Three new species of soil-inhabiting Trichoderma from southwest China

Abstract 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.


Introduction
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 (Gond et al. 2007, Verma et al. 2007, Gazis and Chaverri 2010).Though rarely, they are also found in deserts and freshwater ecosystems.In some woody plants, they are the most abundant endophytes.In addition, a few species of Trichoderma are effective in attacking or inhibiting other fungi through their secondary metabolites and these fungi have been exploited as potential biocontrol agents against plant pathogens (Degenkolb et al. 2008, Cheng et al. 2012, Lopes et al. 2012, Mukherjee et al. 2013).A few Trichoderma species are crop pathogens and can produce toxins to spoil food.For example, T. aggressivum can cause significant crop loss to mushroom production (Oda et al. 2009, Schuster and Schmoll 2010, Kim et al. 2012, 2013).
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 (Bissett et al. 2015).Since then, 71 new Trichoderma species have been reported.Amongst these 71 species, 15 were described based on cultures from ascospores and the remaining 56 were based on asexual morphs in nature (Chen and Zhuang 2016, 2017a, b, c, Qin and Zhuang 2016a, b, c, d, 2017, Zhu et al. 2017a, b, du Plessis et al. 2018).Most of the new species were isolated from soil (Chen and Zhuang 2016, 2017a, b, c, du Plessis et al. 2018), rotten twigs, stems or barks (Qin and Zhuang 2016a, b, c, d, 2017, Zhu et al. 2017a, b).Several were found associated with the attine ants (Montoya et al. 2016) and on the surface of Hypoxylon anthochroum stroma (Sun et al. 2016).
China has an enormous fungal diversity.Amongst the 71 new Trichoderma species reported since 2015, 43 were from China (Chen and Zhuang 2017a,b,c, Qin and Zhuang 2017, Zhu et al. 2017a, b).Of these 43 species, 33 were from the soil of different regions (Chen and Zhuang 2017a, b, c), which shows that soil has a high Trichoderma diversity.In our survey of Trichoderma from soil, 180 Trichoderma strains were collected in southwest China and preserved in the Laboratory for Conservation and Utilization of Bioresources, Yunnan University (YMF) and China General Microbiological Culture Collection Center (CGMCC).Three new species were identified based on morphological features and DNA sequence data at three loci: the genes encoding RNA polymerase II subunit (rpb2) and translation elongation factor 1-α gene (tef1) and the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene cluster.Based on the DNA sequence information, we revealed their phylogenetic positions as belonging to the Viride Clade (two species) and the Harzianum Clade (one species).

Isolates of strains
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, Zhang et al. 2013).All putative strains of Trichoderma were permanently kept in the Herbarium of the Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, Yunnan, P.R.China (YMF).In addition, the holotype strains have been deposited in the China General Microbiological Culture Collection Center (CGMCC).

Morphology characterisation and growth observation
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 (Sutton 1980).Colony diameters were all measured after 3 days for morphological descriptions, diameters at 25 °C and 35 °C and the times when mycelia entirely covered the surface of plate were also recorded.For microscopic morphology, photographs were taken with an Olympus BX51 microscope connected to a DP controller digital camera.

DNA extraction, PCR amplification and sequencing
For each strain, genomic DNA was extracted from mycelium growing on PDA harvested after 3 days of growth, following the method of Wang and Zhuang (2004).For the amplifications of ITS, rpb2 and tef1 gene fragments, three different primer pairs were used: ITS4 and ITS5 for ITS (White et al. 1990), EF1-728F (Carbone and Kohn 1999) and TEF1LLErev (Jaklitsch et al. 2005) for tef1 and tRPB2-5F and tRPB2-7R for rpb2 (Chen and Zhuang 2016).Each 25 μl PCR reaction consisted of 12.5 μl T5 Super PCR Mix (containing Taq polymerase, dNTP and Mg2+, Beijing TsingKe Biotech Co., Ltd., Beijing), 1.25 μl of forward primer (10 μM), 1.25 μl of reverse primer (10 μM), 1μl DNA template, 5 μl of PCR buffer and 4.5 μl sterile water.PCR reactions were run in an Eppendorf Mastercycler following the protocols described by Zhuang and Chen (2016).PCR products were purified with the PCR product purification kit (Biocolor BioScience & Technology Co., Shanghai, China), and sequencing was carried out in both directions on an ABI 3730 XL DNA sequencer (Applied Biosystems, Foster City, California) with primers used during PCR amplification.GenBank accession numbers of sequences generated in this study are provided in Table 1.

Phylogenetic analyses
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 (Thompson et al. 1997) to identify the phylogenetic positions of these species.The three alignments were then combined with BioEdit 7.1.9.0 (Hall 1999).The phylogenetic analyses included 1008 characters for rpb2, 1233 characters for tef1 and 590 characters for ITS.All characters were weighted equally and gaps were treated as missing characters.
Maximum Likelihood (ML) 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.Bayesian Inference (BI) analysis was conducted with MrBayes v3.2.2 (Ronquist and Huelsenbeck 2003).The Akaike information criterion (AIC) implemented in jModelTest 2.0 (Posada and Darriba 2008) was used to select the best fit models after likelihood score calculations were done.The base tree for likelihood calculations was ML-optimised.HKY+I+G was estimated as the best-fit model under the output strategy of AIC, Metropolis-coupled Markov chain Monte Carlo (MCMCMC) searches were run for 2000000 generations, sampling every 500th generation.Two independent analyses with four chains each (one cold and three heated) were run until the average standard deviation of the split frequencies dropped below 0.01.The initial 25% of the generations of MCMC sampling were discarded as burnin.The refinement of the phylogenetic tree was used for estimating Bayesian inference posterior probability (BIPP) values.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 90% at the nodes are shown along branches.

Sequence analyses
The final alignments and the trees obtained have been deposited in TreeBASE (Tree-BASE 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 (Jaklitsch and Voglmayr 2015).Etymology.Latin, speciosum refers to showy and splendid colony on PDA.
Description.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 distrib- uted 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.
Habitat and distribution.In soil from tobacco rhizosphere in part of cultivated land of south-western China.
Teleomorph.Not known Remarks.Trichoderma speciosum is phylogenetically most closed related to three species -T.hispanicum, T. samuelsii and T. junci (Jaklitsch et al. 2012;Jaklitsch 2011).The three species were isolated from ascospores and only T. speciosum was isolated from the anamorph.However, T. speciosum differs from these three species in having verrucose, subglobose to globose conida, while conidia of T. hispanicum and T. samuelsii are oblong and smooth and those of T. junci are ovoid to ellipsoidal with length/width ratio 1.3-1.8(-2.2).
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.

Trichoderma kunmingense Z.F.Yu & J.Y.Li, sp. nov.
MycoBank MB808878 Figure 3 Etymology.Latin, kunmingense, refers to the site in which this species was found.
Habitat and distribution.In garden soil of Kunming city of southwest China.
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 (Yu et al. 2007).Conidia of T. yunnanense (4.0-5.0 ×3.5-4.0 μm) are also larger than those of T. kunmingense.
Description.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.
Habitat and distribution.In soil from tobacco rhizosphere in part of cultivated land of south-western China.
Teleomorph.Not known Remarks.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 (Chaverri et al.2015), while T. zeloharzianum is clearly distinguishable by having verticillated branches and 3-6 terminal whorled phialides.In addition, the conidia of T. harzianum are generally wider [(2.0−)2.5−3.0 (−3.7) μm] than those of T. zeloharzianum.
Trichoderma simmonsii is also distinguished obviously from T. zeloharzianum, except their differences about opposing branches (Chaverri et al. 2015), the phialides are more stout and shorter ((4.2−)5.2−6.5 (−9.0) μm) than those of T. zeloharzianum.Furthermore, T. simmonsii is commonly constricted below the tip to form a narrow neck (Chaverri et al. 2015); however, this character is not found in T. zeloharzianum.

Discussion
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 (Jaklitsch et al. 2012).Rpb2 sequences appeared powerful due to their suitable interspecific variations (Jaklitsch and Voglmayr 2015).ITS sequences are identical or nearly identical for several species of the genus (e.g.those of T. hispanicum, T. koningii, T. viridescens and T. samuelsii), therefore this marker alone is not useful for phylogenetic reconstruction or for barcoding of these fungi (Druzhinina et al. 2005, Jaklitsch et al. 2012).Together, due to their universality and reliability for species in the Trichoderma genus, these three loci were chosen for this study.
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. harzianumlike morphology containing pairs or verticils branches, ampulliform to lageniform phialides and globose to subglobose or broadly ovoid conidia (Chaverri et al. 2015).The T. harzianum species complex is a cosmopolitan and ubiquitous species, playing important roles in ecology and economy.Chaverri et al. (2015) disentangled this species complex recognising 14 species.In the present study, 11 of the 14 species from the Harzianum Clade were included for analyses.T. zeloharzianum is the most closely related to T. harzianum, with the latter being more broadly distributed.The sexual and asexual morphs for T. lixii-T.harzianum have been rejected (Druzhinina et al. 2010, Atanasova et al. 2013) and Chaverri et al. (2015) and also showed that T. lixii and T. harzianum are closely related but represent separate species.Here, we found T. zeloharzianum is more closely to T. harzianum than to T. lixii.
Both T. speciosum and T. kunmingense belong to the Viride Clade.The study of Jaklitsch and Voglmayr (2015) indicated that the structure of the Viride Clade is complex, as there are additional subclades, such as the Hamatum/ Asperellum Clade, the Rogersonii Clade, the Neorufum Clade and several smaller subclades.Samuels et al. (2006) showed that asexual morphs of the Viride Clade often have verrucose conidia.In the present study, T. kunmingense with smooth conidia is found phylogenetically related to T. asperellum and T. asperelloides, two species with verrucose conidia and both belonging to the Asperellum subclade.However, T. speciosum with warted conidia could not be assigned to any specific subclade.
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 (Chen and Zhuang 2016).China is rich in species diversity of the Trichoderma genus.Future studies will likely reveal more new taxa in soil, which could provide a better understanding of the relationship between asexual and sexual states of some species in the genus.

Figure 1 .
Figure 1.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.

Table 1 .
Species, strains and their corresponding GenBank accession numbers of sequences used for phylogenetic analyses.