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Research Article
Two new Trichoderma species (Hypocreales, Hypocreaceae) isolated from decaying tubers of Gastrodia elate
expand article infoChuwen Ye‡, Tingting Jing‡, Yuru Sha‡, Minghe Mo‡, Zefen Yu‡
‡ Yunnan University, Kunming, China

Corresponding author: Minghe Mo ( minghemo@163.com )

Corresponding author: Zefen Yu ( zfyu2021@163.com )

Academic editor: Sajeewa Maharachchikumbura
© 2023 Chuwen Ye, Tingting Jing, Yuru Sha, Minghe Mo, Zefen Yu.
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: Ye C, Jing T, Sha Y, Mo M, Yu Z (2023) Two new Trichoderma species (Hypocreales, Hypocreaceae) isolated from decaying tubers of Gastrodia elate. MycoKeys 99: 187-207. https://doi.org/10.3897/mycokeys.99.109404
Open Access

Abstract

Species of Trichoderma are widely distributed around the world. In this study, two new species in Trichoderma, named as T. albidum and T. variegatum, were introduced and illustrated. These species were isolated from diseased tubers of Gastrodia elata in China and identified based on morphological characteristics and multi-gene sequence analyses of three loci that is the internal transcribed spacer regions of the ribosomal DNA (ITS), the translation elongation factor 1-α encoding gene (tef1-α) and the gene encoding the second largest nuclear RNA polymerase subunit (rpb2). Distinctions between the new species and their close relatives were discussed. According to results of the phylogenetic analyses, T. albidum belonged to the Harzianum clade and T. variegatum are grouped with species of the Spirale clade. The expansion of two clades provided research foundations for the prevention and control of tuber diseases in G. elata.

Key words

Multi-gene phylogeny, plant disease, taxonomy, Trichoderma

Introduction

Trichoderma Pers. is important ecologically and economically. These fungi are widely used in agriculture, industry and medicine, including being used as bio-fungicides to control plant diseases, and as regulators of plant growth, fortifiers of soil fertility, and producers of antibiotics and enzymes (Lorito et al. 2010; Saravanakumar and Kathiresan 2014; Bischof et al. 2016; Adnan et al. 2017; Zhang et al. 2022). Furthermore, some species have great potential to remediate soil and water pollution as well as to manufacture gold or silver nanoparticles (Harman et al. 2004; Anand et al. 2006; Mazyar et al. 2010). However, several species were reported as the causal agents of green mold disease in mushroom cultivation, the disease of Gastrodia elata Bl. 1856 and opportunistic pathogens of humans (Park et al. 2006; Komon-Zelazowska et al. 2007; Sandoval-Denis et al. 2014; Han et al. 2017).

Trichoderma is a hyper-diverse fungal genus. Members of Trichoderma are widely distributed in a variety of ecosystems, including natural soils, decaying wood and bark, and living plant tissues (Samuels et al. 2006; Samuels et al. 2012; Jaklitsch and Voglmayr 2015; Zhang and Zhuang 2017). The initial species-level identification of the genus was based on their morphological characteristics. Nevertheless, as more species were discovered, taxonomic studies of the genus became increasingly complicated due to overlapping morphological traits among species (Druzhinina et al. 2005; Han et al. 2017; Barrera et al. 2021; Zhang et al. 2022). Misidentification species can have profound negative impacts on plant quarantine, industrial applications, and health of human and animal (Sandoval-Denis et al. 2014; Chaverri et al. 2015).

With the development of the times and the progress of science and technology, our research on fungal phylogeny has gradually transitioned from relying on morphological methods to relying on molecular biology methods. DNA sequence analysis was introduced and has provided more reliable identification for Trichoderma species. Numerous loci were considered for use in Trichoderma identifications and phylogenetic analyses, e.g. internal transcribed spacer regions of the ribosomal DNA, the translation elongation factor 1-α encoding gene, the gene encoding the second largest nuclear RNA polymerase subunit, α-actin, calmodulin, chitinase 18-5 (Kullnig-Gradinger et al. 2002; Druzhinina et al. 2012; Chaverri et al. 2015; Chen and Zhuang 2017a; Zhang et al. 2022). Specifically, tef1-α and rpb2 have facilitated rapid and accurate species identifications and have been used in the phylogenetic analyses and identification of novel species of Trichoderma in recent years (Cai et al. 2022). Analyses using only ITS may only be able to identify to the genus level or lead to errors due to fragment length, copy number and other problems, so it is necessary to add rpb2 and tef1 to improve the systematic analysis. It was shown that the multi-gene sequence analysis of ITS, rpb2 and tef1 could identify 60% of the current Trichoderma species, while the other loci were not suitable for gene barcoding due to the small gene size and distribution range (Cai et al. 2022). In contrast, cal and chi18-5 are rarely used due to their missing adequate sequence data or low sequence variability (Druzhinina et al. 2012; Bissett et al. 2015; Jaklitsch and Voglmayr 2015; Zhu and Zhuang 2015; Qin and Zhuang 2016). Furthermore, the large subunit of ATP citrate lyase (acl1) was recently introduced for taxonomic research of the genus, which turns out to be efficient (Jaklitsch et al. 2013). Currently, the combination of multi-loci phylogenetic analyses and phenotypic characteristics have been extensively used for species delineation of Trichoderma. Relying on this method, a number of species which were misclassified previously have been re-identified as new species, so the number of species in the genus has increased dramatically. (Plessis et al. 2018; Innocenti et al. 2019). In addition, morphological approaches remain important to validate and complement the phylogenetic results.

Trichoderma contains more than 400 species belonging to different clades and Harzianum clade and Sprale clade are two of them (Wijayawardene et al. 2020). Since the systematic revision of species in the Harzianum clade was provided by Chaverri et al. (2015), a large number of new species have been described and recorded. The Harzianum clade now contains more than 60 species (Gu et al. 2020). Green ascospores are a common feature of the Harzianum clade (Zhu and Zhuang 2015). Species in Harzianum clade have antifungal properties and bio-control ability, and they can effectively suppress soil-borne plant pathogens. Most of the species can be isolated from soil, rotting wood, other fungi, and plant endophytes (Chaverri et al. 2015). Trichoderma harzianum is one of the most well-known species in Harzianum clade. The Spirale clade is smaller in size compared to the Harzianum clade. The Spirale clade was identified as a separate terminal branch by Jaklitsch and Voglmayr (2015), and was authenticated by later researchers (Chen and Zhuang 2017a). T. hunanense K. Chen & W.Y. Zhuang, T. longisporum K. Chen & W.Y. Zhuang, and T. spirale Bissett are the species of this clade (Chen and Zhuang 2017a). Species in Spirale clade share the following similar features: producing yellow pigments on plates, possessing oblong conidia and forming hairy pustules (Chen and Zhuang 2017a). Identification and complementation of species in two clade is of significance to enrich the species diversity of both branches.

In the present study, 78 isolates obtained from the diseased Gastrodia elata Blume collected from Xiaocaoba, Zhaotong were found to belong to Trichoderma after preliminary identification and classification by ITS sequence. Based on morphological characteristics and DNA sequence data at three loci: the genes encoding RNA polymerase II subunit (rbp2) and translation elongation factor 1-α gene (tef1-α), and ITS regions of the nuclear ribosomal RNA gene, a new species belonging to the Harzianum clade and the other belonging to the Spirale clade were described and illustrated.

Materials and methods

Sample collection and isolation

Tubers of Gastrodia elata with rot symptoms were collected from Xiaocaoba, Yiliang County, Zhaotong city, Yunnan province, China. Samples were placed in sterile plastic bags, labeled, and transported to the laboratory. Infected G. elata were first washed in running tap water and autoclaved water, then surface disinfection with consecutive immersions was conducted for 30 s in 75% ethanol, 2 min in 1.5% sodium hypochlorite, then they were finally rinsed three times with autoclaved water and air-dried. Symptomatic tissues were cut into about 5 × 5 mm slices and placed on potato dextrose agar (PDA; 200 g potato, 20 g dextrose, 18 g agar, 1000 ml distilled water) plates. Petri dishes were sealed, incubated at 25 °C, and examined periodically. A small amount of hyphal tip cells was picked up and transferred to PDA medium when fungi grew out from infected tissues. The pure strains were further transferred and incubated on PDA, cornmeal agar (CMA; 20 g cornmeal, 18 g agar, 1000 ml distilled water) and synthetic low nutrient 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, 1000 ml distilled water) at 25 °C. After incubation, the colony and the microscopic morphology on PDA, CMA and SNA plates were observed, measured and photographed. Microscopic observations were performed using a BX51 microscope (Olympus) and with sterile water as a mounting medium for microscopy. Microscopic structures such as mycelium, conidiophores, conidia and phialides were observed and photographed, and at least 30 individuals of data were measured for each structure. Colony colors (surface and reverse) were confirmed based on Rayner’s color charts (Rayner 1970).

The pure cultures and dried cultures were deposited in the Herbarium of the Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, Yunnan, P. R. China ( YMF).

DNA extraction, amplification and sequencing

DNA was extracted from fresh mycelia harvested from PDA plates after 4 days of incubation at 25 °C. 0.5g fungal mycelia we collected was transferred into a 1.5ml microcentrifuge tube with 0.7–0.8ml lysis buffer (7 mol/L Urea, 50 mmol/L Tris-HCl, 62.5 mmol/L NaCl, 1% SDS). The mixture was spun at 12000 r/min for 5 min and the aqueous phase was transferred into a new 1.5 ml tube. An equal volume of DNA extract (phenol/chloroform/ isoamyl alcohol, 25:24:1) was added into the homogenates. The mixture was spun at 12000 r/min for 5 min and the aqueous phase was transferred into a new 1.5 ml tube. The homogenates containing DNA were re-extracted by adding an equal volume of isopropanol and 1/10 volume of 3 mol/L NaAc. The mixture was placed at -20 °C for 20 min and then centrifuged at 12000 r/min for 5 min, and the aqueous phase was discarded. The DNA pellet was washed with 70% ethanol twice in order to precipitate them, dried, and re-suspended in 50 μl H2O for PCR (Sun et al. 2000; Liu et al. 2005). Fragments of the internal transcribed spacers (ITS), RNA Polymerase II subunit B (rpb2), and translation elongation factor 1-alpha (tef1-α) were amplified with the three primer pairs: 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, respectively. A 25 μl reaction volume contained 1.0 μl DNA template, 1.0 μl of each forward and reverse primers, 12.5 μl 2× MasterMix (Tiangen Biotech) and 9.5 μl dd H2O. The PCR thermal cycle programs of the amplification followed Chaverri (Chaverri et al. 2011) and Chen (Chen and Zhuang 2017a). PCR products were purified with the PCR product purification kit (Biocolor BioScience and Technology Co., Shanghai, China), and forward and reverse sequencing was carried out on an ABI 3730 XL DNA sequencer (Applied Biosystems, Foster City, California) with primers used during PCR amplification. The sequences were deposited in the GenBank database at the National Center for Biotechnology Information (NCBI) and the accession numbers were listed in Table 1.

Table 1.
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Strains and the GenBank accession numbers analyzed in this study.

Species Strain GenBank accession number
ITS RPB TEF
Protocrea farinosa CBS 121551 MH863119 EU703935 EU703889
Protocrea pallida CBS 299.78 MH861137 EU703948 EU703900
T. achlamydosporum YMF 1.06226* MN977791 MT052180 MT070156
T. afarasin DIS 314F FJ442259 FJ442778 FJ463400
T. afroharzianum CBS 124620* FJ442265 FJ442691 FJ463301
T. afroharzianum GJS 04-193 FJ442233 FJ442709 FJ463298
T. aggregatum HMAS 248863* KY687946 KY688001 KY688062
T. aggregatum HMAS 248864 KY687947 KY688002 KY688063
T. aggressivum CBS 100525 AF057600 AF545541 AF348095
T. aggressivum DAOM 222156* AF456924 FJ442752 AF348098
T. alni CBS 120633* EU518651 EU498349 EU498312
T. alni CPK 2494 EU518652 EU498350 EU498313
T. alpinum HMAS 248821* KY687906 KY687958 KY688012
T. alpinum HMAS 248830 KY687912 KY687961 KY688015
T. anaharzianum YMF 1.00241 MH262584 MH262577 MH236493
T. anaharzianum YMF 1.00383* MH113931 MH158995 MH183182
T. asiaticum YMF 1.00168 MH262582 MH262575 MH236492
T. asiaticum YMF 1.00352* MH113930 MH158994 MH183183
T. azevedoi CEN 1422* MK714902 MK696821 MK696660
T. azevedoi CEN 1423 MK714903 MK696822 MK696661
T. bannaense HMAS 248840* KY687923 KY687979 KY688037
T. bannaense HMAS 248865 KY687948 KY688003 KY688038
T. breve HMAS 248844* KY687927 KY687983 KY688045
T. breve HMAS 248845 KY687928 KY687984 KY688046
T. brunneoviride CBS 120928 EU518661 EU498358 EU498318
T. brunneoviride CBS 121130* EU518659 EU498357 EU498316
T. camerunense CBS 137272* AY027780 NA AF348107
T. camerunense GJS 99-231 AY027783 NA AF348108
T. ceraceum GJS 95-159 AF275332 AF545508 AY937437
T. cerinum DAOM 230012* KC171336 KJ842184 KJ871242
T. christiani CBS 132572* NA KJ665244 KJ665439
T. christiani S93 NA KJ665245 KJ665442
T. concentricum HMAS 248833* KY687915 KY687971 KY688027
T. concentricum HMAS 248858 KY687941 KY687997 KY688028
T. dacrymycellum WU 29044 FJ860749 FJ860533 FJ860633
T. epimyces CBS 120534* EU518663 EU498360 EU498320
T. epimyces CPK 2487 EU518665 EU498361 EU498322
T. guizhouense HGUP 0038* JN191311 JQ901400 JN215484
T. guizhouense S628 NA KJ665273 KJ665511
T. hainanense HMAS 248837* KY687920 KY687976 KY688033
T. hainanense HMAS 248866 KY687949 KY688004 KY688034
T. harzianum CBS 226.95* AJ222720 AF545549 AF348101
T. harzianum GJS 05-107 FJ442679 FJ442708 FJ463329
T. helicolixii CBS 133499* NA KJ665278 KJ665517
T. helicolixii CBS 135583 NA KJ665277 KJ665516
T. hengshanicum HMAS 248852* KY687935 KY687991 KY688054
T. hengshanicum HMAS 248853 KY687936 KY687992 KY688055
T. hirsutum HMAS 248834* KY687916 KY687972 KY688029
T. hirsutum HMAS 248859 KY687942 KY687998 KY688030
T. hunanense HMAS 248841* NR_154571 KY687980 KY688039
T. hunanense HMAS 248867 KY687950 KY688005 KY688040
T. ingratum HMAS 248822* KY687917 KY687973 KY688018
T. ingratum HMAS 248827 KY687909 KY687966 KY688021
T. italicum CBS 132567* NA KJ665282 KJ665525
T. italicum S15 NA KJ665283 KJ665526
T. koreanum SFC20130926-S008 NA MH025989 MH025983
T. koreanum SFC20131005-S066* MH050352 MH025988 MH025979
T. lentinulae CGMCC 3.19848 MN594470 MN605868 MN605879
T. lentinulae HMAS 248256* MN594469 MN605867 MN605878
T. liberatum HMAS 248831* KY687913 KY687969 KY688025
T. liberatum HMAS 248832 KY687927 KY687970 KY688026
T. linzhiense HMAS 248846* KY687929 KY687985 KY688047
T. linzhiense HMAS 248874 KY687957 KY688011 KY688048
T. longisporum HMAS 248843* KY687926 KY687982 KY688043
T. longisporum HMAS 248868 KY687951 KY688006 KY688044
T. neotropicale CBS 130633* MH865818 NA HQ022771
T. parepimyces CBS 122768 FJ860801 FJ860563 FJ860665
T. parepimyces CBS 122769* MH863234 FJ860562 FJ860664
T. peberdyi CEN1425 MK714905 MK696824 MK696663
T. peberdyi CEN1426* MK714906 MK696825 MK696664
T. pinicola KACC 48486 * MH050354 MH025993 MH025981
T. pinicola SFC20130926-S014 NA MH025991 MH025978
T. pleuroti CBS 124387* HM142363 HM142372 HM142382
T. pleuroti CPK 2117 NA NA EU279975
T. pleuroticola CBS 124383* HM142362 HM142371 HM142381
T. pleuroticola TRS70* KP009264 KP009172 KP008951
T. polypori HMAS 248855* KY687938 KY687994 KY688058
T. polypori HMAS 248861 KY687944 KY688000 KY688059
T. propepolypori YMF 1.06199 MN977790 MT052182 MT070157
T. propepolypori YMF 1.06224* MN977789 MT052181 MT070158
T. pseudodensum HMAS 248828* KY687910 KY687967 KY688023
T. pseudodensum HMAS 248829 KY687911 KY687968 KY688024
T. rifaii CBS 130746* FJ442663 NA FJ463324
T. rifaii DIS 337F FJ442621 FJ442720 FJ463321
T. rufobrunneum HMAS 266614* KF729998 KF730010 KF729989
T. rufobrunneum isolate 8155 NA KF730007 KF729992
T. rugulosum SFC20180301-001* MH050353 MH025986 MH025984
T. rugulosum SFC20180301-002 NA MH025987 MH025985
T. simile YMF 1.06201* MN977793 MT052184 MT070154
T. simile YMF 1.06202 MN977794 MT052185 MT070153
T. simplex HMAS 248842* KY687925 KY687981 KY688041
T. simplex HMAS 248860 KY687943 KY687999 KY688042
T. solum HMAS 248847 KY687930 KY687986 KY688049
T. solum HMAS 248848* KY687931 KY687987 KY688050
T. spirale DIS 173A FJ442217 FJ442705 FJ463371
T. spirale E425 NA MK044189 MK044096
T. spirale E510 NA MK044198 MK044105
T. stramineum CBS 114248* AY737765 AY391945 AY737746
T. stramineum TAMA 0425 AB856609 AB856748 AB856675
T. subazureum YMF 1.6185 MN977799 MT052190 MT070148
T. subuliforme YMF 1.6182 MN977796 MT052187 MT070151
T. subuliforme YMF 1.6183 MN977797 MT052188 MT070150
T. subuliforme YMF 1.6184 MN977798 MT052189 MT070149
T. vermifimicola CGMCC 3.19850 MN594472 MN605870 MN605881
T. vermifimicola HMAS 248255* MN594473 MN605871 MN605882
T. xixiacum CGMCC 3.19698 MN594477 MN605875 MN605886
T. xixiacum HMAS 248253* MN594476 MN605874 MN605885
T. zayuense HMAS 248835* KY687918 KY687974 KY688031
T. zayuense HMAS 248836 KY687919 KY687975 KY688032
T. zelobreve CGMCC 3.19696 MN594475 MN605873 MN605884
T. zelobreve HMAS 248254* MN594474 MN605872 MN605883
T. albidum YMF 1.7530* OQ517962 OQ559127 OQ559118
T. albidum YMF 1.7531 OQ517963 OQ559128 OQ559119
T. variegatum YMF 1.7532 OQ517964 OQ559129 OQ559120
T. variegatum YMF 1.7533* OQ517965 NA OQ559121
T. variegatum YMF 1.7534 OQ517966 OQ559130 OQ559122

Notes: NA, not applicable. *, type strains.

Phylogenetic analyses

Sequences of ITS, rbp2, and tef1-α of 111 strains, representing 59 species with close phylogenetic relation to two new species based on blast result of ITS sequence were downloaded from GenBank. Among them, 98 strains belong to the Harzianum clade and 11 strains belong to the Spirale clade, with Protocrea farinosa Berk. & Broome (CBS 121551) and P. pallida Ellis & Everh. Jaklitsch et al. (CBS 299.78) as the outgroups. Both the reference sequences and newly generated sequences in this study were listed in Table 1. DNA sequence data of each locus were aligned, respectively, by Clustalx 1.83 (Thompson et al. 1997) with the default parameters. Aligned sequences of multiple loci were manually adjusted and concatenated using BioEdit v.7.0 (Hall 1999). Finally, we obtained the combined sequence matrix (Fasta file) generated by BioEdit v.7.0, containing 2530 characters from three genes (522 from ITS, 833 from rpb2, 1178 from tef1-α).

Maximum Likelihood (ML) and Bayesian inference (BI) analyses were conducted to allocate the phylogenetic positions of the new species. Maximum Likelihood analysis was computed by RAxML (Stamatakis 2006) with the PHY files generated with ClustalX 1.83 (Thompson et al. 1997), using the GTR-GAMMA model. Maximum likelihood bootstrap proportions (MLBP) were computed with 1000 replicates. Under the best fit model, Bayesian Inference (BI) analysis was performed with MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001) with the NEXUS file converted by MEGA7 (Kumar et al. 2016). The best fit evolutionary model for each dataset was determined using MrModeltest 2.3 and incorporated into the analyses. A Markov Chain Monte Carlo (MCMC) algorithm of four chains was started in parallel from a random tree topology with the heating parameter set to 0.3. The MCMC analysis was run until the average standard deviation of the split frequencies dropped below 0.01 with trees saved each 1,000 generations. The initial 25% of the generations of MCMC sampling were discarded as the “burn-in” and posterior probabilities determined from the remaining trees. The Tree was viewed in FigTree v1.4 (Rambaut 2012), values of Maximum likelihood bootstrap proportions (MLBP) greater than 70% and Bayesian inference posterior probabilities (BIPP) greater than 85% at the nodes are shown along branches.

Results

Phylogenetic analyses

Phylogenetic positions of the new species were determined by analyses of the combined tef1, rpb2 and ITS dataset containing 2533 characters. In our analyses, the 116 strains included 100 strains belonging to the Harzianum Clade, 14 strains belonging to the Spirale Clade and two outgroup taxa.

The ML analysis showed similar tree topology and was congruent with that obtained in the BI analysis (Fig. 1). The tree topology showed that our strains belonged to two new species, one new species classified in the Harzianum clade and the other one in the Spirale clade. Two strains were grouped together in an independent clade in the Harzianum clade and associated with T. epimyces Jaklitsch, T. rufobrunneum Z.X. Zhu & W.Y. Zhuang and T. aggressivum Samuels & W. Gams, designated as T. albidum (BIPP/MLBP = 100%/100%). In the Spirale clade, our three isolates formed one corresponding to a new species, designated as T. variegatum (BIPP/MLBP = 100%/100%).

Figure 1. 

Phylogenetic tree of Trichoderma species based on the combined ITS, tef1-α and rpb2 gene sequences constructed using Maximum-likelihood (ML) analysis and Bayesian inference (BI) analysis. The former values near nodes represent Bayesian posterior probabilities over 85% and the latter represent bootstrap support from ML bootstrap support over 70%. Protocrea farinose CBS 121551 and P. pallida CBS 299.78 were used as outgroups. Bold font indicates newly described species.

Taxonomy

Trichoderma albidum Z.F. Yu & T.T. Jing, sp. nov.

MycoBank No: MycoBank No: 847677
Fig. 2

Etymology

Referring to the rare white, whitish colonies on cultures media.

Description

Sexual morph : Unknown. Asexual morph: Conidiophores straight or slightly curved, branches mostly asymmetrically arranged, also paired, sometimes at irregular intervals along the main axis, closely-spaced, often orientated toward the apex, rarely forming secondary branches. Phialides lageniform to somewhat subulate, straight or slightly curved, often with a narrow neck, often in whorls of 2-5, (6.0–) 8.3–13.5 (–15.7) × (2.9–) 3.2–4.6 (–4.8) µm, l/w ratio (1.5–) 2.0–4.0 (–4.5), (1.5–) 1.9–3.2 (–3.8) µm wide at the base, widest around the middle. Conidia ovoid to subglobose, sometimes oblong, hyaline, smooth, (3.5–) 3.7–5.3 (–6.3) × (3.2–) 3.3–4.2 (–4.8) µm (mean = 4.4 × 3.8 μm, n=50), l/w ratio 1.0–1.3 (–1.8).

Culture characteristics

Optimum temperature for growth 25 °C. No growth at 35 °C in CMA, PDA and SNA.

Colony radius on CMA after 3 days: 9–11 mm at 25 °C, 5–6 mm after 6 days at 30 °C, covering the plate after 11 days at 25 °C. Colony white to whitish, radial, not zonate, aerial hyphae sparse, arachnoid. Conidiation start after 4 days. Chlamydospores rare. No distinct odor noted, no diffusing pigment observed.

Colony radius on PDA after 3 days: 22 mm at 25 °C, 11 mm at 30 °C, covering the plate after 7 days at 25 °C. Colony dense, pale white, finely zonate, circular, aerial hyphae abundant, fluffy. Conidiation start after 8 days, formed numerously on aerial hyphae. No distinct odor noted, no diffusing pigment observed.

Colony radius on SNA after 72 h: 14 mm at 25 °C, 4 mm at 30 °C, covering the plate after 8 days at 25 °C. Colony hyaline, indistinctly zonate, aerial hyphae scarcely degenerating. Conidiation start after 7 days. Chlamydospores rare. No distinct odor noted, no diffusing pigment observed.

Materials examined

China. Yunnan province, Zhaotong city, Yiliang county, Xiaocaoba Town, on diseased Gastrodia elata, 25 Oct. 2021, T.T. Jing (holotype YMF 1.7530). Ibid. (culture: YMF 1.7531).

Notes

Phylogenetically, T. albidum is associated with T. aggressivum. In comparison, T. aggressivum grows faster on PDA (50.5–56.0 mm after 3 days at 25 °C) and SNA (58.5–62.2 mm after 3 days at 25 °C), and produces shorter and narrower phialides ((4.0–) 5.7–7.8 (–21.0) × (1.3–) 2.7–3.5 (–4.3) µm) and much smaller green conidia (3.2–3.3 × 2.8–2.9 μm) (Samuels et al. 2002).

Figure 2. 

Morphology of Trichoderma albidum (YMF 1.7530) A–C cultures on PDA, 7d; SNA, 8d; CMA, 11d D–H conidiophores and phialides I conidia. Scale bars: 10 μm (D–I).

Trichoderma variegatum Z.F. Yu & T.T. Jing, sp. nov.

MycoBank No: MycoBank No: 847676
Fig. 3

Etymology

Referring to the luteous, orange to amber, variable coloration of the colonies on cultures media.

Description

Sexual morph : Unknown. Asexual morph: Conidiophores straight or curved, asymmetry, sparsely branches, cylindrical mostly sterile to the apex. Often with a main axis, frequently tips sterile, disposed, relatively distant distribution at right angles to the axis or slightly oriented towards the conidiophore terminus, often solitary, not or rebranched once. Phialide lageniform to subulate, sometimes cylindrical, often with a narrow neck, discrete or integrated, solitary or in whorls of 2–3 (–4), (9.5–) 9.8–14.6 (–15.4) × (3.2–) 3.5–5.4 (–6.7) µm, l/w ratio (1.9–) 2.1–4.3 (–4.6), 2.2–3.4 µm wide at the base, widest around the middle. Conidia ellipsoidal to oblong, sometimes obovate, green, smooth, (4.3–) 4.7–7.4 (–8.6) × (2.7–) 3.0–4.1 (–4.3) µm (mean = 6.1 × 3.5 μm, n=50), l/w ratio (1.0–) 1.4–2.0 (–2.2).

Culture characteristics

Optimum temperature for growth 25 °C. No growth at 35 °C in CMA, PDA and SNA.

Colony radius on CMA after 72 h: 20–22 mm at 25 °C, 14–15 mm at 30 °C, covering the plate after 8 days at 25 °C. Colony hyaline, indistinctly zonate, aerial hyphae nearly lacking. Conidiation starting after 8 days, formed on aerial hyphae. Chlamydospores common, subglobose to globose, smooth, terminal and intercalary, 5.3–13.4 × 5.0–11.3 µm. No distinct odor noted, yellow pigment noted.

Colony radius on PDA after 72 h: 30–32 mm at 25 °C, 27–29 mm at 30 °C, cover the plate after 6 days at 25 °C. Colony dense, aerial hyphae abundant, margin slightly lobed, forming numerous small yellow pigment droplets on the surface in the mature phase. Conidiation started after 8 days, formed on aerial hyphae. Chlamydospores abundant, subglobose to globose, smooth, terminal and intercalary, 5.5–10.6 × 5.3–10.1 µm. No distinct odor noted, yellow to brownish pigment diffusing into the agar.

Colony radius on SNA after 72 h: 18–22 mm at 25 °C, 17–19 mm at 30 °C, covering the plate after 8 days at 25 °C. Colony hyaline, not zonate, aerial hyphae sparse, relatively abundant at margin, arachnoid. Conidiation formed after 7 days, formed on aerial hyphae. Chlamydospores common, subglobose to globose, smooth, terminal and intercalary, 5.8–12.3 × 5.7–11.4 µm. No distinct odor noted, yellow pigment noted.

Materials examined

China. Yunnan province, Zhaotong city, Yiliang county, Xiaocaoba Town, on diseased Gastrodia elata, 25 Oct 2021, T.T. Jing (holotype YMF 1.7533). Ibid. (cultures: YMF 1.7532 and YMF 1.7534).

Notes

Phylogenetically, T. variegatum is closely related to T. hunanense in the Spirale clade. T. hunanense can be easily distinguished by much shorter conidia ((3.6–) 4.2–5.6 (–7.5) µm) and not producing chlamydospore (Chen and Zhuang 2017a). Moreover, T. hunanense grows faster on PDA (46–47 mm after 3 days at 25 °C) and SNA (27–28 mm after 3 days at 25 °C) and forms green pustules in culture compared with T. variegatum (Chen and Zhuang 2017a).

Figure 3. 

Morphology of Trichoderma variegatum (YMF 1.7533) A–D cultures on PDA, 8d; SNA, 9d; CMA, 9d; PDA, 4d E, F, H, I conidiophores and phialides G conidia. Scale bars: 10 μm (E–I).

Discussion

At present, the combination of phylogenetic, morphological, ecological, and biogeographic data has effectively resolved all the known species within the genus Trichoderma. Specifically, two genes, rpb2 and tef1-α, are widely deployed for identifications of new Trichoderma species. The present study employed a multilocus phylogenetic analysis for three molecular markers (ITS, rpb2 and tef1-α) and morphological comparisons to delimit and recognize species within two clades of Trichoderma. Our analyses showed that our two novel Trichoderma species belonged to the Spirale clade and the Harzianum clade.

New specie, T. variegatum, is described here as a member of the Spirale clade, which is newly introduced by Chen and Zhuang to accommodate three Trichoderma species, T. hunanense, T. longisporum and T. spirale (Chen and Zhuang 2017a). T. spirale was first described by Bissett (Bissett 1991). However, the phylogenetic position of T. spirale was variable in the initial analyses. Chaverri and Samuels reported that T. spirale was closest to T. polysporum Rifai in the Polysporum clade (Chaverri and Samuels 2003). Subsequently, T. spirale was placed into the Strictipile clade by Jaklitsch and was closely related to T. longipile Bissett and T. strictipile Bissett (Jaklitsch 2009). Whereas, T. spirale was considered as a separate terminal branch in Jaklitsch and Voglmayr’s study (Jaklitsch and Voglmayr 2015). Afterwards, Chen and Zhuang introduced the Spirale clade to accommodate three aforementioned Trichoderma species in 2017 (Chen and Zhuang 2017a). Zheng et al. later added two species, T. subuliforme and T. subazureum, into the Spirale clade (Zheng et al. 2021). Members of the Spirale clade generally form hairy pustules, produce yellow pigments in culture and have more or less oblong conidia (Chen and Zhuang 2017a). T. variegatum is morphologically different from others in the Spirale clade in that it does not form hairy pustules in culture. Previously reported species of the Spirale clade were all isolated as saprobes from soil (Zhang and Zhuang 2017; Zheng et al. 2021). It is the first time that species of the clade have been found in plant tissues, confirming that species in the Spirale clade have flexible nutrition modes and potentially novel diversity in plants.

T. albidum belongs to the Harzianum clade, which is a cosmopolitan and ubiquitous group. The T. harzianum species complex is well known for its antifungal properties and effective bio-control capacity, often applied to restrain soil-borne plant pathogens (Chaverri et al. 2015; Degenkolb et al. 2015; Bunbury-Blanchette and Walker 2019; Gu et al. 2020). The Harzianum clade displays a complicated speciation history and heterogeneous morphology (Atanasova et al. 2010; Druzhinina et al. 2010; Qin and Zhuang 2017). Members of the Harzianum clade generally exhibit great variation in the number and size of pustules formed in culture, type of conidiophores and shape of phialides and conidia (Chaverri and Samuels 2003; Jaklitsch 2009; Qin and Zhuang 2017; Zheng et al. 2021). The taxonomy of species in the Harzianum clade was revised and the identity of commercial strains of T. harzianum was performed by Chaverri et al. in 2015 (Chaverri et al. 2015). Since then, multitudes of new species of the Harzianum clade have been reported and more than 70 species have been placed in the clade (Jaklitsch and Voglmayr 2015; Chen and Zhuang 2017a, b; Qiao et al. 2018; Zhang and Zhuang 2018; Phookamsak et al. 2019; Gu et al. 2020; Inglis et al. 2020; Barrera et al. 2021; Bustamante et al. 2021; Nuangmek et al. 2021). No doubt many species of this clade remain to be discovered.

The habitat of Trichoderma is highly heterogeneous, including agricultural fields, prairies, forests, salt marshes, and even desert (Gond et al. 2007; Verma et al. 2007; Gazis and Chaverri 2010). Some taxa of Trichoderma are contributing in suppressing or attacking other plant pathogens through their secondary metabolites, which have been explored as potential biological control agents (Degenkolb et al. 2008; Cardoso Lopes et al. 2012; Cheng et al. 2012; Degenkolb et al. 2015; Zhu et al. 2017; Bunbury-Blanchette and Walker 2019). On the contrary, a few species, such as T. atrobrunneum F.B. Rocha et al., T. pleuroti and T. pleuroticola S.H. Yu & M.S. Park were considered as causal agents of “Green mold” disease of the cultivated mushroom Agaricus bisporus (J.E. Lange) Imbach (Sandoval-Denis et al. 2014; Innocenti et al. 2019). T. aggressivum, which is closely related to T. albidum, also was reported to cause enormous damages to mushroom production (Oda et al. 2009; Schuster and Schmoll 2010; Kim et al. 2012; Kim et al. 2013). In our survey, we isolated and identified the fungi from diseased G. elata tissues, and obtained more than 250 isolates. 78 isolates were determined to be Trichoderma after preliminary identification by ITS barcoding. At present, the potential effects of these fungi on G. elata cultivation remain largely unknown. However, they likely represent a (yet to be confirmed) but growing number of fungal pathogens capable of infecting crop plants (Xu 2022). This study sets the foundation for future pathogenicity and epidemiological studies of these three Trichoderma species on G. elata, contributing to the future prevention and controls of tuber diseases in G. elata crop fields

Acknowledgement

We are grateful to editors and reviewers for critically reviewing the manuscript providing helpful suggestions to improve this paper. Additionally, we would like to thank our team for help and support with this experiment.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was financed by the National Key R & D Program of China (2022YFD1400700), the National Natural Science Foundation Program of PR China (32170017, 31970013) and Yunnan University Research and Innovation Fund for Postgraduates (2021Y294).

Author contributions

M.M. and Z.Y. conceived and designed the study; C.Y. and T.J. wrote the manuscript and revised; C.Y., T.J. and Y.S. conducted the experiments. All authors have read and agreed to the published version of the manuscript.

Data availability

All of the data that support the findings of this study are available in the main text. All sequences have been deposited in GenBank at the accession numbers given in the text.

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