Research Article |
Corresponding author: ZenFen Yu ( zfyu2021@163.com ) Academic editor: Cecile Gueidan
© 2018 Min Qiao, Xing Du, Zhe Zhang, JianPing Xu, ZenFen 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:
Qiao M, Du X, Zhang Z, Xu JP, Yu ZF (2018) Three new species of soil-inhabiting Trichoderma from southwest China. MycoKeys 44: 63-80. https://doi.org/10.3897/mycokeys.44.30295
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Fungi in the genus Trichoderma are widely distributed in China, including in Yunnan province. In this study, we report three new soil-inhabiting species in Trichoderma, named as T. kunmingense, T. speciosum and T. zeloharzianum. Their colony and mycelial morphology, including features of asexual states, were described. For each species, their DNA sequences were obtained from three loci, the internal transcribed spacer (ITS) regions of the ribosomal DNA, the translation elongation factor 1-α encoding gene (tef1) and the gene encoding the second largest nuclear RNA polymerase subunit (rpb2). Our analyses indicated that the three new species showed consistent divergence amongst each other and from other known and closely related species. Amongst the three, T. speciosum and T. kunmingense belong to the Viride Clade. Specifically, T. speciosum is related to three species – T. hispanicum, T. samuelsii and T. junci and is characterised by tree-like conidiophores, generally paired branches, curved terminal branches, spindly to fusiform phialides and subglobose to globose conidia. In contrast, T. kunmingense morphologically resembles T. asperellum and T. yunnanense and is distinguished by its pyramidal conidiophores, ampulliform to tapered phialides, discrete branches and ovoidal, occasionally ellipsoid, smooth-walled conidia. The third new species, T. zeloharzianum, is a new member of the Harzianum Clade and is closely associated with T. harzianum, T. lixii and T. simmonsii but distinguished from them by having smaller, subglobose to globose, thin-walled conidia.
Rhizospheric fungi, diversity, Hypocreales , taxonomy
The genus Trichoderma Pers. (Ascomycota, Sordariomycete, Hypocreales, teleomorph Hypocrea Fr.) is cosmopolitan, often existing as saprophytes in a diversity of ecosystems, such as agricultural fields, prairies, forests and salt marshes (
As multilocus molecular phylogeny enables rapid and accurate identification of Trichoderma species, a significant number of Trichoderma species have been recently reported based on molecular phylogenetic evidence. Following the guidelines of the International Code of Nomenclature (ICN) for algae, fungi and plants (Melbourne Code, Art. 14.13), 254 names of Trichoderma species and two names of varieties in Trichoderma were accepted in 2015 (
China has an enormous fungal diversity. Amongst the 71 new Trichoderma species reported since 2015, 43 were from China (
Soil samples were collected from Luliang and Kunming in Yunnan Province, southwest China. All the samples were stored at 4 °C before use. Trichoderma strains were obtained by serial dilutions (1,000 to 1,000,000 fold) and spread on to the surface of Rose Bengal agar with antibiotics (40 mg streptomycin, 30 mg ampicillin per litre) added in a 9-cm-diam. Petri dish, followed by incubation under 25 °C for 5 days. Representative colonies were picked up with a sterilised needle and transferred to new plates containing potato dextrose agar (PDA,
For morphological studies, we used three different media: cornmeal dextrose agar CMD (40 g cornmeal, 2% (w/v) dextrose, 2% (w/v) agar), PDA and synthetic low nutrient agar (SNA). Each strain was first cultured on a PDA plate for 3 days and a small agar piece of 0.5 cm diam. with mycelium was then transferred respectively to new CMD, PDA and SNA plates. Strains were incubated in 9 cm diam. Petri dishes at 25 °C with a 12 h natural light and 12 h darkness interval (
For each strain, genomic DNA was extracted from mycelium growing on PDA harvested after 3 days of growth, following the method of
Species, strains and their corresponding GenBank accession numbers of sequences used for phylogenetic analyses.
Name | Strain | GenBank accession number | ||
---|---|---|---|---|
ITS | rpb2 | tef1 | ||
Trichoderma afarasin P. Chaverri & Branco-Rocha | Dis 314F | FJ442259 | FJ442778 | FJ463400 |
T. afroharzianum P. Chaverri, F.B. Rocha & Druzhinina | GJS 04-186 | FJ442265 | FJ442691 | FJ463301 |
T. asperelloides Samuels | GJS 04-187 | JN133553 | JN133560 | JN133571 |
GJS 04-116 | GU198301 | GU248411 | GU248412 | |
GJS 08-87 | – | GU198272 | GU198241 | |
T. asperellum Samuels, Lieckf. & Nirenberg | GJS 90-7 | EU330956 | EU338337 | EU338333 |
GJS 01-294 | EU856297 | FJ150788 | EU856323 | |
GJS 06-294 | GU198307 | GU198266 | GU198235 | |
|
KF425754 | KF425755 | KF425756 | |
GJS 05-328 | GU198318 | EU248614 | EU248627 | |
T. atrobrunneum F.B. Rocha, P. Chaverri & Jaklitsch | GJS 04-67 | FJ442273 | FJ442724 | FJ463360 |
T. atroviride P. Karst | DAOM 222144 | AF456916 | FJ442754 | AF456889 |
T. gamsii Samuels & Druzhinina | GJS 04-09 | DQ315459 | JN133561 | DQ307541 |
T. guizhouense Q.R. Li, McKenzie & Yong Wang | S628 | – | KJ665273 | KJ665511 |
T. harzianum Rifai | T55 | KX632511 | KX632568 | KX632625 |
T18 | KX632492 | KX632549 | KX632606 | |
T2 | FJ884174 | KX632534 | KX632591 | |
CBS 226.95 | AY605713 | AF545549 | AF348101 | |
T11 | KX632600 | KX632543 | KX632486 | |
T. hispanicum Jaklitsch & Voglmayr | S453 | JN715595 | JN715600 | JN715659 |
T. inhamatum Veerkamp & W. Gams | CBS 273.78 | FJ442680 | FJ442725 | AF348099 |
T. junci Jaklitsch | CBS 120926 | FJ860761 | FJ860540 | FJ860641 |
T. kunmingense Y. Zhang |
|
KJ742800 | KJ742801 | KJ742802 |
T. lentiforme P. Chaverri, Samuels & F.B. Rocha | Dis 218E | FJ442220 | FJ442793 | FJ463310 |
T. lieckfeldtiae Samuels | GJS 00-14 | DQ109528 | EU883562 | EU856326 |
T. lixii P. Chaverri | GJS 97-96 | AF443920 | KJ665290 | AF443938 |
T. pleuroti S.H. Yu & M.S. Park | CBS 124387 | HM142363 | HM142372 | HM142382 |
T. pleuroticola S.H. Yu & M.S. Park | CBS 124383 | HM142362 | HM142371 | HM142381 |
T. pyramidale Jaklitsch & P. Chaverri | S73 | – | KJ665334 | KJ665699 |
T. rifaii F.B. Rocha, P. Chaverri & Samuels | Dis 337F | FJ442621 | FJ442720 | FJ463321 |
T. samuelsii Jaklitsch & Voglmayr | S5 | JN715596 | JN715599 | JN715651 |
T. simmonsii P. Chaverri, F.B. Rocha, Samuels & Jaklitsch | S7 | – | KJ665337 | KJ665719 |
T. speciosum Z.F. Yu & X. Du |
|
MH113929 | MH155270 | MH183184 |
T. theobromicola Samuels & H.C. Evans | Dis 85f | DQ109525 | FJ007374 | EU856321 |
T. valdunense Jaklitsch | CBS 120923 | FJ860863 | FJ860605 | FJ860717 |
T. viride Pers | CBS 119325 | DQ677655 | EU711362 | DQ672615 |
T. yunnanense Z.F. Yu & K.Q. Zhang | CBS 121219 | GU198302 | GU198274 | GU198243 |
T. zeloharzianum Z.F. Yu & X. Du |
|
MH113932 | MH158996 | MH183181 |
Nectria eustromatica Jaklitsch & Voglmayr | CBS 125578 | HM534897 | HM534887 | HM534876 |
Preliminary BLAST searches with tef1, rpb2 and ITS gene sequences of the new isolates against NCBI and UNITE databases identified species closely related to our three isolates. Based on this information, we downloaded tef1, rpb2 and ITS sequences of 40 strains, representing 25 species. To show the phylogenetic position of T. zeloharzianum, 11 of the 14 species belonging to the T. harzianum complex were included. The remaining three species in this complex were not included because their rpb2 sequences are not available in NCBI.
Three alignment files were generated, one for each gene and converted to NEXUS files with ClustalX 1.83 (
Maximum Likelihood (ML) analysis was computed by RAxML (
The final alignments and the trees obtained have been deposited in TreeBASE (TreeBASE accession number: 23172). Phylogenetic positions of the new species were ascertained by analyses of the combined tef1, rpb2 and ITS dataset containing 2831 characters, of which 487 characters were constant, 2344 were variable.
In our analyses, sequences from 41 strains including 21 strains of the Harzianum Clade, 19 strains of the Viride Clade and an outgroup taxa, Nectria eustromatica were used to construct the phylogenetic tree. Of the three new species, T. speciosum and T. kunmingense belonged to the Viride Clade, whereas T. zeloharzianum were located in the Harzianum Clade. These two clades formed a monophyletic group, which is generally consistent with what was found in a previous study (
Phylogenetic tree based on Bayesian analysis of the combined tef1, rpb2 and ITS sequences. Nectria eustromatica is used as the outgroup. Bayesian posterior probabilities greater than 0.90 are given at the nodes (left). Maximum likelihood bootstrap values greater than 70% are given at the nodes (right). The scale bar shows the expected changes per site. New species proposed are in boldface.
Latin, speciosum refers to showy and splendid colony on PDA.
Characterised by tree-like conidiophores, branches paired or in whorls of 3–4, spindly to fusiform phialides (5.0–10.0 × 2.0–3.0 μm), subglobose to globose conidia (3.7–4.9 × 3.1–3.8 μm). Differs from T. hispanicum by paired branches, whorled and thinner phialides, subglobose to globose conidia. Differs from T. samuelsii by paired and compact branches, subglobose to globose conidia and the character of pustules on SNA. Differs from T. junci by whorled, smaller phialides and subglobose to globose conidia.
CHINA. From soil of tobacco rhizosphere, Luliang, Yunnan Province, 24°57'22"N, 103°46'30"E, 1800 m alt., Jul 2007, Z.F. Yu (
Mycelium covers plate after 72 h at 25 °C and 30 °C on CMD, no growth at 35 °C. Colony homogenous, pale yellowing, not zonate, outline circular. Aerial hyphae sparse, relatively abundant at margin, distinctly radial, arachnoid. Conidial production noted after 4 days.
On PDA, mycelium covers the plate after 72 h at 25 °C and 30 °C, no growth at 35 °C. Colony circular, typically zonate, yellow-green colony homogeneous distributed around the point of inoculation, forming a coarse circle. Whitish aerial hyphae distributed on the agar surface in external zone, hairy, dense and radial. Conidial production noted after 3 days.
On SNA after 72 h, colony radius 37–38 mm at 25 °C, mycelium covers the plate after 120 h, 56–59 mm at 30 °C after 72 h, no growth at 35 °C. Colony hyaline, thin, fan-shaped with indistinct outline. Aerial hyphae scarcely degenerating. Conidial production noted after 5 days, minute white pustules formed around central part of the colony, turning green after 6 days. Conidiophores tree-like, comprising a main axis with second branches, base 3.0–4.0 μm wide, second branches paired or in whorls of 3, sometimes second branches branched again, the distance between neighbouring second branches is (12.0–) 15.0–29.0 (–30.0) μm, main axis and branches terminating in whorls of up to five phialides. Conidiogenous cells phialides lageniform or ampulliform, arising singly or in 2–4; 5.0–10.0 × 2.0–3.0(–3.5) μm, length/width ratio 1.7–3.6 (–4.2), non-equilateral when curved. Conidia ovoid to short ellipsoidal, verrucose (3.6–)3.7–4.9(–5.0) × (3.0–)3.1–3.8(–4.2) μm, length/width ratio (1.0)1.1–1.4(–1.5).
In soil from tobacco rhizosphere in part of cultivated land of south-western China.
Not known
Trichoderma speciosum is phylogenetically most closed related to three species – T. hispanicum, T. samuelsii and T. junci (
In addition, side branches of T. hispanicum are often unpaired, phialides often singly, whereas branches of T. speciosum are generally paired or in whorls of 3–5. For T. samuelsii, branches are sparser and phialides with l/w of (1.7–)2.5–4.6(–7.1) are more slender than those of T. speciosum. Phialides of T. junci are also more slender than those of T. speciosum, which are narrowly lageniform.
Latin, kunmingense, refers to the site in which this species was found.
Characterised by pyramidal fashion conidiophores, ampulliform to tapered phialides (6.0–9.0 × 2.5–4.5 µm), discrete branches and ovoid, occasionally ellipsoid, smooth-walled conidia (3.4–4.4 × 2.7–3.4 µm). Differs from T. asperellum by slightly shorter and sometimes more whorled phialides, mostly obovoid conidia. Differs from T. yunnanense by sparser branches and more whorled, smaller phialides and conidia.
CHINA. Kunming, Yunnan, 24°52'28"N, 102°49'34"E. 1929 m alt, in soil, Aug 2007, Y. Zhang (
Colony on CMD after 72 h radius 35–50 mm, mycelium covering the plate after 96 h at 25 °C, 55–59 mm at 30 °C and 41–46 mm at 35 °C after 72 h. Colony hyaline, margin distinctly noted. Aerial hyphae are indistinctly observed, radiate and sparse, white pustule formed from inner zone, asymmetrical to pulvinate, loosely arranged. Conidial production noted after 48 h. No diffusing pigment produced.
Mycelium covers plate after 72 h at 25 °C and 35 °C on PDA and radius 52–56 mm at 30 °C. Colony layered distinctly, margin conspicuous and radial. Aerial hyphae, hairy to floccose, dense internal zone, but relative sparse on margin, abundantly and flat in a large green disc around the inoculums, turning green after 24 h of conidiation.
Colony on SNA after 72 h radius 48–50 mm, mycelium covering the plate after 96 h at 25 °C, 53–56 mm at 35 °C and covering the plate at 30 °C after 72 h. Colony and pustules are similar to that on CMD, colony hyaline and smooth, the shape of pustules more regular, sometimes hemispherical, loosely distributed around the point of inoculation. Conidiophores well defined, branching 2–3 times in a pyramidal fashion, with the longest branches verticillate on the discrete main axis, the base 2.2–3.9(–4.4) μm wide, branched toward the tip, the distance between neighbouring second branches are 11.0–38.5 μm. Phialides arising generally 1–3 times repetition on each branches or in whorls of 3–5, ampulliform to tapered, slightly constricted at the base, often straight or less sinuous or curved toward apex of conidiophore, mostly (5.0–) 6.0–9.0(–10.0) × 2.5–4.5 µm, length/width ratio (1.3–)1.4–3.4(–3.6). Conidia obovoid, sometimes ellipsoidal, smooth-walled, both ends broadly rounded or at the base slightly narrower, 3.4–4.4 × 2.7–3.4 µm, length/width ratio (1.1–)1.2–1.6, pale green when viewed singly, usually greenish in mass.
Specimen examined. PR China, Kunming, Yunnan Province, 24°52'N, 102°49'E, elev. 1929 m, isolated from soil samples, Aug. 2007, by Y. Zhang (Holotype,
In garden soil of Kunming city of southwest China.
Not known
Trichoderma kunmingense can be distinguished from T. asperellum Samuels, Lieckfeldt and Nirenberg, by having more crowded branches and phialides. T. asperellum typically forms whorls of 2–4 phialides, whereas phialides of T. kunmingense sometimes attain 5 phialides. Although the phialides are ampulliform in both species, the phialides of T. asperellum are slightly longer (type strain: 7.2–11.5 µm) than those of T. kunmingense. Moreover, conidia of T. asperellum have inconspicuous and small ornamentation, but those of T. kunmingense are smooth and conidia are slightly longer (type strain: 3.5–4.5 × 2.7–4.0 µm) (
Trichoderma kunmingense and T. yunnanense Yu and Zhang are also closely related in the phylogenetic tree, but branches and phialides of T. yunnanense are more crowded than those of T. kunmingense. Phialides in T. yunnanense arising separately or more often paired with branches, rarely in whorls of 3 (
Greek zelo-, meaning emulation + harzianum, referred to Trichoderma harzianum
Characterised by pyramidal conidiophores, verticillate branches, ampulliform to lageniform phialides (5.5–10.0 × 2.5–3.5 μm) and subglobose to globose, thin-walled conidia (2.7–3.1 × 2.4–2.6 μm). Differs from T. harzianum by verticillate branches, 3–6 whorled phialides on terminal of each branch and thinner conidia. Differs from T. lixii by verticillate and compact branches, more terminal phialides on main axis and smaller conidia. Differs from T. simmonsii by verticillate branches and longer conidia.
CHINA. Yunnan: Qujing City, Luliang county, 25°05'25"N, 103°56'42"E, 1800 m alt., in soil, Jul 2007, Z.F. Yu (
On CMD after 72 h, colony radius 59–62 mm, mycelium covers the plate after 96 h at 25 °C; 43–45 mm at 30 °C and 46–52 mm at 35 °C after 72 h. Colony yellowing, margin distinct. Aerial hyphae fertile and conspicuous, hairy radial, distributed on surface, green conidial production noted after 4 days.
On PDA after 72 h, colony radius 57–58 mm, mycelium covers the plate after 96 h at 25 °C. Covering the plate at 30 °C and 38–42 mm at 35 °C after 72 h. Colony white, margin distinct. Aerial hyphae abundant, hairy to floccose, denser around central disc. Green conidiation noted after 3 days.
On SNA after 72 h, radius 59–65 mm, mycelium covers the plate after 144 h at 25 °C, 64–65 mm at 30 °C and 29–37 mm at 35 °C after 72 h. Aerial hyphae sparsely, slightly radial and conspicuous zonate. Conidiophores well defined, branching 2–3 times in a pyramidal fashion. Branches paired or a whorl of 3–4, the distance between neighbouring second branches is 16.0–39.0 μm, base 3.0–4.0 μm wide, each branch terminating in a whorl of 3–6 phialides, phialides ampulliform to lageniform, often verticillated up to 5 around the main axis near the apex, rarely singly arising, (4.5)5.5–10.0(–11.0) × 2.5–3.5(–4.0) μm, length/width ratio (1.4–)1.8–3.4(–3.6). Conidia smooth on surface, subglobose to globose, sometimes obovoid, (2.6–) 2.7–3.1(–3.2) × (2.3–) 2.4–2.6(–2.7) μm, length/width ratio (1.0–)1.1–1.3(–1.4).
In soil from tobacco rhizosphere in part of cultivated land of south-western China.
Not known
Trichoderma zeloharzianum forms a single branch with T. harzianum Rifai as sister clade. Morphologically, T. harzianum is similar to T. zeloharzianum in their shape of conidiophores and phialides, but the branches of T. harzianum are opposite of each other and each branch terminating in a whorl of 2–5 phialides (
Trichoderma lixii differs from T. zeloharzianum also by having opposing pairs of branches and fewer terminal phialides (2–4) on main axis. Beyond that, closely spaced branches are common in T. lixii (
Trichoderma simmonsii is also distinguished obviously from T. zeloharzianum, except their differences about opposing branches (
The application of molecular barcode for fungal taxonomy has led to a re-evaluation of morphology-based taxonomy of Trichoderma. A recent study suggested that tef1 introns could provide a high resolution to this genus and is shown to be superior to other phylogenetic markers (
Based on the combined analysis of sequences from three genes, phylogenetic positions of three species were ascertained, amongst which T. zeloharzianum belonged to the Harzianum clade. T. zeloharzianum has the characteristic of typical T. harzianum-like morphology containing pairs or verticils branches, ampulliform to lageniform phialides and globose to subglobose or broadly ovoid conidia (
Both T. speciosum and T. kunmingense belong to the Viride Clade. The study of
Species of the Harzianum and Viride Clades were commonly isolated from soil. However, the number of published soil-inhabiting Trichoderma species is limited compared with that on woody substrates. Furthermore, the sexual states of most soil-inhabiting species are unknown (
This work was financed by the National Natural Science Foundation Program of PR China (31760012, 31570023). We are grateful to two reviewers for critically reviewing the manuscript and for providing helpful suggestions to improve this paper.