescent light/darkness. Microscopic observations were conducted with an Olympus BX53 microscope and a MicroPublisher 5.0 RTV digital camera (Olympus Corp., Tokyo, Japan). Continuous characters, such as length and width, were measured with the CellSens Standard Image software (Olympus Corp., Tokyo, Japan). Continuous measurements were based on 10–30 measured units and were reported as the extremes (maximum and minimum) in brackets separated by the mean plus and minus one standard deviation. Colour standards were from Kornerup and Wanscher (1978). Growth-rate trials were performed on 9 cm Petri dishes with 20 ml of CMD, PDA, MEA and SNA at 15 °C, 20 °C, 25 °C, 30 °C, and 35 °C. Petri dishes were incubated in darkness up to 1 week or until the colony covered the agar surface. Colony radii were measured daily. Trials were replicated three times.

DNA extraction, polymerase chain reaction (PCR) and sequencing

Strains were grown in 9 cm-diameter Petri dishes containing PDA (potato dextrose agar, Difco). Cultures were incubated at 25 °C for ca. 3–5 days. Genomic DNA was extracted from the mycelial mat harvested from the surface of the broth with the Fungal Genomic DNA Extraction Kit (Aidlab Biotechnologies Co. Ltd., Beijing, China). The amplification of ITS was performed using the primer pair ITS5 and ITS4 (White et al. 1990), for tef1-α, primer pair EF1-728F (Carbone and Kohn 1999) and tef1-ΑLLErev (Jaklitsch et al. 2005) was used and, for rpb2, primer pair frpb2-5f and frpb2-7cr (Liu et al. 1999) was used. PCR amplification of each gene was performed as described by Park et al. (2014) and Chaverri et al. (2011). PCR products were purified and sequenced by ABI3730 Gene Analyzer at Sangon (Sangon Biotech (Shanghai) Co., Ltd.).

Molecular identification and phylogenetic analyses

We followed the molecular identification protocol for a single Trichoderma isolate (Cai and Druzhinina 2021; www.trichoderma.info) and estimated the pairwise similarity between the ITS sequence of the query strain and the sequences that are given in the ITS56 datasets (Cai and Druzhinina 2021). Tef1-α and rpb2 sequences were subjected to Multiloci Identification System for Trichoderma (MMIT) (mmit.china-cctc.org) (Dou et al. 2020) and NCBI nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to detect the most closely related species. A sufficient number of representative sequences (n > 6) of Trichoderma species (Bissett et al. 2015; Cai and Druzhinina 2021) that are closely related to the new species were chosen for phylogenetic analyses. Protocrea illinoensis and Protocrea farinose were selected as outgroups.

Sequences were aligned with ClustalW (Thompson et al. 1994) and adjusted manually. Gaps were treated as missing data. Phylogenetic analyses were performed with tef1-α or rpb2 with MEGA-X software (Kumar et al. 2018). Model testing was used to find the best DNA model for ML analyses. The stability of clades was evaluated by bootstrap tests with 1000 replications. Bootstrap values above 50% were indicated on the corresponding branches. Maximum Parsimony (MP) analyses were performed with MEGA-X software (Kumar et al. 2018) using 1000 replicates of heuristic search with the random addition of sequences and tree bisection reconnection as the MP search method. All molecular characters were weighted equally and gaps were treated as missing data. Bootstrap proportions were calculated from 1000 replicates, each with 10 replicates of random addition of taxa.

Results

Molecular identification and sequence analyses

We estimated the pairwise similarity between the ITS sequence of the query strain and the sequences that are given in the ITS56 datasets. All the query strain belongs to the genus Trichoderma spp. with similarity value > 81% compared to the sequences in the datasets. The query strain has unique sequences of tef1-α and rpb2 (does not meet the sp∃!(rpb299≅tef197) standard for known Trichoderma species).

Tef1-α or rpb2 sequences of new taxon were subjected to MMIT and NCBI nucleotide BLAST and 34 representative sequences of Trichoderma species (all the species with similarity rpb2 and tef1-α ≥ 92% in the Viride clade) that are closely related to the new species, were chosen for phylogenetic analyses of T. hailarense, T. nordicum and T. vadicola. The accession numbers for the sequences are provided in Table 1. Model testing suggested using the Hasegawa-Kishino-Yano model (HKY; Hasegawa 1985) with gamma distributed with invariant sites (HKY+G+I) for ML analyses of tef1-α and the Tamura-Nei model (TN93; Tamura 1993) with gamma distributed substitution rates (TN93+G) for rpb2. The phylogenetic trees from rpb2 or tef1-α analyses are shown in Figs 1 and 2, respectively. Sequence alignments and the trees obtained were deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S29166). Twenty representative sequences of closely-related Trichoderma species (all the Trichoderma species in the Polysporum clade) were chosen for phylogenetic analyses of T. macrofasciculatum and T. shangrilaense (Table 1). Model testing suggested using the Hasegawa-Kishino-Yano model (HKY; Hasegawa 1985) with gamma distributed substitution rates (HKY+G) for ML analyses of tef1-α and the Kimura 2-parameter (K2; Kimura 1980) with gamma distributed substitution rates (K2+G) for rpb2. The phylogenetic trees from rpb2 or tef1-α analyses are shown in Figs 3 and 4, respectively. Sequence alignments and the trees obtained were deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S29166).

Table 1.
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Strain numbers and GenBank accession numbers of sequences used for phylogenetic analyses.

Species Clade Strain GenBank accession numbers
ITS tef1-α rpb2
T. adaptatum Viride HMAS 248800 – KX428024 KX428042
T. albofulvopsis Viride HMAS 273760 – KU529127 KU529138
T. alutaceum Polysporum CBS 120535 FJ860725 FJ179567 FJ179600
T. appalachiense Viride GJS 97-243 DQ315419 DQ307503 DQ307503
T. atlanticum Polysporum CBS 120632 FJ860781 FJ860649 FJ860546
T. atroviride Viride CBS 119499 FJ860726 FJ860611 FJ860518
T. bavaricum Polysporum CBS 120538 FJ860737 FJ860621 FJ860527
T. beijingense Viride HMAS 248804 – KX428025 KX428043
T. bifurcatum Viride HMAS 248795 – KX428018 KX428036
T. caerulescens Viride S195 JN715589 JN715621 JN715604
T. composticola Viride S590=CBS 133497 – KC285631 KC285754
T. europaeum Polysporum S611 – KJ665489 KJ665268
T. foliicola Polysporum Hypo 645 JQ685871 JQ685862 JQ685876
T. gamsii Viride S488 – JN715613 KJ665270
T. hailarense Viride WT17901*= ACCC 39711 MH287485 MH287505 MH287506
T. hailarense Viride WT17803 MH606226 MH606229 MH606232
T. hispanicum Viride S453=CBS 130540 JN715595 JN715659 JN715600
T. istrianum Viride S123 – KJ665521 KJ665280
T. laevisporum Viride HMAS 273756 – KU529128 KU529139
T. lacuwombatense Polysporum GJS 99-198 – KJ665547 KJ665286
T. leucopus Polysporum CBS 122499 FJ860764 FJ179571 FJ179605
T. luteffusum Polysporum CBS 120537 FJ860773 FJ860645 FJ860543
T. macrofasciculatum Polysporum WT37805* = ACCC 39712 MH287487 MH287509 MH287493
T. macrofasciculatum Polysporum WT37810 MH287488 MH287510 MH287494
T. mediterraneum Polysporum S190 – KJ665568 KJ665296
T. minutisporum Polysporum GJS 90-82 – KJ665618 KJ665316
T. neokoningii Viride CBS 120070=GJS 04-216 DQ841734 KJ665620 KJ665318
T. nordicum Viride WT13001* =ACCC 39713 MH287483 MH287501 MH287502
T. nordicum Viride WT61001 MH287484 MH287503 MH287504
T. nybergianum Polysporum CBS 122500 FJ860791 FJ179575 FJ179611
T. ochroleucum Viride CBS 119502 FJ860793 FJ860659 FJ860556
T. olivascens Viride S475=CBS 132574 – KC285624 KC285752
T. pachypallidum Polysporum CBS 122126 FJ860798 FJ860662 JQ685879
T. palidulum Viride HMAS 275665 – MG383493 MG383487
T. paratroviride Viride CBS136489 – KJ665627 KJ665321
T. paraviridescens Viride CBS 119321 DQ677651 DQ672610 KC285763
T. parapiluliferum Polysporum CBS 120921 FJ860799 FJ179578 FJ179614
T. piluliferum Polysporum CBS 120927 FJ860810 FJ860674 FJ179615
T. placentula Polysporum CBS 120924 – FJ179580 FJ179616
T. polysporum Polysporum CPK 3131 – FJ860661 FJ860558
T. pruinosum Polysporum HMAS 247217 – MF371227 MF371212
T. samuelsii Viride S5=CBS 130537 JN715593 JN715651 JN715599
T. sempervirentis Viride S599=CBS 133498 – KC285632 KC285755
T. seppoi Polysporum CBS 122498 – FJ179581 FJ179617
T. shangrilaense Polysporum WT34004*= ACCC 39714 MH287489 MH287495 MH287496
T. shangrilaense Polysporum WT40502 MH606224 MH606227 MH606230
T. shaoguanicum Viride HMAS 248809 – KX428031 KX428049
T. sinoluteum Polysporum HMAS 252868 – KJ634777 KJ634744
T. speciosum Viride CGMCC 3.19079 MH113929 MH183184 MH155270
T. sphaerosporum Viride HMAS 273763 – KU529134 KU529145
T. subviride Viride HMAS 273761 – KU529131 KU529142
T. tardum Viride HMAS 248798 – KX428020 KX428038
T. trixiae Viride ATCC 32630 DQ315445 DQ307526 KC285770
T. vadicola Viride WT10708*= ACCC 39716 MH287491 MH287499 MH287511
T. vadicola Viride WT32801 MH606225 MH606228 MH606231
T. valdunense Viride CBS 120923 FJ860863 FJ860717 FJ860605
T. vinosum Viride GJS 99-158=CBS 119087 AY380904 AY376047 KC285779
T. viridarium Viride S136=CBS 132568 – KC285658 KC285760
T. viride Viride CBS 119327 DQ677655 DQ672617 EU711362
T. viridescens Viride S452=CBS 132573 – KC285646 KC285758
T. viridialbum Viride S250=CBS 133495 – KC285706 KC285774
T. virilente Viride S281=CBS 132569 – KC285692 KC285767
T. vulgatum Viride HMAS 248796 – KX428019 KX428037
Protocrea illinoensis Outgroup TFC 96-98 EU703930 EU703905 EU703952
Protocrea farinosa Outgroup CPK 3144 EU703917 EU703894 EU703938

Newly-sequenced material is indicated in bold type.

The MP analyses using tef1-α and rpb2 (Fig. 1) resulted in topologically similar trees with minor differences. Each new taxon of Trichoderma formed a distinct clade and meets the Trichoderma new species standard (does not meet the sp∃!(rpb299≅tef197) standard for known Trichoderma species) (Cai and Druzhinina 2021). The similarity value between the new species and the reference strain is shown in the number on the right side of the phylogenetic trees.

Trichoderma hailarense clearly separated from T. gamsii S488 (with similarity rpb2 = 97.32% and tef1-α = 97.43%) and T. neokoningii CBS120070 (with similarity rpb2 = 96.86% and tef1-α = 96.66%). Trichoderma nordicum was associated, but clearly separated from T. paratroviride CBS136489 with similarity rpb2 = 98.15% and tef1 = 94.43%. Trichoderma vadicola was associated, but clearly separated from T. caerulescens S195 (with similarity 95.26%), T. tardum HMAS 248798 (with similarity 95.57%) and T. bifurcatum S195 (with similarity 95.76%) in the phylogenetic tree of the rpb2. However, there were differences in the phylogenetic tree of the tef1-α; T. vadicola was associated and separated from T. palidulum HMAS 275665 (with similarity 94.52%), T. istrianum S123 (with similarity 96.14%), T. ochroleucum CBS 119502 (with similarity 93.49%) and T. albofulvopsis HMAS 273760 (with similarity 93.16%) (Fig. 1). The strains of T. macrofasciculatum were associated, but clearly separated from T. polysporum C.P.K. 3131 with similarity rpb2 = 96.41% and tef1-α = 92.81%; T. shangrilaense was closely related and separated from T. parapiluliferum CBS 120927 with similarity rpb2 = 98.93% and tef1-α = 96.35% (Fig. 2).

Figure 1. 

Phylogenetic tree, based on the Maximum Likelihood analysis of the rpb2 (left; InL = -5930.92) and tef1-α (right; InL = -7681.95) dataset. Bootstrap values of Maximum Likelihood (left) and Maximum Parsimony (right) above 50% are indicated at the nodes. The tree is rooted with Protocrea illinoensis TFC 9698 and P. farinose CPK 3144. New species proposed here are indicated in bold. The type strains are indicated with an asterisk (*) after the strain number. Results of the pairwise sequence similarity are illustrated on the dashed lines between the query strain and its closely-related species (arrows point to the reference strains).

Figure 2. 

Phylogenetic tree based on the Maximum Likelihood analysis of the rpb2 (left; InL = -5912.02) and tef1-α (right; InL = -9060.53) dataset. Maximum Likelihood bootstrap values (left) and MPBP (right) above 50% are indicated at the nodes. The tree is rooted with Protocrea illinoensis TFC 9698 and P. farinose CPK 3144. New species proposed here are indicated in bold. The type strains are indicated with an asterisk (*) after the strain number. Results of the pairwise sequence similarity are illustrated on the dashed lines between the query strain and its closely-related species (arrows point to the reference strains).

Taxonomy

Trichoderma hailarense G.Z. Zhang, sp. nov.

MycoBank No: 821318
Fig. 3

Etymology

The specific epithet “hailarense” refers to the locality, the Hailar River Basin in Inner Mongolia of China where the holotype was found.

Typification

China. Inner Mongolia, Hailar River Basin, 618 m (altitude), isolated from soil, 17 September 2016, G.Z. Zhang (Holotype WT 17901).

Diagnosis

Phylogenetically, Trichoderma hailarense formed a distinct clade and is related to T. gamsii and T. neokoningii (Fig. 1). The sequence similarity of rpb2 with T. gamsii S488 and T. neokoningii CBS120070 was 97.32% and 96.86%, respectively and the sequence similarity of tef1-α with T. gamsii S488 and T. neokoningii CBS120070 was 97.43% and 96.66%, respectively. Colonies of T. hailarense did not form conidia on PDA and conidia of T. hailarense on other media were obovoid, delicately roughened and easily distinguished from those of T. gamsii and T. neokoningii.

Teleomorph

Unknown.

Growth optimal at 30 °C, slow at 35 °C on all media. Colony radius after 72 h at 30 °C 53–56 mm on PDA, 54–56 mm on CMD, 33–37 mm on MEA and 33–36 mm on SNA. Colony radius after 72 h at 35 °C 13–15 mm on PDA, 10–14 mm on CMD, 9–12 mm on MEA and 10–12 mm on SNA. Aerial mycelia abundant, arachnoid on PDA after 72 h at 25 °C under 12 h photoperiod. Conidiation started around the inoculation point after 7 days on PDA, with relatively few or small conidia. Diffusing pigment or distinctive odour absent. Conidiation started around the inoculation point after 7 days on MEA, forming a few large pustules, cream yellow. On SNA, aerial mycelia were few, forming a few large pustules around the inoculation point in age, cream-yellow. Conidiophores and branches narrow and flexuous, tending to be regularly verticillate, forming a pyramidal structure, with each branch terminating in a cruciate whorl of up to five phialides. Phialides, lageniform, (8.0–)9.4–13.1(–15.5) × (2.5–)3.0–3.5(–3.6) μm (mean = 11.2 × 3.3 μm), base 1.8–2.5 μm (mean = 2.1 μm); phialide length/width ratio (2.33–)2.7–4.4(–5.9) (mean = 3.4). Conidia obovoid, (4.2–)4.3–4.7(–4.9) × (3.4–)3.6–3.9(–4.1) μm (mean = 4.5 × 3.7 μm), length/width ratio 1.1–1.4 (mean = 1.2), delicately roughened. Chlamydospores: (7.0–)7.5–8.2(–8.5) × (6.5–)7.0–7.5(–8.3) μm.

Figure 3. 

Trichoderma hailarense A–D cultures on different media incubated at 25 °C for 14 days (A on PDA B on MEA C on CMD D on SNA) E, G–K conidiophores and phialides F chlamydospores L, M conidia. Notes: E on MEA F–M on PDA A–M from WT17901. Scale bars: 10 μm (E–J).

Distribution

China. Inner Mongolia.

Additional specimen examined

China. Inner Mongolia, Hulun Buir, 610 m (altitude), isolated from soil, 17 September 2016, J.D. Hu (WT17905).

Notes

Phylogenetically Trichoderma hailarense is related to T. gamsii and T. neokoningii (Fig. 1) and does not meet the sp∃!(rpb299≅tef197) standard for T. gamsii or T. neokoningii. Morphologically, colonies of T. gamsii and T. neokoningii on PDA formed conidia sporadically or in hemispherical pustules and conidia of T. gamsii and T. neokoningii were ellipsoidal to oblong, smooth-walled (Jaklitsch et al. 2006). However, colonies of T. hailarense did not form conidia on PDA and conidia of T. hailarense on other media were obovoid, delicately roughened and easily distinguished from those of T. gamsii and T. neokoningii.

Trichoderma macrofasciculatum G.Z. Zhang, sp. nov.

MycoBank No: 821299
Fig. 4

Etymology

The specific epithet “macrofasciculatum” refers to the morphological feature of the conidiation, conidiophores aggregated into large fascicles in concentric rings.

Typification

China, Sichuan, Nine-Village Valley, 2405 m (altitude), isolated from soil, 24 September 2016, G.Z. Zhang (Holotype WT 37805).

Diagnosis

Phylogenetically, Trichoderma macrofasciculatum WT37805 and WT37810 formed a distinct clade and is related to T. polysporum C.P.K. 3131 in the Polysporum clade, but the similarities of rpb2 and tef1-α between these two species were only 96.41% and 92.81%, respectively. Trichoderma macrofasciculatum cannot grow at 35 °C as T. polysporum and the former formed large and white pustules in concentric rings at 25 °C, elongations were rarely observed and conidia had few guttules, which are distinct from T. polysporum.

Teleomorph

Unknown.

Growth optimum at 20 °C, slow or limited at 30 °C, absent at 35 °C. Colony radius after 72 h at 25 °C 21–24 mm on PDA, 23–27 mm on CMD, 17–20 mm on MEA and 12–16 mm on SNA. Aerial mycelia abundant on PDA and MEA after incubation for 72 h at 25 °C under a 12 h photoperiod. Conidiation typically in pustules in concentric rings on PDA, solitary or aggregated, producing a farinose to granular mat. Diameter of pustules up to 2.2 mm, pompon-like, white. Diffusing pigment and distinct odour absent. Conidiation on MEA typically in pustules in concentric rings, pompon-like as on PDA. On CMD, aerial mycelia sparsely developed. Conidiation aggregated in sporadic pustules near the colony margin, white. On SNA, aerial mycelia few and conidiation not observed. Conidiophores and branches irregularly branched in a dendriform structure, with each branch terminating in a cruciate whorl of up to five phialides. Hyphal septa clearly visible. Phialides flask-shaped, often curved, (4.9–)5.6–7.8(–8.8) × (2.8–)3.0–3.2(–3.4) μm (mean = 6.7 × 3.1 μm), 1.8–2.6 μm (mean = 2.2 μm) near the base; phialide length/width ratio (1.5–)1.8–2.4(–2.8) (mean = 2.1). Conidia subglobose to ellipsoid, hyaline, smooth, with one or few distinctly verrucose, (2.6–)2.8–3.3(–3.6) × (2.4–)2.5–2.7(–2.9) μm (mean = 3.0 × 2.6 μm), length/width ratio 1.0–1.3 (mean = 1.2). Chlamydospores not observed.

Figure 4. 

Trichoderma macrofasciculatum A–C cultures on different media incubated at 25 °C for 7 days (A on PDA B on MEA C on CM) D–G conidiophores and phialides H conidia with guttules. Notes: A, D, E from WT37810 B, C, F, G from WT37805. Scale bars: 10 μm (D–H).

Distribution

China, Sichuan Province.

Additional material examined

China, Sichuan, Nine-Village Valley, 2405 m (altitude), isolated from soil, 24 September 2016, G.Z. Zhang (WT 37810).

Notes

Phylogenetically Trichoderma macrofasciculatum WT 37805 is related to T. polysporum C.P.K. 3131 in the Polysporum clade (Fig. 1), but the similarities of rpb2 and tef1-α between these two species were only 96.41% and 92.81% respectively, with 94 and 41 bp differences amongst 1311 and 1152 bp. Trichoderma macrofasciculatum cannot grow at 35 °C as T. polysporum and the former formed large and white pustules in concentric rings at 25 °C, elongations were rarely observed and conidia had few guttules, which are distinct from T. polysporum (Lu et al. 2004).

Trichoderma nordicum G.Z. Zhang, sp. nov.

MycoBank No: 8212301
Fig. 5

Etymology

“nord” means found in the north of China.

Holotype

China, Beijing, Yu-yuan-tan Park, 43 m (altitude), isolated from soil, 27 October 2016, G.Z. Zhang (Holotype WT 13001), ex-type culture ACCC 39713.

Diagnosis

Phylogenetically Trichoderma nordicum is related to T. paratroviride, but the sequence similarities of rpb2 and tef1-α were 98.15% and 94.43%, respectively. That does not meet the sp∃!(rpb299≅tef197) standard for T. paratroviride or other known Trichoderma species. Morphologically, conidiophores of T. paratroviride consisting of a main axis and often distantly-spaced side branches, not re-branching. Conidiophores of T. nordicum are branched in a more complex manner; conidia are larger than those of T. paratroviride.

Teleomorph

Unknown.

Growth optimal at 25 °C, slow or limited at 30 °C, absent at 35 °C. Colonies grew fast on PDA, CMD and MEA and slow on SNA. Colony radius after 72 h at 25 °C 67–71 mm on PDA, 68–71 mm on CMD, 51–55 mm on MEA and 21–24 mm on SNA. Aerial mycelia sparse on PDA after 72 h at 25 °C under 12 h photoperiod and conidiation developed within 48 h beginning at the inoculation point and progressed around, grey-white at first and slowly turning green. Diffusing pigment or distinctive odour absent. Aerial mycelia sparse and flocculence on MEA after 72 h at 20 °C under 12 h photoperiod. Conidia developed within 48 h beginning near the colony margin on MEA, grey-white at first and slowly turning green, transparent liquid secreted. Aerial mycelia few on SNA and CMD after 72 h at 25 °C, conidia formed around the inoculation point and in distinct concentric rings after 96 h under 12 h photoperiod on SNA and CMD, diffusing pigment not produced. Conidiophores and branches narrow and flexuous, tending to be regularly verticillate forming a pyramidal structure, each branch terminating in a cruciate whorl of up to five phialides. Phialides, lageniform, (6.2–)7.2–10.3(–12.9) × (2.6–)2.9–3.2(–3.4) μm (mean = 8.8 × 3.1 μm), 1.6–2.3 μm (mean = 1.9 μm) near the base; phialide length/width ratio (2.1–)2.4–3.4(–4.3) (mean = 2.9). On PDA, phialides curved, distinguished from those on other media. Conidia, globose to obovoidal, (4.1–)4.4–4.8(–5.0) × (4.0–)4.1–4.4(–4.6) μm (mean = 4.6 × 4.3 μm), length/width ratio 1.0–1.2 (mean = 1.1). Chlamydospores sometimes present, (8.7–)9.8 × 10.4(–12.5) μm.

Figure 5. 

Trichoderma nordicum A–D cultures on different media at 25 °C after 10 days (A on PDA B on MEA C on CMD D on SNA) E–G, I, J conidiophores and phialides H conidia. Notes: E on PDA F–J on MEA A–D from WT13001 E–J from WT61001. Scale bars: 10 μm (E–J).

Distribution

China, Beijing and Hebei.

Additional specimen examined

China. Hebei, Bai-yang Lake, 19 m (altitude), isolated from soil, 15 September 2016, J.S. Li (WT 61001).

Notes

Phylogenetically, Trichoderma nordicum is related to T. paratroviride (Fig. 1), but the sequence similarities of rpb2 and tef1-α were 98.15% and 94.43%, respectively. That does not meet the sp∃!(rpb299≅tef197) standard for T. paratroviride or other known Trichoderma species. Morphologically, conidiophores of T. paratroviride consist of a main axis and often distantly-spaced side branches, not re-branching. Conidiophores of T. nordicum are branched in a more complex manner; conidia are larger than those of T. paratroviride (Jaklitsch and Voglmayr 2015).

Trichoderma shangrilaense G.Z. Zhang, sp. nov.

MycoBank No: 821300
Fig. 6

Etymology

“shangrilaense” was originally found at Shangrila in Yunnan Province of China.

Typification

China. Yunnan, Pudacuo National Park, 3611 m (altitude), isolated from soil, 21 June 2016, G.Z. Zhang (Holotype WT 34004), Ex-type culture ACCC 39714.

Diagnosis

Phylogenetically, Trichoderma shangrilaense is related to T. parapiluliferum (CBS 120921) (Fig. 1), but the sequence similarity of rpb2 between these two species was 98.93% and the sequence similarity of tef1-α was 96.35%. That does not meet the sp∃!(rpb299≅tef197) standard for T. parapiluliferum or other known Trichoderma species. Conidiophore main axis of T. shangrilaense fertile to apex, conidia obovoid to ellipsoid, easily distinguished from that of T. parapiluliferum.

Teleomorph

Unknown.

Growth optimal at 20 °C, slow, limited at 25 °C and absent at 30 °C or 35 °C. Colony radius after 72 h at 20 °C 19–21 mm on PDA, 23–24 mm on CMD, 19–21 mm on MEA and 8–11 mm on SNA. Aerial mycelia abundant, compact on PDA after 7 days at 20 °C under 12 h photoperiod, conidiation not easily formed and a yellow diffusing pigment developed near the inoculation point; conidiation formed unequal in size, white pustules after 14 days. Conidiophores and branches narrow and flexuous, forming a dendriform structure and irregularly branched, not rebranched, main axis to 4.3–5.0 µm wide, fertile to apex. Phialides, flask-shaped, often curved, (4.5–)5.7–9.0(–11.1) × (2.9–)3.2–3.5(–4.1) μm (mean = 7.4 × 3.4 μm), 1.6–3.4 μm wide (mean = 2.6 μm) near the base; phialide length/width ratio (1.5–)2.0–2.6(–3.0) (mean = 2.3). Conidia, obovoid to ellipsoidal, smooth, (3.3–)3.5–4.0(–4.4) × (2.8–)3.0–3.3(–3.5) μm (mean = 3.8 × 3.19 μm), length/width ratio 1.1–1.4 (mean = 1.2). Chlamydospores not observed.

Figure 6. 

Trichoderma shangrilaense A–D cultures (A on PDA, 25 °C, 10 days B on PDA, 25 °C, 21 days C on MEA, 25 °C, 21 days D on CMD, 25 °C, 21 days) E–G, I–K conidiophores and phialides H conidia A–K from WT34004. Scale bars: 10 μm (E–K).

Colony radius 28–33 mm, aerial mycelia abundant and floccose after 7 days at 20 °C under 12 h photoperiod. Conidiation slowly developing on MEA. After about 14 days, pompon-like, white fascicles developed. No diffusing pigment observed. On CMD after 7 days at 20 °C under 12 h photoperiod, colony radius 28–33 mm, aerial mycelia few. Conidiation formed flat or cushion-shaped pustules near the colony margin after 21 days and a yellow diffusing pigment developed near the inoculation point. On SNA after 7 days at 20 °C under 12 h photoperiod, colony mycelia sparse and no conidiation formed. After 10 days, pustules scattered around the periphery of the colony. Diffusing pigment not developed.

Distribution

China. Yunnan and Sichuan.

Additional specimen examined

China. Sichuan, Huanglong Nature Reserve, 3561 m (altitude), isolated from soil, 25 September 2016, Z. Li (WT 34012).

Notes

Phylogenetically, Trichoderma shangrilaense is related to T. parapiluliferum (CBS 120921) (Fig. 1), but the sequence similarity of rpb2 between these two species was 98.93% and the sequence similarity of tef1-α was 96.35%. The sequence similarity of tef1-α with the ex-type culture G.J.S. 91-60 (GenBank accession no. AY937444) was only 92%. Optimum temperature for growth of T. shangrilaense was 20 °C, no growth occurred at 30 °C as in T. parapiluliferum and conidiation structures consist of flat or cushion-shaped pustules, formed near the colony margin on MEA, SNA and CMD. Conidiophore main axis of Trichoderma parapiluliferum has conspicuous spiral sterile apical elongations, conidia ellipsoidal to oblong (Lu et al. 2004). Conidiophore main axis of T. shangrilaense fertile to apex, conidia obovoid to ellipsoid, easily distinguished from that of T. parapiluliferum.

Trichoderma vadicola G.Z. Zhang, sp. nov.

MycoBank No: 821316
Fig. 7

Etymology

The specific epithet “vadicola”, from the noun “vadum”, reflects the ecological environment and means that the species inhabits shallow water.

Typification

China. Shandong, 2 m (altitude), isolated from soil, 13 August 2016, G.Z. Zhang (Holotype WT 10708), Ex-type culture ACCC 39716.

Diagnosis

Phylogenetically, Trichoderma vadicola is related to T. caerulescens in the Viride clade (Fig. 1), but the sequence similarity of tef1-α and rpb2 between these species was all 95%. Morphologically, colonies of T. vadicola and T. caerulescens on PDA have similar features, such as abundant aerial hyphae, forming strands and a whitish hairy or floccose mat. However, the former Trichoderma vadicola formed no or relatively few conidia and the latter forming greyish-bluish patches around the plug. On CMD, T. caerulescens peculiar greyish-blue pigment formed after 1–2 months and conidiophores simply or slightly branched; the former had no observed diffusing pigment and conidiophores branched in a complex manner in pyramidal structure or tree-like.

Teleomorph

Unknown.

Growth optimal at 25 °C, no grow at 35 °C on all media. Colony radius after 72 h at 25 °C 25–29 mm on PDA, 24–27 mm on CMD, 23–26 mm on MEA and 22–26 mm on SNA. Aerial mycelia abundant on PDA after 72 h at 25 °C under 12 h photoperiod, forming strands and floccose mat. Conidiation not formed or relatively few. No diffusing pigment or distinctive odour was produced. On MEA after 72 h at 25 °C under 12 h photoperiod, aerial mycelia abundant, floccose. After 7 days, mycelia covered the plate and conidia appeared, effuse, granuliform. On CMD after 72 h at 25 °C under 12 h photoperiod, aerial mycelia not observed. After 7 days, mycelia covered the plate and conidia developed near the colony margin. On SNA after 72 h at 25 °C under 12 h photoperiod, aerial mycelia not observed. After 7 days, mycelia covered the plate, aerial mycelia floccose and conidia formed, effuse. Conidiophores and branches regularly verticillate, formed a pyramidal structure, each branch terminating in a cruciate whorl of 3–5 phialides. Phialides lageniform, (8.3–)9.9–12.3(–15.1) × (2.0–)2.6–3.2(–3.4) μm (mean = 11.1 × 2.9 μm), 1.1–2.9 μm wide (mean = 1.9 μm) near the base; phialide length/width ratio (2.7–)3.2–4.6(–6.6) (mean = 3.9). Conidia subglobose or obovoidal, (3.5–)3.7–4.3(–4.8) × (3.2–)3.4–3.6(–3.8) μm (mean = 4.0 × 3.5 μm), length/width ratio 1.0–1.3 (mean = 1.1). Chlamydospores not observed.

Figure 7. 

Trichoderma vadicola A–D cultures on different media at 25 °C (A on PDA after10 days B on MEA, after 7 days C on CMD after 7 days D on SNA after 7 days) E–I conidiophores and phialides J conidia. Notes: E, F, H–J on MEA G on SNA A–J from WT10708. Scale bars: 10 μm (E–J).

Distribution

China. Shandong and Yunnan Provinces.

Additional specimen examined

China. Yunnan, Shangri-La, Pudacuo National Park, 3551 m (altitude), isolated from soil, 21 September 2016, H.T. Yang (WT 10713).

Notes

Phylogenetically, Trichoderma vadicola is related to T. caerulescens in the Viride clade (Fig. 1), but the sequence similarity of tef1-α and rpb2 between these species was all 95%, with 62 and 60 bp differences amongst 1218 and 1130 bp, respectively. Morphologically, colonies of T. vadicola and T. caerulescens on PDA have similar features, such as abundant aerial hyphae, forming strands and a whitish hairy or floccose mat. However, the former Trichoderma vadicola formed no or relatively few conidia, with the latter forming greyish-bluish patches around the plug. On CMD, T. caerulescens formed peculiar greyish-blue pigment after 1–2 months and conidiophores simply or slightly branched (Jaklitsch et al. 2012); the former had no observed diffusing pigment and conidiophores branched in a complex manner in pyramidal structure or tree-like.

Discussion

In this paper, five new species of Trichoderma were described from wetland soils. An ML tree was reconstructed, based on individual tef1-α and rpb2, to explore the taxonomic positions of the new species. Our phylogenetic analyses showed that the five new Trichoderma species belong to the Polysporum clade or the Virde clade. Trichoderma macrofasciculatum and T. shangrilaense belong to the Polysporum clade (as Pachybasium core group; Jaklitsch 2011) (Fig. 2). Here, we added two new species, T. macrofasciculatum and T. shangrilaense, which are close to T. polysporum and T. parapiluliferum. Morphologically, species in this clade are heterogeneous, comprising teleomorphs with upright, stipitate or small pulvinate stromata. The teleomorphs of T. macrofasciculatum and T. shangrilaense have not been found at present, but their asexual characteristics, such as conidiation in white pustules, resemble other species in this clade.

Trichoderma nordicum, T. vadicola, and T. hailarense belong to the Viride clade (formerly section Trichoderma) (Fig. 1). Here, we added three new species, T. hailarense T. nordicum and T. vadicola, which are all located in the unnamed branches and close to T. gamsii/T. neokoningii, T. paratroviride and T. caerulescens, respectively. Phenotypically, phialides of three new species are lageniform and have green conidia, which is consistent with the characteristics of Trichoderma species in the Viride clade. Only T. hailarense has coarsely warted conidia, two other species being smooth-walled.

At present, the identification of Trichoderma species is mainly based on phylogenetic analysis and morphological characteristics. The new species hypothesis needs to be supported by the topology of both phylograms (rpb2 and tef1-α). However, there are no numerical standards of the similarity threshold at the level which is sufficient for identification for most of the existing species (Cai and Druzhinina 2021) and this has led to many inaccuracies in the original identification of Trichoderma. In the phylogenetic tree constructed in this paper, some Trichoderma species combinations showed low bootstrap values (Figs 1 and 2) and have high similarity, which meet the sp∃!(rpb299≅tef197) standard developed by Cai and Druzhinina (2021). They may be identified as the same Trichoderma species: for example, T. viridialbum, T. viridarium and T. sempervirentis, which belong to the Trichoderma viridescens complex (Jaklitsch et al. 2013), may still be identified as T. viridescens. T. paraviridescens, T. trixiae and T. appalachiense may be identified as the same Trichoderma species.

Trichoderma species cannot be identified by phylogenetic analysis without considering the sequence similarity values. Therefore, Cai and Druzhinina (2021) developed a protocol for molecular identification of Trichoderma that requires analysis of the three DNA barcodes (ITS, tef1-α and rpb2). Molecular identification of Trichoderma species can be achieved, based on the analysis of sequence similarities between the query strain and the reference strains that are analysed for tef1-α (≥ 97%) and rpb2 (≥ 99%). If this condition is not met, the query strain may be a new species of Trichoderma and the new species hypothesis can be made, based on sequence similarities and phylogenetic concordance, i.e. analysis of single loci tree topologies for tef1-α and rpb2 and must be verified, based on morphology. In the identification process of the new species, we made full reference to this protocol and there were sufficient differences in sequence similarity between the newly-identified species and the reference species, as well as significant differences in morphological characteristics. According to Jaklitsch et al. (2013), the morphology of T. viridialbum, T. viridarium and T. sempervirentis (meeting the sp∃!(rpb299≅tef197) standard) shows a high degree of similarity and should still be identified as T. viridescens. This also fully verified that the identification protocol developed by Cai and Druzhinina (2021) is helpful to ensure the accuracy of Trichoderma species identification, which is worth promoting and applying, especially for the identification of Trichoderma species.

Acknowledgements

The authors sincerely thank Jin Dong Hu, Zhe Li and Ji Shun Li for providing the soil specimens. The authors are grateful to Konstanze Bensch for advising on the Latin names. This work was financed by the Shandong Key Research and Development Project (2014GSF121028; 2019GSF107086), Shandong Major Science and Technology innovation project (2019JZZY020610) and National Natural Science Foundation of China (Project no. 31700426; 31901928).

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Supplementary material

Supplementary material 1 

Five new species of Trichoderma from moist soils in China

Guang-Zhi Zhang, He-Tong Yang, Xin-Jian Zhang, Fang-Yuan Zhou, Xiao-Qing Wu, Xue-Ying Xie, Xiao-Yan Zhao, Hong-Zi Zhou

Data type: COL

Explanation note: Trichoderma hailarense G.Z. Zhang, sp. nov.; Trichoderma macrofasciculatum G.Z. Zhang, sp. nov.; Trichoderma nordicum G.Z. Zhang, sp. nov.; Trichoderma shangrilaense G.Z. Zhang, sp. nov.; Trichoderma vadicola G.Z. Zhang, sp. nov.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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