Mycologia

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Three new species of Trichoderma in the

Harzianum and Longibrachiatum lineages from
Peruvian cacao crop soils based on an integrative
approach

Danilo E. Bustamante, Martha S. Calderon, Santos Leiva, Jani E. Mendoza,

Marielita Arce & Manuel Oliva

To cite this article: Danilo E. Bustamante, Martha S. Calderon, Santos Leiva, Jani E. Mendoza,
Marielita Arce & Manuel Oliva (2021): Three new species of Trichoderma in the Harzianum and
Longibrachiatum lineages from Peruvian cacao crop soils based on an integrative approach,
Mycologia, DOI: 10.1080/00275514.2021.1917243

To link to this article: https://doi.org/10.1080/00275514.2021.1917243

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MYCOLOGIA
https://doi.org/10.1080/00275514.2021.1917243

Three new species of Trichoderma in the Harzianum and Longibrachiatum

lineages from Peruvian cacao crop soils based on an integrative approach
a,b a,b a a a
Danilo E. Bustamante , Martha S. Calderon , Santos Leiva , Jani E. Mendoza , Marielita Arce ,
and Manuel Oliva a
a
Instituto de Investigación para el Desarrollo Sustentable de Ceja de Selva, Universidad Nacional Toribio Rodríguez de Mendoza, Chachapoyas,
Amazonas, Peru; bFacultad de Ingeniería Civil y Ambiental, Universidad Nacional Toribio Rodríguez de Mendoza, Chachapoyas, Amazonas, Peru

ABSTRACT ARTICLE HISTORY

The hyperdiverse genus Trichoderma is one of most useful groups of microbes for a number of Received 15 January 2021
human activities, and their accurate identification is crucial. The structural simplicity and lack of Accepted 12 April 2021
distinctive phenotypic variation in this group enable the use of DNA-based species delimitation KEYWORDS
methods in combination with phylogenies (and morphology when feasible) to establish well- Cacao farm; fungal diversity;
supported boundaries among species. Our study employed a multilocus phylogeny and four DNA- integrative approach;
based methods (automated barcode gap discovery [ABGD], statistical parsimony [SPN], generalized multilocus phylogeny; Peru;
mixed Yule coalescent [GMYC], and Bayesian phylogenetics and phylogeography [BPP]) for four species delimitation;
molecular markers (acl1, act, rpb2, and tef1) to delimit species of two lineages of Trichoderma. Trichoderma; 3 new taxa
Although incongruence among these methods was observed in our analyses, the genetic distance
(ABGD) and coalescence (BPP) methods and the multilocus phylogeny strongly supported and
confirmed recognition of 108 and 39 different species in the Harzianum and Longibrachiatum
lineages, including three new species associated with cacao farms in northern Peru, namely, T.
awajun, sp. nov., T. jaklitschii, sp. nov., and T. peruvianum, sp. nov. Morphological distinctions
between the new species and their close relatives are primarily related to growth rates, colony
appearance, and size of phialides and conidia. This study confirmed that an integrative approach
(DNA-based methods, multilocus phylogeny, and phenotype) is more likely to reliably verify
supported species boundaries in Trichoderma.

INTRODUCTION
Trichoderma is a hyperdiverse fungal genus that com­
prises species that are frequently found on dead wood
and bark, on other fungi, in soil, and living within
healthy plant roots, and occasionally on stems and
leaves (Jaklitsch and Voglmayr 2015; Zhu et al.
2017). The species of Trichoderma belong to one of
the most useful groups of microbes and have had
a considerable impact on human welfare, serving as
effective biocontrol agents and producers of enzymes,
antibiotics, and heterologous proteins for the food,
feed, textile, and biofuel industries (Mukherjee et al.
2013; Zhu et al. 2017). Under controlled conditions,
Trichoderma cultures usually grow quickly and form
sparse aerial mycelia with green or white pustules
(Bissett 1991). Microscopically, Trichoderma species
usually display highly ramified conidiophores and
cylindrical to nearly subglobose phialides bearing sin­
gle-celled conidia that are green or hyaline (Chaverri
and Samuels 2003).
After the discontinuation of the dual nomenclature for
different morphs of the same fungus in 2011 (McNeill et al.
2012) and the prioritization of Trichoderma (typified by an
asexual type species) over Hypocrea (typified by a sexual
type species) (Rossman et al. 2013), it is estimated that
approximately 438 species of Trichoderma were legiti­
mated (www.indexfungorum.org, www.mycobank.org/).
Contemporary approaches for Trichoderma classification
grouped these species into 10 phylogenetic lineages (i.e.,
Brevicompactum, Deliquescens, Harzianum, Hypocrea-
num, Longibrachiatum, Polysporum, Psychrophilum,
Semiorbis, Stromaticum, and Viride) based on analyses
of sequence alignments of tef1, rpb2, and acl1 (Chaverri
et al. 2015; Jaklitsch and Voglmayr 2015). Among these
lineages, Harzianum and Longibrachiatum (Jaklitsch and
Voglmayr 2015) encompass a large number of species with
agricultural applications such as biological control and
plant growth- and defense-enhancing products
(Atanasova et al. 2013; Zhang et al. 2016; Zhang and

CONTACT Danilo E. Bustamante danilo.bustamante@untrm.edu.pe

Danilo E. Bustamante and Martha S. Calderon contributed equally to this work.
Supplemental data for this article can be accessed on the publisher’s Web site.
© 2021 The Mycological Society of America

Published online 15 Jun 2021

2 BUSTAMANTE ET AL.: NEW SPECIES OF TRICHODERMA FROM NORTHERN PERU
Zhuang 2017; Bunbury-Blanchette and Walker 2019;
Filizola et al. 2019; Marik et al. 2019; Alfiky and
Weisskopf 2021). The Longibrachiatum lineage mostly
contains clonal species that reproduce exclusively asexually
and is characterized by ornamented conidia (Druzhinina &
Kubicek 2005; Druzhinina et al. 2010; Samuels et al. 2012;
Yabuki et al. 2014; Jaklitsch and Voldgmyr 2015), whereas
the Harzianum lineage includes species with both asexual
and sexual morphs, and conidiophores that have fewer
than five phialides and are typically divergent or “cruciate”
(Chaverri et al. 2015; Jaklitsch and Voldgmyr 2015).
In cacao-producing areas of Peru, frosty pod rot (caused
by Moniliophthora roreri H.C. Evans) is one of the most
important fungal diseases affecting Theobroma cacao
(cacao) and is responsible for significant losses in bean
production (Evans et al. 1998; Phillips-Mora and
Wilkinson 2007; Ortega-Andrade et al. 2017; Bailey et al.
2018). Accordingly, species of Trichoderma (i.e.,
T. asperellum, T. longibrachiatum, T. ovalisporum,
T. stromaticum, and T. virens) have been investigated as
viable biological controls against M. roreri (Evans et al.
2003; Krauss et al. 2006; Bailey et al. 2018; Crozier et al.
2015). Native isolates of these biocontrol agents have been
tested in several countries (e.g., Costa Rica, Cameroon,
Panama, and Peru) to reduce major diseases of cacao and
have yielded promising results (Krauss and Soberanis 2002;
Holmes et al. 2006; Krauss et al. 2006, 2010; Tondje et al.
2007; Crozier et al. 2015).
Species are a fundamental unit in biological research,
but they can be challenging to delimit and recognize
objectively (Kuchta et al. 2016; Bustamante et al. 2019;
Lücking et al. 2020). Species identification in
Trichoderma has been perceived as difficult due to inter­
specific morphological similarities (Chaverri and
Samuels 2003; Jaklitsch 2009). Previous studies have
applied an integrative taxonomy that combined phylo­
geny, phenotype, and reproductive biology (when feasi­
ble) to describe new taxa within Trichoderma (Chaverri
et al. 2011, 2015; Jaklitsch and Voglmayr 2012, 2015;
Yamaguchi et al. 2012; Kim et al. 2013; Yabuki et al.
2014; Zhu and Zhuang 2015; Qin and Zhuang 2016,
2017; Chen and Zhuang 2017; Zhang and Zhuang
2017; Zhu et al. 2017; du Plessis et al. 2018; Qiao et al.
2018; Gu et al. 2020; Lücking et al. 2020), whereas others
have recognized taxa based on a phylogenetic species
concept alone (Hanada et al. 2008; Samuels and Ismaiel
2009; Sun et al. 2016; Inglis et al. 2020; Tomah et al.
2020). Under the integrative approach, DNA-based
methods are being used to delimit and recognize species
in fungi using distinct strategies such as genetic distance,
coalescent, and genealogical concordance for framing
reliably supported species boundaries (Goldstein et al.
2000; Powell et al. 2011; Parnmen et al. 2012; Carstens
et al. 2013; Fujisawa and Barraclough 2013; Liu et al.
2016; Haelewaters et al. 2018; Luo et al. 2018;
Bustamante et al. 2019).
Due to the potential of native Trichoderma strains to
control cacao diseases, we studied Trichoderma isolates
obtained from cacao crop soils in northern Peru. Three
new species belonging to the Harzianum and
Longibrachiatum lineages were identified based on an
integrative approach (i.e., morphological observations,
phylogenetic inferences, and DNA species delimitation
methods). This study may represent a valuable resource
for ongoing biocontrol research in northern Peru.
MATERIALS AND METHODS
Collection of specimens and isolation.—Soil samples
were collected during the rainy season (Feb–Apr 2017)
from four districts in the province of Bagua, Amazonas,
Peru. These sandy clay loam soils were collected from
20 cm depth corresponding to the H and O horizons
(Friedl and Druzhinina 2012) approximately 1 m from
cacao plant stems (Chaverri and Samuels 2003). The
fungal strains were isolated from these soil samples
according to Chaverri and Samuels (2003). Briefly, 10
g of soil was suspended in 90 mL of sterile distilled
water, and the mixture was stirred. After the precipita­
tion of soil particles, dilutions were transferred to four
different media containing 0.1 g/L chloramphenicol:
cornmeal dextrose agar (CMD; Merck, Darmstadt,
Germany), Czapek Dox agar (CDA; Merck), dichloran
rose bengal chloramphenicol (DRBC; Merck), and
potato dextrose agar (PDA; Merck), and incubated at
three different temperatures (25, 30, and 35 C) for 15
days with 12-h photoperiods (white fluorescent light/
darkness). Preliminary tests revealed poor mycelial
growth in CMD. Therefore, this medium was not uti­
lized for further isolation procedures or morphological
analyses.
Morphological characterization of isolates.—Eighty-
one fungal strains were incubated as monosporic cul­
tures on CDA, DRBC, and PDA at 25, 30, and 35 C for
15 days with 12-h photoperiods (white fluorescent light/
darkness). Morphological characterization of the fungi
was performed as described by Chaverri and Samuels
(2003). Observation of fungal mycelia and other struc­
tures stained with methylene blue (0.1–0.5%) was per­
formed by microscopy. Photomicrographs were taken
under an inverted microscope (IX83; Olympus, Tokyo,
Japan) with an integrated camera (Nikon D810; Tokyo,
Japan). Quantitative characters represent average values
with standard deviations from approximately 30

measurements from three different biological replicates
(cultures per specimen). Dried and living cultures of
fungal strains were deposited in the herbarium of
Universidad Nacional Toribio Rodriguez de Mendoza
de Amazonas, Peru (CHAX).
Molecular phylogenetic analyses.—Genomic DNA
was extracted from single-spore-derived PDA cultures
using the NucleoSpin Plant II kit (Macherey-Nagel,
Düren, Germany) following the manufacturer’s instruc­
tions. Four markers were sequenced: acl1, act, rpb2, and
tef1. Each marker was amplified using polymerase chain
reaction (PCR) with MasterMix (Promega, Madison,
Wisconsin, USA) in the following reaction mixture: 10
ng of DNA and 0.25–0.5 pmol of the forward and
reverse primers in a total volume of 10 μL. The PCR
protocols and primer combinations for acl1 (230up,
1220low; Jaklitsch and Voldgmyr 2015), act (Tact1,
Tact2; Samuels et al. 2006a), rpb2 (fRPB2-5F, fRPB2-
7cR; Chaverri and Samuels 2003), and tef1 (983F,
1567RintB; Stielow et al. 2015) were in keeping with
the protocols described by Jaklitsch and Voldgmyr
(2015) and Bustamante et al. (2019). The sequences of
the forward and reverse strands were determined by the
commercial service of Macrogen (Seoul, South Korea).
New acl1, act, rpb2, and tef1 sequences were deposited in
GenBank (SUPPLEMENTARY TABLE 1). These
sequences and others obtained from GenBank corre­
sponding to the Harzianum and Longibrachiatum
lineages were initially aligned using MUSCLE algo­
rithms (Thompson et al. 1994) and adjusted manually
with MEGA6 (Tamura et al. 2013).
A phylogeny based on concatenated data combining
acl1, act, rpb2, and tef1 was generated (178 sequences;
SUPPLEMENTARY TABLE 1). The selection of the
best-fitting nucleotide substitution model was con­
ducted using PartitionFinder (Lanfear et al. 2012) with
four partitions (acl1, act, rpb2, and tef1). The best parti­
tioning strategy and model of sequence evolution were
selected based on the Bayesian information criterion
(BIC). The general time-reversible nucleotide substitu­
tion model with a gamma distribution and a proportion
of invariable sites (GTR+Γ+I) was selected for all parti­
tions. Maximum likelihood (ML) analyses were con­
ducted with raxmlHPC-AVX (Stamatakis 2014)
implemented in the raxmlGUI 1.3.1 interface (Silvestro
and Michalak 2012) using a GTRGAMMAI model with
1000 bootstrap replications. Bayesian inference (BI) was
performed with MrBayes 3.2.6 (Ronquist et al. 2012)
using Metropolis-coupled Markov chain Monte Carlo
(MCMC) and the GTR+Γ+I model. We conducted two
runs each with four chains (three hot chains and one
MYCOLOGIA 3
cold chain) for 10 000 000 generations, sampling trees
every 1000 generations. We plotted likelihood vs. gen­
eration using Tracer 1.6 (Rambaut et al. 2014) until
a likelihood plateau was reached and set the burn-in
value. The four-locus data set was deposited in
TreeBASE (Submission ID 27561).
DNA-based species delimitation.—Four different
DNA-based delimitation methods were explored using
the acl1, act, rpb2, and tef1 data sets to assess species
boundaries in Trichoderma. Two of these DNA-based deli­
mitation methods were based on genetic distance (statistical
parsimony [SPN; Hart and Sunday 2007] and automated
barcode gap discovery [ABGD; Puillandre et al. 2012]), and
the other two were based on coalescence (generalized
mixed Yule coalescent [GMYC; Pons et al. 2006] and
Bayesian phylogenetics and phylogeography [BPP;
Rannala and Yang 2003]).
For the SPN analyses, data sets were generated in TCS
1.21 (Clement et al. 2000) with a maximum connection
probability set at 95% statistical confidence (Tineo et al.
2020). The ABGD method was tested via a Web interface
(ABGD web, http://www.abi.snv.jussieu.fr/public/abgd/
abgdweb.html). Prior to analysis, the model criteria were
set as follows: variability (P) between 0.001 (Pmin) and 0.1
(Pmax), minimum gap width (X) of 0.5, Kimura 2-para­
meter, and 50 screening steps (Bustamante et al. 2019).
To perform GMYC delimitation, an ultrametric tree
was constructed in BEAST 2.0.2 (Drummond et al.
2012), relying on the uncorrelated lognormal relaxed
clock, the GTR+Γ+I model, and a coalescent tree prior.
A Bayesian Markov chain Monte Carlo simulation was
run for 50 million generations, and trees and para­
meters were sampled every 1000 generations (Tineo
et al. 2020). Log files were visualized in Tracer 1.6
(Rambaut et al. 2014) to assess the stationary state of
parameters on the basis of the estimated effective sam­
ple size (ESS). After removing 25% of trees as burn-in,
the remaining trees were utilized to generate a single
summarized tree in TreeAnnotator 2.0.2 (part of the
BEAST 2.0.2 package) as an input file for GMYC ana­
lyses. GMYC analyses with single a threshold model
were performed in R (R Development Core Team 3.6.3;
http://www.R-project.org) in the SPLITS package using
the “gmyc” function (R-Forge, http://r-forge.r-project.
org/projects/splits/).
To validate the outcomes of single-locus species deli­
mitation (i.e., ABGD, GMYC, and SPN), a multilocus
BPP was applied using BP&P 2.0 (Yang and Rannala
2010). The four markers were used as inputs for BPP
under the A11 model (A11: species delimitation = 1,
species tree = 1). Specimens were a priori assigned to
4 BUSTAMANTE ET AL.: NEW SPECIES OF TRICHODERMA FROM NORTHERN PERU
species based on the number of species from the results
of the phylogenetic analysis. Five variables (ε1–ε5) were
automatically fine-tuned following the instructions of
BP&P (Yang and Rannala 2010). Analyses were per­
formed with three different prior combinations
(Leaché and Fujita 2010). Each analysis was performed
five times to confirm consistency between runs. Two
independent MCMC analyses were run for 100 000
generations with the “burn-in” = 20 000.
RESULTS
Molecular phylogeny.—In the phylogeny of the selected
Trichoderma species, the analyzed data matrix included
a total of 4842 base pairs (bp) (1198 bp for acl1, 742 bp
for act, 1322 bp for rpb2, and 1580 bp for tef1) from 178
individuals embedded into two evolutionary lineages,
namely, Harzianum (124) and Longibrachiatum (54).
The multilocus phylogeny obtained from ML and BI ana­
lyses strongly supported the monophyly of the lineages
Harzianum and Longibrachiatum (FIGS. 1, 2). These phy­
logenies showed distinctiveness in the interspecific rela­
tionships among species. The genetic divergence
comparisons showed a minimum threshold (p-distance)
for acl1, act, and rpb2 for distinguishing species of
Trichoderma embedded in these two lineages (TABLE 1);
however, there was no minimum threshold (p-distance)
for tef1 because different species had identical sequences
(e.g., T. danicum–T. pyramidale). The genetic distances
among the unidentified Peruvian species (e.g.,
Trichoderma sp. 1, Trichoderma sp. 2, and Trichoderma
sp. 4) and their sister taxa were greater than the minimum
thresholds (TABLE 2).
Species delimitation.—The species delimitation meth­
ods based on genetic distance (ABGD and SPN) and
coalescence (GMYC and BPP) yielded incongruent results
for the four markers (TABLE 3). The greatest number of
species was delimited in the BPP analyses for the lineages
Harzianum (108) and Longibrachiatum (39) (TABLE 3).
This method confirmed the species number recognized in
the multilocus phylogeny (FIGS. 1, 2). The genetic dis­
tance method based on barcode gaps (ABGD) demon­
strated slightly lower species numbers than those obtained
in the BPP and phylogenetic analyses for the rpb2 and tef1
markers for both lineages (TABLE 3). Conversely, con­
servative results were observed under SPN and GMYC
(SUPPLEMENTARY TABLE 2) for the four genes in both
lineages.
In our study, DNA-based delimitation methods (i.e.,
ABGD, SPN, and BPP) and multilocus phylogenetic
analyses demonstrated the presence of isolates from
Harzianum (i.e., T. afroharzianum, Trichoderma sp. 2,
Trichoderma sp. 3, and Trichoderma sp. 4) and
Longibrachiatum (i.e., T. orientale, T. parareesei, and
Trichoderma sp. 1) lineages from cacao farms in Peru.
The status of Trichoderma sp. 3 needs to be reevaluated
with the amplification of additional markers.
Morphological observations.—Differences in mor­
phological characteristics and colony growth/appearance
were observed in Trichoderma sp. 1, Trichoderma sp. 2,
and Trichoderma sp. 4. Trichoderma sp. 1 is distinguished
from its close relatives (T. bissettii and T. longibrachiatum)
by growth rates in PDA. Trichoderma sp. 2 differs from
T. breve and T. zelobreve by the growth rate and pattern
and phialide dimensions. Trichoderma sp. 2 is different
from Trichoderma sp. 3 by the colony growth and appear­
ance on CDA. These features distinguish these taxa from
their close relatives within the Harzianum and
Longibrachiatum lineages. Our results based on DNA-
based approach, multilocus phylogeny, and morphologi­
cal differences recognized the proposal of the following
taxa as new species: T. awajun, sp. nov. (= Trichoderma
sp. 1), T. jaklitschii, sp. nov. (= Trichoderma sp. 2), and
T. peruvianum, sp. nov. (= Trichoderma sp. 4).
TAXONOMY
Trichoderma awajun M.S. Calderon, D.E. Bustamante
& S. Leiva, sp. nov. FIG. 3
MycoBank MB835463
Typification: PERU. AMAZONAS: Prov. Bagua, Dist.
Copallin, Lluhuana, 78°24′39″W, 5°41′1″S, alt. 820 m, 11
Mar 2017, S. Leiva CP24-7 (holotype CHAX CP24-7).
Ex-type culture CHAXC CP24-7. GenBank: acl1 =
MW480155; act = MW480163; rpb2 = MW480147;
tef1 = MW480138.
Etymology: This species is named after the autochtho­
nous aboriginal Awajun people, who are found in con­
siderable numbers at the Copallin type locality and
sustainably farm cacao trees. The cultural center of
these people is close to the type locality.
Description: Cultures and asexual morph: Optimum
growth at 30 C on all media. On PDA, colony radius after
48 h at 30 C of 31.3 mm, covering the Petri dish on day 3. At
25 C, colony radius after 48 h of 18.2 mm, covering the Petri
dish on day 6. At 35 C, colony radius after 48 h of 19.8 mm,
covering the Petri dish on day 5. On day 4 (at 30 C), colonies
semidense, mycelia white, sporulation effuse and scant,
forming a reduced green lawn, zonation inconspicuous.
Autolytic excretions and hyphal coiling absent. No distinct
odors or exudates noted. Reverse pale. Conidiation on day
2, dense, heavily sporulating regions in the zonation rings.
 MYCOLOGIA 5

ABGD GMYC SPN

rpb2 tef1 rpb2 tef1 rpb2 tef1 BPP
(n=39) (n=33) (n=01) (n=01) (n=28) (n=16) (n=39)
74/1.0 Trichoderma subalpinum (CBS119128) Austria ▲
-/0.88 Trichoderma flavipes (GJS-92-102) USA ▲
* Trichoderma sambuci (WU29467) Austria
Trichoderma tremelloides (CBS121140) Germany ▲
Trichoderma leguminosarum (CBS130014) Spain ▲
*
Trichoderma britdaniae (8020) England ▲
84/1.0 Trichoderma patella (GJS91-141) USA
* Trichoderma alboviride (TC916) China ▲
*
Trichoderma novae-zelandiae (GJS81-265) NZ ▲
93/1.0 Trichoderma saturnisporopsis (S19) Italy
* -/0.84 Trichoderma flagellatum (CPK3345) Ethiopia
* Trichoderma sinense (DAOM230004) Taiwan
* Trichoderma gillesii (GJS-00-72) France ▲
Trichoderma konilangbra (CBS100808) Uganda ▲
* Trichoderma solani (GJS08-81) Mexico ▲
Trichoderma effusum (CPK-254) India ▲
28/-
83/1.0 Trichoderma capillare (GJS06-66) Vietnam
96/0.89
Trichoderma pseudokoningii (GJS-NS19) Australia ▲
60/-
Trichoderma citrinoviride (S20-32173) Italy
* Trichoderma andinense (GJS90-140) Venezuela ▲
Trichoderma euskadiense (CBS130013) Spain ▲
Trichoderma saturnisporum (18903) USA ▲
58/0.84
Trichoderma ghanense (GJS95-137) Ghana ▲
82/0.99
Trichoderma kunigamense (TAMA193) Japan ▲
* Trichoderma flavescens (HMJAU-34730) China ▲
Trichoderma centrosinicum (HMAS252910) China ▲
Trichoderma tsugarense (TAMA203) Japan ▲
Trichoderma gracile (GJS-10-263) Malaysia ▲
* Trichoderma reesei (0089) Brazil
* Trichoderma rugosum (11325) China ▲
72/0.83
Trichoderma thermophilum (HMAS252912) China ▲
88/0.90 Trichoderma parareesei (CP10-3) Peru
73/0.90
Trichoderma parareesei (CBS125925) Argentina ▲
* Trichoderma parareesei (CP55-3) Peru
80/0.98 Trichoderma parareesei (CP14-3) Peru
Trichoderma parareesei (CP14-5) Peru
76/1.0
Trichoderma aethiopicum (PPRC-H5) Ethiopia
* * Trichoderma pinnatum (GJS04-100) Vietnam ▲
Trichoderma xanthum (TC714) China ▲
Trichoderma sp. 1 (CP24-7) Peru ▲
* T. awajun sp. nov.
* Trichoderma sp. 1 (CP24-8) Peru
* Trichoderma bissettii (SZMC25718) Croatia
* Trichoderma longibrachiatum (CBS816-68) USA ▲
Trichoderma orientale (CP15-1) Peru
* Trichoderma orientale (CP63-2) Peru
Trichoderma orientale (CP63-1) Peru
Trichoderma orientale (GJS-88-81) China ▲
95/1.0 Trichoderma orientale (CP62-1) Peru
Trichoderma orientale (CP61-1) Peru
* Trichoderma orientale (CP61-4) Peru
Trichoderma orientale (CP01-3) Peru
Trichoderma orientale (CP52-2) Peru
Trichoderma orientale (CP64-1) Peru
Trichoderma orientale (CP01-5) Peru
* Harzanium clade
0.08

Figure 1. Phylogenetic tree of the Longibrachiatum lineage based on maximum likelihood inference of combined acl1, act, rpb2, and
tef1. Values above branches = maximum likelihood bootstrap (BS) values/Bayesian posterior probabilities. Bars represent species
delimitation results from ABGD-, GMYC-, SPN-, and BPP-based algorithmic methods based on rpb2 and tef1 sequences (sky blue:
delimited as the different species; green: delimited as the same species). Type strains are indicated by ▲. Specimens from this study
are shown in blue bold type. The scale bar indicates the number of nucleotide substitutions per site.

At 25 C, colonies hyaline and sterile, mycelium white to
transparent, no soluble pigments. At 30 C, colonies similar
to those forming at 25 C and less conspicuous.
Chlamydospores scarce, terminal and intercalary, 6.2–10.4
× 7.3–11.2 μm, globose to subglobose. Conidiation notice­
able and greenish after 3–4 days, initially short-effuse,
abundant, in shrubs or loose. Pustules comprising dense
clusters and peripheral conidiophores, thick-walled stipes;
primary branches forming a reticulum, from which
numerous main axes originate more or less radially.
Peripheral conidiophores straight or slightly curved, with
a distinct main axis; branches 2.5–6.5 μm wide, at right
angles or slightly inclined upward, typically unpaired at
lower levels, tending to be paired or in whorls at upper
levels; moniliform branches uncommon at the periphery.
Phialides commonly in whorls of 2–5, 3.1–10.0 × 6.4–9.6
μm, inequilateral, often slightly curved, distinctly enlarged,
rather plump with a mostly short neck and base, sometimes
6 BUSTAMANTE ET AL.: NEW SPECIES OF TRICHODERMA FROM NORTHERN PERU

ABGD GMYC SPN

rpb2 tef1 rpb2 tef1 rpb2 tef1 BPP
* Longibrachiatum Clade (n=93) (n=91) (n=01) (n=01) (n=71) (n=54) (n=108)
Trichoderma sulawesense (GJS85-228) Indonesia
Trichoderma lycogaloides (SL) French Guiana
* Trichoderma hunanense (HMAS248841) China ▲
Trichoderma spirale (DAOM183974) Thailand ▲
Trichoderma strictipile (CPK1601) Austria
* Trichoderma longipile (DAOM177227)
72/1.0
Trichoderma longipile (GJS91-93) Canada ▲
Trichoderma phyllostachydis (CBS114071) France
* * Trichoderma sp. (CBS133494) Spain
Trichoderma sp. (CBS137002) Greece
Trichoderma costaricense (PC21) CostaRica ▲
* Trichoderma thelephoricola (CBS120925) Austria
* Trichoderma chlorosporum (GJS98-1-33) Costa Rica
77/0.99
Trichoderma surrotundum (GJS88-73) ▲
Trichoderma sinuosum (PC8) Austria ▲
71/0.83 20/0.64
* Trichoderma brevicrassum (HMAS248871) ▲
Trichoderma cremeoides (S112) Italy ▲
40/0.84 Trichoderma cremeum (GJS91-125) USA igual ▲
* Trichoderma danicum (CBS121273) Denmark ▲
* Trichoderma spinulosum (CBS311-50) England
* Trichoderma aerugineum (CBS120541) Germany ▲
* Trichoderma britannicum (SB1) England ▲
Trichoderma sp. (CBS137006) Greece
67/- * Trichoderma aureoviride (CPK2848) Netherlands
Trichoderma pseudocandidum (PC59) Costa Rica ▲
* Trichoderma virescentiflavum (PC278) Costa Rica
Trichoderma thailandicum (GJS97-61) Thailand ▲
* Trichoderma estonicum (CBS121556) Sweden
Trichoderma ceramicum (CBS114576) USA
72/-
Trichoderma parestonicum (CBS120636) Austria igual ▲
Trichoderma chromospermum (GJS94-67) USA
* Trichoderma gliocladium (CBS130009) Italy ▲
74/0.99 Trichoderma gelatinosum (CPK1618) Austria
* Trichoderma crassum (DAOM164916) Canada igual Tana
Trichoderma virens (DAOM167652) USA igual ▲
87/0.94
Trichoderma helicolixii (CBS133499) Greece ▲
Trichoderma hausknechtii (CBS133493) France ▲
Trichoderma sp. (CBS136473) Spain
* Trichoderma pseudogelatinosum (CNUN309) Korea E▲
* Trichoderma catoptron (GJS02-76) SriLanka ▲
71/0.89 Trichoderma hirsutum (HMAS248834) China ▲
* Trichoderma pinicola (SFC20130926) Korea ▲
94/1.0 Trichoderma simplex (HMAS248842) China ▲
Trichoderma sp. (CBS136994) Spain
* * Trichoderma cerinum (S357) France
Trichoderma cerinum (DAOM230012) Nepal ▲
Trichoderma peberdyi (CEN1426) Brazil ▲
Trichoderma ceraceum (GJS95-159) USA
89/0.93 Trichoderma tomentosum (DAOM178713a) Canada ▲
80/0.91
* Trichoderma viridulum (HMAS273865) China ▲
Trichoderma velutinum (DAOM230013) Nepal igual ▲
82/0.93 * Trichoderma polypori (HMAS248855) China ▲
* 82/0.91 Trichoderma stramineum (GJS02-84) SriLanka igual ▲
Trichoderma cinnamomeum (GJS97-237) USA
* Trichoderma cinnamomeum (GJS97-230) USA E▲
Trichoderma dacrymycellum (WU29044) Germany
81/0.91
Trichoderma atrogelatinosum (DAOM167632) NZ
Trichoderma brunneoviride (CBS12092830) Austria E▲
Trichoderma brunneoviride (CBS121130) Germany ▲
* Trichoderma amazonicum (CBS126898) Peru E▲
* Trichoderma amazonicum (IB95) Peru
* Trichoderma pleuroticola (TRS70) Poland E▲
Trichoderma pleuroticola (CBS124383) Korea ▲
* Trichoderma pleuroti (CBS124387) Korea ▲
90/0.91
Trichoderma solum (HMAS248848) China ▲
76/0.91 Trichoderma compactum (CBS121218) China ▲
Trichoderma parepimyces (CBS122769) Austria igual ▲
Trichoderma italicum (CBS132567) Italy igual ▲
* * Trichoderma corneum (GJS97-82) Thailand E▲
* Trichoderma ingratum (HMAS248822) China ▲
Trichoderma concentricum (HMAS248833) China ▲
* Trichoderma perviride (HMAS273786) China
Trichoderma christiani (CBS132572) Spain ▲
* Trichoderma sp. (S138) Italy
86/0.91 * Trichoderma alni (CBS120633) England ▲
Trichoderma alpinum (HMAS248821) China ▲
72/0.81
Trichoderma pseudodensum (HMAS248828) China ▲
Trichoderma zayuense (HMAS248835) China
Trichoderma linzhiense (HMAS248832) China ▲
* Trichoderma liberatum (HMAS248831) China ▲
Trichoderma tawa (DAOM232841) Thailand igual ▲
Trichoderma hengshanicum (HMAS248852) China ▲
Trichoderma priscilae
* Trichoderma tenue (HMAS273785)(CBS131487) Spain igual ▲
China ▲
Trichoderma rufobrunneum (HMAS266614) China ▲
* Trichoderma epimyces (CBS120534) Austria igual ▲
Trichoderma purpureum (HMAS273787) China ▲
* Trichoderma aggressivum (DAOM222156) E▲
* Trichoderma aggressivum (CBS100526) England
Trichoderma sp. (CBS135582) Spain
* * Trichoderma guizhouense (S278) Croatia
Trichoderma guizhouense (HGUP0038) China ▲
* Trichoderma atrobrunneum (Hypo4) Germany
Trichoderma pyramidale (CBS135574) Italy igual ▲
* Trichoderma
Trichoderma
sp. 2 (CP62-2) Peru
sp. 2 (CP01-6) Peru
* * Trichoderma sp. 2 (CP61-2) Peru ▲ T. jaklitschii sp. nov.
* Trichoderma sp. 2 (CP18-9) Peru
Trichoderma sp. 2 (CP18-2) Peru
* Trichoderma sp. 3 (CP18-3) Peru
Trichoderma sp. 3 (CP45-1) Peru
* Trichoderma zelobreve (CGMCC3-19695) China ▲
Trichoderma breve (HMAS248844) China ▲
Trichoderma sp. 4 (CP15-9) Peru
* Trichoderma sp. 4 (CP15-2) Peru ▲ T. peruvianum sp. nov.
Trichoderma afroharzianum (CP38-2) Peru
* Trichoderma afroharzianum (GJS04-186) Peru E▲
78/0.99
Trichoderma afroharzianum (CP24-6) Peru
Trichoderma sp. (CBS135585) Greece
Trichoderma rugulosum (SFC20180301-001) Korea ▲
Trichoderma rifaii (DIS337F) Panama E▲
Trichoderma lentiforme (GJS98-6) French Guiana
Trichoderma azevedoi (CEN1422) Brazil ▲
90/1.0 Trichoderma afarasin (DIS314F) Camaron
Trichoderma zeloharzianum (XD2018k) China
Trichoderma harzianum (CBS226-95) England ▲
79/1.0 * Trichoderma lixii (CBS110080) Thailand igual ▲
* Trichoderma lentinulae (CGMCC3-19847) China ▲
Trichoderma xixiacum (CGMCC3-19697) China ▲
* Trichoderma vermifimicola (CGMCC3-19694) China ▲
76/0.97 Trichoderma simmonsii (S7) Italy
85/1.0 Trichoderma inhamatum (CBS273-78) Colombia ▲
Trichoderma bannaense (HMAS248840) China ▲
0.08 Trichoderma sp. (CBS135581) Spain

Figure 2. Phylogenetic tree of the Harzianum lineage based on maximum likelihood inference of combined acl1, act, rpb2, and tef1.
Values above branches = maximum likelihood bootstrap (BS) values/Bayesian posterior probabilities. Bars represent species delimita­
tion results from ABGD-, GMYC-, SPN-, and BPP-based algorithmic methods based on rpb2 and tef1 sequences (sky blue: delimited as
the different species; green: delimited as the same species). Type strains are indicated by ▲. Specimens from this study are shown in
blue bold type. The scale bar indicates the number of nucleotide substitutions per site.

Table 1. Lowest genetic distances (p-distances) in percentage for narrower with a long neck on young conidiophores.
species embedded into the Harzianum and Longibrachiatum Conidia 1.5–3.0 × 1.3–2.6 μm, green, subglobose to globose,
lineages of Trichoderma for tree markers. rarely oblong, smooth to faintly verruculose.
p-distance Colony radius on CDA after 72 h of 12–15 mm at 30 C,
Lineage Marker Sister species (%)
Harzanium acl1 T. atrobrunneum–T. pyramidale 0.2
2.5–3 mm at 25 C. Colonies circular, dense, with uniform
act T. rifaii–T. azevedoi 0.2 hyphae; margin wavy. Aerial hyphae present, in widely
rpb2 T. alni–T. alpinum 0.2 spaced fascicles, forming a more or less radial flat greenish
Longibrachiatum acl1 T. longibrachiatum–T. orientale 0.6
act T. bissettii–T. orientale 0.2 farinose structure. Autolytic excretions and coiling com­
rpb2 T. bissettii–T. longibrachiatum 0.3 mon. Diffusing pigment green, agar greenish, odor
 MYCOLOGIA 7

Table 2. Lowest genetic distances (p-distances) in percentage for the new species embedded into the
Harzianum and Longibrachiatum lineages of Trichoderma from northern Peru for four markers.
Lineage Marker Sister species p-distance (%)
Harzanium acl1 Trichoderma sp. 2–T. afroharzanium 1.6
Trichoderma sp. 4–T. afroharzanium 2.3
act Trichoderma sp. 2–T. lixii 0.3
Trichoderma sp. 4–T. lixii 0.4
rpb2 Trichoderma sp. 2–T. afarasin 0.3
Trichoderma sp. 4–T. zelobreve 0.6
tef1 Trichoderma sp. 2–T. peruvianum 0.3
Trichoderma sp. 4–T. zelobreve 0.2
Longibrachiatum acl1 Trichoderma sp. 1–T. longibrachiatum 0.8
act Trichoderma sp. 1–T. orientale 0.6
rpb2 Trichoderma sp. 1–T. longibrachiatum 0.3
tef1 Trichoderma sp. 1–T. longibrachiatum 0.2

Table 3. Number of species embedded into the Harzianum and Longibrachiatum lineages of
Trichoderma identified under DNA-based species delimitation methods and phylogenetic analysis.
Genetic distance Coalescence
Lineage Marker ABGD SPN GMYC BPP Multilocus phylogeny
Harzanium acl1 32 39 1 108 108
act 24 30 1
rpb2 93 71 1
tef1 91 54 1
Longibrachiatum acl1 15 14 1 39 39
act 8 6 1
rpb2 39 28 1
tef1 33 16 1

transient. Conidiation initiated after 2 weeks in central
floccules, effuse, scarce.
Colony radius on DRBC after 72 h of 16–19 mm at 30 C,
3.5–5 mm at 25 C. Colonies hyaline, dense, circular, with
conspicuously curved hyphae of variable widths. Aerial
hyphae common. Autolytic excretions scant, coiling fre­
quent. Diffusing pigment yellow, odor indistinct.
Chlamydospores scant, terminal and intercalary.
Conidiation noticeable after 3 days, turning green
after day 5, initially short-effuse from the center, soon
forming pustules. Conidia forming on loose, regularly tree-
like conidiophores.
Ecology and distribution: T. awajun is present in the
cacao agricultural ecosystem at low altitudes
(500–900 m elev.) in the Aramango, Copallin, and La
Peca districts of the Amazon region in northern Peru.
T. awajun was isolated from sandy clay loam soils
(0–20 cm depth) approximately 1 m from cacao plant
stems.
Additional specimens examined: PERU. AMAZONAS:
Prov. Bagua, Dist. Aramango, El Mirador, 78°26′42″W, 5°
25′52″S, alt. 515 m, 20 Feb 2017, S. Leiva CP24-8; Dist. La
Peca, San Francisco, 78°27′41″W, 5°37′36″S, alt. 647 m, 8
Mar 2017, S. Leiva CP25-1.
Notes: T. awajun is currently only known as an asexual
morph from three isolates. Phylogenetic analyses placed
T. awajun in a clade that included T. bissettii and
T. longibrachiatum (FIG. 1). The sequence divergence
among T. awajun, T. bissettii, and T. longibrachiatum was
over 0.8% for acl, 2.0% for act, 0.5% for rpb2, and 0.2% for
tef1.
The new species can be distinguished from its close
relatives by growth rates in PDA and other morphological
characteristics. Colonies of T. bissettii reach a radius of
84–112 mm after 48 h of incubation at 30–35 C (Sandoval-
Denis et al. 2014); in contrast, T. awajun only forms colo­
nies that measure 28.9–33.7 mm after 48 h at 30 C. In
addition, T. awajun forms subglobose to globose conidia
and scant chlamydospores, whereas T. bissettii produces
conidia that are broadly ellipsoidal to nearly oblong and
abundant chlamydospores (Sandoval-Denis et al. 2014).
Moreover, T. longibrachiatum fills a Petri plate within
72 h after inoculation at 25 C (Samuels et al. 2012), whereas
T. awajun covers it after 144 h under the same conditions.
T. awajun and T. longibrachiatum are also distinguished
based on the arrangement of phialides and conidia size.
T. awajun has phialides in whorls and small conidia
(1.5–3.0 × 1.3–2.6 μm), whereas T. longibrachiatum has
solitary phialides along the main axis and larger conidia
(3–5 × 2.5–3.0 μm) (Samuels et al. 2012).
Trichoderma jaklitschii M.S. Calderon, D.E. Bustamante
& S. Leiva, sp. nov. FIG. 4
MycoBank MB835465
Typification: PERU. AMAZONAS: Prov. Bagua,
Dist. Aramango, Pomabamba, 78°25′7″W, 5°26′12″
8 BUSTAMANTE ET AL.: NEW SPECIES OF TRICHODERMA FROM NORTHERN PERU

Figure 3. Morphological features of Trichoderma awajun (CP24-7). A–B. Photographs of colonies on CDA at 4 (A) and 8 (B) days. C–D.
Photographs of colonies on DRBC at 4 (C) and 8 (D) days. E–F. Photographs of colonies on PDA at 4 (E) and 8 (F) days. G. Conidia.
H. Conidiophores. Bars: A–F = 2 cm; G–H = 10 µm.

S, alt. 874 m, 6 Apr 2017, S. Leiva CP61-2 (holotype
CHAX CP61-2). Ex-type culture CHAXC CP61-2.
GenBank: acl1 = MW480162; act = MW480165;
rpb2 = MW480149; tef1 = MW480140.
Etymology: The specific epithet “jaklitschii” honors
Walter M. Jaklitsch for his valuable contributions to
the understanding of the systematics of Trichoderma
associated with cacao plants.
Description: Cultures and asexual morph: Optimum
growth at 30 C on all media. On PDA, colony radius
after 48 h of 35.7 mm, covering the Petri dish on day 3 at
30 C. At 25 C, colony radius after 48 h of 19.4 mm,
covering the Petri dish on day 6. At 35 C, colony radius
after 48 h of 19.5 mm, covering the Petri dish on day 6.
On day 4, colonies dense, mycelia white, sporulation
effuse and scant, forming a reduced reddish lawn,
 MYCOLOGIA 9

Figure 4. Morphological features of Trichoderma jaklitschii (CP61-2). A–B. Photographs of colonies on CDA at 4 (A) and 8 (B) days. C–D.
Photographs of colonies on DRBC at 4 (C) and 8 (D) days. E–F. Photographs of colonies on PDA at 4 (E) and 8 (F) days.
G. Chlamydospore. H. Conidiophores. Bars: A–F = 2 cm; G–H = 10 µm.

zonation inconspicuous. Autolytic excretions and
hyphal coiling absent. No distinct odors or exudates
noted. Reverse pale. Conidiation on day 2, dense, heavily
sporulating regions in the zonation rings. At 25 C,
colonies hyaline and sterile, mycelium white, no soluble
pigments. At 30 C, colonies similar to those that form at
25 C and less conspicuous. Chlamydospores scarce,
terminal and intercalary, 7.5–9.2 × 6.2–8.1 μm, globose
to subglobose. Conidiation noticeable and light reddish
after 3–4 days, initially short-effuse, abundant, loose.
Pustules comprising dense clusters and peripheral con­
idiophores, thick-walled stipes; primary branches form­
ing a reticulum, from which numerous main axes
originate more or less radially. Peripheral conidiophores
10 BUSTAMANTE ET AL.: NEW SPECIES OF TRICHODERMA FROM NORTHERN PERU
straight or slightly curved, with a distinct main axis;
branches 2.1–5.3 μm wide, at right angles or slightly
inclined upward, typically unpaired at lower levels, tend­
ing to be paired or in whorls at upper levels; moniliform
branches uncommon at the periphery. Phialides solitary,
6.1–10.2 × 3.9–6.7 μm, inequilateral, often slightly
curved, distinctly enlarged, rather plump with a mostly
short neck and base, sometimes narrower with a long
neck on young conidiophores. Conidia 2.2–4.0 × 1.7–2.9
μm, greenish, subglobose to globose, smooth.
Colony radius on CDA after 72 h of 15.1–17.6 mm at
30 C, 4.6–5.8 mm at 25 C. Colonies circular, dense, with
uniform hyphae; margin wavy. Aerial hyphae present, in
widely spaced fascicles, forming a more or less radial flat
whitish farinose structure. Autolytic excretions and coil­
ing common. Diffusing pigment greenish yellow, agar
slightly green, odor transient. Conidiation initiated after
2 weeks in central floccules, effuse, remaining white.
Colony radius on DRBC after 72 h of 18.1–21.5 mm
at 30 C, 4.2–6.4 mm at 25 C. Colonies hyaline, dense,
circular, with conspicuously curved hyphae of variable
widths. Aerial hyphae present. Autolytic excretions
scant, coiling present. Diffusing pigment yellow, odor
indistinct. Chlamydospores scant, terminal and interca­
lary. Conidiation noticeable after 3 days, turning green
after day 5, initially short-effuse from the center, soon
forming pustules. Conidia forming on loose, regularly
tree-like conidiophores.
Ecology and distribution: T. jaklitschii is present in the
cacao agricultural ecosystem at low altitudes
(500–900 m elev.) in the Aramango and La Peca districts
of the Amazon region in northern Peru. T. jaklitschii was
isolated from sandy clay loam soils (0–20 cm depth)
approximately 1 m from cacao plant stems.
Additional specimens examined: PERU. AMAZONAS:
Prov. Bagua, Dist. Aramango, El Mirador, 78°26′42″W, 5°
25′52″S, alt. 515 m, 20 Feb 2017, S. Leiva CP01-6;
Pomabamba, 78°25′8″W, 5°26′13,4″S, alt. 891 m, 6
Apr 2017, S. Leiva CP62-2; Dist. La Peca, Arrayan, 78°26′
47″W, 5°35′59″S, alt. 897 m, 9 Mar 2017, S. Leiva CP18-2;
San Francisco, 78°27′41″W, 5°37′36″S, alt. 647 m, 20
Feb 2017, S. Leiva CP18-9.
Notes: T. jaklitschii is currently only known as an asexual
morph from five isolates. T. jaklitschii is phylogenetically
closely related to T. breve, T. peruvianum, and T. zelobreve.
The sequence divergences between T. jaklitschii and
T. breve, T. peruvianum, and T. zelobreve were 2.9%,
3.2%, and 2.6% for rpb2 and 1.3%, 0.3%, and 1.2% for
tef1, respectively. Morphologically, T. jaklitschii is distin­
guished from T. breve by the growth pattern and phialide
dimensions. The former has a reduced reddish lawn on
PDA plates and wider phialides (6.1–10.2 × 3.9–6.7 μm),
whereas the latter has concentric zones of colonies and
slender phialides (6.7–10.0 × 2.8–3.9 μm) (Chen and
Zhuang 2017). T. jaklitschii and T. zelobreve can be readily
distinguished based on their growth rates. On PDA, colo­
nies of T. zelobreve only grow 8–9 mm after 72 h at 35 C, in
contrast to the growth shown by T. jaklitschii, which forms
a radius of 19.5 mm after 48 h at 35 C. In addition, conidial
production starts on day 2 in T. jaklitschii, whereas it was
observed 3 days after the inoculum in T. zelobreve (Gu et al.
2020).
T. jaklitschii and T. peruvianum are morphologically
similar; however, these two species differ slightly in
colony growth and appearance on CDA. Colonies of
T. jaklitschii measure 15.1–17.6 mm in radius after 72
h at 30 C and diffusing pigments are greenish yellow,
whereas T. peruvianum grows 10.4–14.7 mm under the
same conditions and pigments are dark greenish. The
conidia of T. jaklitschii are smaller (2.2–4.0 × 1.7–2.9
μm) than those of T. peruvianum (3.1–4.3 × 2.4–3.7 μm).
Plates display a light reddish color after 3–4 days of
conidiation in T. jaklitschii, whereas a greenish color is
observed in T. peruvianum.
Trichoderma peruvianum M.S. Calderon, D.E.
Bustamante & S. Leiva, sp. nov. FIG. 5
MycoBank MB835466
Typification: PERU. AMAZONAS: Prov. Bagua, Dist. La
Peca, San Francisco, 78°27′41″W, 5°37′36″S, alt. 647 m, 20
Feb 2017, S. Leiva CP15-2 (holotype CHAX CP15-2). Ex-
type culture CHAXC CP15-2. GenBank: acl1 =
MW480157; act = MW480169; rpb2 = MW480153; tef1 =
MW480145.
Etymology: The specific epithet “peruvianum” is
derived from the country where the samples of this
species were collected.
Description: Cultures and asexual morph: Optimum
growth at 30 C on all media. On PDA, colony radius after
48 h of 28.8 mm, covering the Petri dish on day 3 at 30 C. At
25 C, colony radius after 48 h of 16.3 mm, covering the Petri
dish on day 6. At 35 C, colony radius after 48 h of 19.8 mm,
covering the Petri dish on day 6. On day 4, colonies semi­
dense, mycelia white, sporulation effuse and scant, forming
a reduced white lawn, zonation inconspicuous. Autolytic
excretions and hyphal coiling absent. No distinct odors or
exudates noted. Reverse pale. Conidiation on day 2, dense,
heavily sporulating regions in the zonation rings. At 25 C,
colonies hyaline and sterile, mycelium white to transparent,
no soluble pigments. At 30 C, colonies similar to those that
form at 25 C and less conspicuous. Chlamydospores scarce,
terminal and intercalary, 4.3–9.1 × 3.5–8.2 μm, globose to
subglobose. Conidiation noticeable and greenish after 3–4
days, initially short-effuse, abundant, in shrubs. Pustules
 MYCOLOGIA 11

Figure 5. Morphological features of Trichoderma peruvianum (CP15-2). A–B. Photographs of colonies on CDA at 4 (A) and 8 (B) days. C–D.
Photographs of colonies on DRBC at 4 (C) and 8 (D) days. E–F. Photographs of colonies on PDA at 4 (E) and 8 (F) days. G. Conidia.
H. Conidiophores. Bars: A–F = 2 cm; G–H = 10 µm.

comprising dense clusters and peripheral conidiophores,
thick-walled stipes; primary branches forming a reticulum,
from which numerous main axes originate more or less
radially. Peripheral conidiophores straight or slightly
curved, with a distinct main axis; branches 3.1–5.9 μm
wide, at right angles or inclined upward, typically unpaired
at lower levels; moniliform branches uncommon at the
periphery. Phialides solitary or in whorls, 6.6–11.4 ×
5.8–9.0 μm, inequilateral, often slightly curved, distinctly
enlarged, rather plump with a mostly short neck and base.
Conidia 3.1–4.3 × 2.4–3.7 μm, green, subglobose to globose.
Colony radius on CDA after 72 h of 10.4–14.7 mm at 30
C, 4.6–6.8 mm at 25 C. Colonies circular, dense, with uni­
form hyphae; margin wavy. Aerial hyphae present, in
widely spaced fascicles, forming a more or less radial flat
whitish farinose structure. Autolytic excretions and coiling
12 BUSTAMANTE ET AL.: NEW SPECIES OF TRICHODERMA FROM NORTHERN PERU
common. Diffusing pigment dark greenish, agar yellowish
green, odor transient. Conidiation initiated after 2 weeks in
central floccules, effuse, remaining white.
Colony radius on DRBC after 72 h of 16–19 mm at 30 C,
3.5–5 mm at 25 C. Colonies hyaline, dense, circular, hyphae
with variable widths. Aerial hyphae present. Autolytic
excretions scant, coiling present. Diffusing pigment yellow,
odor indistinct. Chlamydospores scant, terminal and inter­
calary. Conidiation noticeable after 3 days, turning green
after day 5, initially short-effuse from the center, soon
forming pustules. Conidia forming on loose, regularly tree-
like conidiophores.
Ecology and distribution: T. peruvianum is present in
the cacao agricultural ecosystem at low altitudes
(250–950 m elev.) in the Imaza and La Peca districts of
the Amazon region in northern Peru. T. peruvianum
was isolated from sandy clay loam soils (0–20 cm
depth) approximately 1 m from cacao plant stems.
Additional specimen examined: PERU. AMAZONAS:
Prov. Bagua, Dist. Imaza, Pakun, 78°17′6″W, 5°10′33″S,
alt. 284 m, 28 Feb 2017, S. Leiva CP15-9.
Notes: T. peruvianum is currently only known as an
asexual morph from two isolates. Trichoderma peruvia­
num is phylogenetically related to T. breve, T. jaklitschii,
and T. zelobreve. The sequence divergences between
T. peruvianum and T. breve and T. zelobreve are 0.6%
and 1.7% for rpb2 and 0.3% and 0.2% for tef1.
The distinctions between T. jaklitschii and T. peruvianum
were mentioned above. T. peruvianum produces wider
phialides (6.6–11.4 × 5.8–9.0 μm) and larger conidia
(3.1–4.3 × 2.4–3.7 μm) than those of T. breve (6.7–10.0 ×
2.8–3.9 μm and 3.0 × 2.8 μm, respectively) and T. zelobreve
(4.0–6.0 × 2.6–3.2 μm and 2.4 × 2.0 μm, respectively) (Chen
and Zhuang 2017; Gu et al. 2020). Colonies of T. zelobreve
only grow 8–9 mm after 72 h at 35 C on PDA, contrary to
the growth shown by T. peruvianum, which forms a radius
of 19.8 mm after 48 h at 35 C.
DISCUSSION
The dependence of fungal identification on species concepts,
delimitation, and recognition approaches was recently
recommended, motivating researchers to employ the
approach of integrative taxonomy (i.e., multilocus phylo­
geny, phenotype) for species identification (Lumbsch and
Leavitt 2011; Liu et al. 2016; Santos et al. 2017; Pozzi et al.
2018; Bustamante et al. 2019; Lücking et al. 2020). The
hyperdiverse genus Trichoderma is one of the most useful
groups of microbes for various human activities, and their
accurate identification is crucial (Druzhinina et al. 2010).
The taxonomy of Trichoderma has been updated with new
combinations and descriptions of new species based primar­
ily on phylogenies and morphological comparisons among
closely related species (Jaklitsch 2009, 2011; Chaverri et al.
2015; Jaklitsch and Voglmayr 2015; du Plessis et al. 2018;
Zhang and Zhuang 2017; Zhu et al. 2017; Gu et al. 2020;
Inglis et al. 2020). However, the structural simplicity and
lack of distinctive phenotypic variation in this group enable
the use of DNA-based species delimitation methods in
addition to phylogenies (and morphology when feasible)
to establish well-supported boundaries among species
(Carstens et al. 2013; Bustamante et al. 2019).
Our study used a multilocus phylogeny and four DNA-
based methods targeting four molecular markers to delimit
and recognize species within two lineages of Trichoderma.
Although incongruence among these methods was
observed in our analyses, the genetic distance method
(ABGD), coalescence method (BPP), and multilocus phy­
logeny strongly supported and confirmed the recognition
of 108 and 39 different species in the Harzianum and
Longibrachiatum lineages, respectively (FIGS. 1–2;
TABLE 3). These 147 species (including the three new
species associated with northern Peruvian cacao farms)
were recognized and identified through diagnostic barcode
gaps for the rpb2 marker (TABLE 1). The tef1 marker was
not diagnostic for the recognition of species in these
lineages, since there were different species that had identical
sequences. Incomplete lineage sorting and recombination
are common events in fungi that support this gene discor­
dance (Stewart et al. 2014; Matute and Sepúlveda 2019);
nevertheless, gene duplication and loss, horizontal gene
transfer, and hybridization might also explain this phenom­
enon (Degnan and Rosenberg 2009; Pozzi et al. 2018).
Additionally, several species of both lineages lack of acl1
and act sequences; thus, these markers were not evaluated
as diagnostic.
The methods based on genetic distance (ABGD and
SPN) showed slightly different results from the multi­
locus phylogeny in delimiting species embedded in the
Harzianum and Longibrachiatum lineages. The fewer
putative species indicated by ABGD and SPN may pri­
marily be due to the merging among some species that
have identical sequences (e.g., tef1) or low p-distance
values (e.g., rpb2). Conversely, one of the coalescence
methods (GMYC) showed the lowest putative species
results (2) for the four loci, leading to conservative infer­
ences. Coalescent models usually produce false positives
and complex false positives when delimiting different
taxa showing low or high magnitudes of gene flow,
respectively (Zhang et al. 2011). This suggests that the
negative impacts observed when delimiting species in the
Harzianum and Longibrachiatum lineages are due to
gene flow rates among their species (Luo et al. 2018), as
occurred in another genus with structural simplicity,
namely, Beauveria (Bustamante et al. 2019). Finally, the
other coalescence method (BPP) supports the status of

the species recognized by the multilocus phylogeny (pos­
terior probabilities [PPs] 0.94–0.95; SUPPLEMENTARY
TABLE 3) and did not support those merged taxa by SPN
or GMYC (PPs lower than 0.02; SUPPLEMENTARY
TABLE 3). The performance in empirical studies of the
genetic and coalescent methods tends to undersplit and
oversplit species, respectively (Paz and Crawford 2012;
Pentinsaari et al. 2017; Renner et al. 2017; Luo et al.
2018). However, our results suggest that ABGD (for the
rpb2 marker) and BPP are appropriate for determining
diversity in the Harzianum and Longibrachiatum
lineages by recognizing the species delimited by the mul­
tilocus phylogeny. Limited insights were obtained for
these methods by Lücking et al. (2020), although these
approaches have been further recommended to delimit
and recognize species among fungal taxa (Powell et al.
2011; Parnmen et al. 2012; Fujisawa and Barraclough
2013; Haelewaters et al. 2018; Bustamante et al. 2019).
DNA-based methods and a set of morphological fea­
tures support the recognition of three new species,
namely, T. awajun, T. jaklitschii, and T. peruvianum.
The genetic divergence among these species and other
Trichoderma is higher than the minimum threshold
observed in different species of this genus, as well as
other species belonging to the Harzianum lineage (e.g.,
T. beinartii, T. caeruleimontis, and T. chetii; du Plessis
et al. 2018), that were not included in our analyses due to
the short length of their sequences (tef1 and rpb2).
Ecologically, the three new species present sympatric dis­
tributions in Bagua Province, since T. awajun,
T. jaklitschii, and T. peruvianum are found across all
collection sites. However, the limited area of this study
might suggest that further sampling is needed to confirm
the current distribution of these species.
Furthermore, the strongly supported cluster contain­
ing specimens identified as T. jaklitschii and T. orientale
was delimited with 2 and 3–9 putative species, respec­
tively, by ABGD (for tef1 and rpb2). This result confirms
that T. jaklitschii and T. orientale have higher sequence
diversity than other Trichoderma species and that their
putative species might be recognized as different entities;
however, multilocus delimitation (phylogeny and BPP)
highly supports the recognition of these clades as unique
species. Further analysis with additional sequences and
markers of new specimens might confirm their status
because few individuals often fail to represent the species
as a whole (Davis and Nixon 1992; Walsh 2000).
Conversely, although the specimens labeled as
Trichoderma sp. 3 diverged from T. breve and
T. zelobreve by 1.4% and 1.5%, respectively, for the tef1
marker, this species was not recognized as a different
species in this study, since additional markers are
needed to confirm this sister relationship.
MYCOLOGIA 13
The identification of these three new species increases
the number of isolates associated with Theobroma spp.
(e.g., T. lieckfeldiae, T. evansii, T. koningiopsis, T. martiale,
T. ovalisporum, T. paucisporum, T. stromaticum, and
T. theobromicola embedded into Harzianum, Hamatum,
and Viride lineages), which have been proposed as poten­
tial biological controls for cacao diseases (Chaverri et al.
2003; Holmes et al. 2006; Samuels et al. 2006a, 2006b;
Hanada et al. 2008; Samuels and Ismaiel 2009). Our
results might represent important progress in agronomic
programs regarding the exploration of antagonists for
biocontrol and pathogen eradication (Paterson 1991;
Bidochka et al. 2001; Krauss et al. 2010; Inglis et al.
2020). However, the possible use of T. awajun as
a mycoparasite and biocontrol agent should be evaluated
with caution, because its close relative species T. bissettii
and T. longibrachiatum, as well as other members of the
Longibrachiatum lineage, have been isolated from clinical
samples and are known to be human pathogenic/oppor­
tunistic species (Druzhinina et al. 2010; Samuels et al.
2012; Sandoval-Denis et al. 2014; Hatvani et al. 2019).
The natural classification of species within many impor­
tant fungal genera has been facilitated by the use of inte­
grative approaches, including morphology, metabolomics,
phylogenetics, genetic distance, and coalescent methods
(DNA-based approaches) (Milic et al. 2012; Liu et al.
2016; Bustamante et al. 2019, Lücking et al. 2020). This
study recognized 108 and 39 species embedded into the
Harzianum and Longibrachiatum lineages, including three
new species. These results were supported by congruence
among genetic distance (ABGD), coalescence (BPP), multi­
locus phylogeny, and morphology, suggesting that the use
of an integrative approach is more likely to identify reliably
supported species boundaries in Trichoderma.
FUNDING
This study was supported by National Institute of Agricultural
Innovation (INIA) grant 004-2016-INIA-PNIA-UPMSI/IE
through the National Program for Agricultural Innovation of
Peru (PNIA), by grant 10-2018-FONDECYT-BM-ADT-AV
from National Foundation for Scientific, Technological
Development and Technological Innovation (Fondecyt) of
Peru, and by grant SNIP no. 352641–CEINCACAO.
ORCID
Danilo E. Bustamante http://orcid.org/0000-0002-5979-
6993
Martha S. Calderon http://orcid.org/0000-0003-3611-140X
Santos Leiva http://orcid.org/0000-0003-1710-1994
Jani E. Mendoza http://orcid.org/0000-0001-8324-039X
Marielita Arce http://orcid.org/0000-0002-2916-0089
Manuel Oliva http://orcid.org/0000-0002-9670-0970
14 BUSTAMANTE ET AL.: NEW SPECIES OF TRICHODERMA FROM NORTHERN PERU
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