Microbiological Research 275 (2023) 127469

Contents lists available at ScienceDirect

Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Trichoderma agriamazonicum sp. nov. (Hypocreaceae), a new ally in the

control of phytopathogens
Thiago Fernandes Sousa a, b, Bruna Nayara Pantoja Vieira Reça c, Gleucinei Santos Castro d,
Ingride Jarline Santos da Silva a, b, Fernanda Fátima Caniato e, Moysés Batista de Araújo Júnior f,
Michel Eduardo Beleza Yamagishi g, Hector Henrique Ferreira Koolen d,
Giovana Anceski Bataglion h, Rogério Eiji Hanada i, *, Gilvan Ferreira da Silva b, *
a
Programa de Pós-graduação em Biotecnologia, Universidade Federal do Amazonas (UFAM), 69080-900 Manaus, Brazil
b
Embrapa Amazônia Ocidental, 69010-970 Manaus, Brazil
c
Programa de Pós-graduação em Agricultura no Trópico Úmido (ATU), Instituto Nacional de Pesquisas da Amazônia (INPA), 69067-375 Manaus, Brazil
d
Grupo de Pesquisas em Metabolômica e Espectrometria de Massas, Universidade do Estado do Amazonas (UEA), 690065-130 Manaus, Brazil
e
Departamento de Ciências Fundamentais e Desenvolvimento Agrícola, Faculdade de Ciências Agrárias, Universidade Federal do Amazonas (UFAM), 69080-900
Manaus, Brazil
f
Programa de Pós-graduação em Química, Universidade Federal do Amazonas (UFAM), 69080-900 Manaus, Brazil
g
Embrapa Agricultura Digital, 13083-970 Campinas, Brazil
h
Departamento de Química do Instituto de Ciências Exatas, Universidade Federal do Amazonas (UFAM), 69080-900 Manaus, Brazil
i
Instituto Nacional de Pesquisas da Amazônia (INPA), 69067-375 Manaus, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: The genus Trichoderma comprises more than 500 valid species and is commonly used in agriculture for the
Amazon fungi control of plant diseases. In the present study, a Trichoderma species isolated from Scleronema micranthum
Peptaibols (Malvaceae) has been extensively characterized and the morphological and phylogenetic data support the
Post-harvest
proposition of a new fungal species herein named Trichoderma agriamazonicum. This species inhibited the
Biocontrol
mycelial growth of all the nine phytopathogens tested both by mycoparasitism and by the production of VOCs,
with a highlight for the inhibition of Corynespora cassiicola and Colletotrichum spp. The VOCs produced by
T. agriamazonicum were able to control Capsicum chinense fruit rot caused by Colletotrichum scovillei and no
symptoms were observed after seven days of phytopathogen inoculation. GC-MS revealed the production of
mainly 6-amyl-α-pyrone, 1-octen-3-ol and 3-octanone during interaction with C. scovillei in C. chinense fruit. The
HLPC-MS/MS analysis allowed us to annotate trikoningin KBII, hypocrenone C, 5-hydroxy-de-O-methyl­
lasiodiplodin and unprecedented 7-mer peptaibols and lipopeptaibols. Comparative genomic analysis of five
related Trichoderma species reveals a high number of proteins shared only with T. koningiopsis, mainly the en­
zymes related to oxidative stress. Regarding the CAZyme composition, T. agriamazonicum is most closely related
to T. atroviride. A high protein copy number related to lignin and chitin degradation is observed for all Tricho­
derma spp. analyzed, while the presence of licheninase GH12 was observed only in T. agriamazonicum. Genome
mining analysis identified 33 biosynthetic gene clusters (BGCs) of which 27 are new or uncharacterized, and the
main BGCs are related to the production of polyketides. These results demonstrate the potential of this newly
described species for agriculture and biotechnology.

1. Introduction metabolites and volatile organic compounds (VOCs), which can be used
in crop growth and protection (Sharma et al., 2019; Sood et al., 2020;
Trichoderma species are ubiquitous and commonly found in soil, Poveda, 2021; Woo et al., 2022). Trichoderma spp. also are great pro­
plants or decaying wood. They have been widely used in agriculture due ducers of peptaibols, membrane-active α-aminoisobutyric acid rich
to their ability to secrete lytic enzymes and produce bioactive secondary polypeptides, which have many properties including antimicrobial and

* Corresponding authors.
E-mail addresses: rhanada.inpa@gmail.com (R.E. Hanada), gilvan.silva@embrapa.br (G.F. da Silva).

https://doi.org/10.1016/j.micres.2023.127469
Received 28 March 2023; Received in revised form 23 July 2023; Accepted 1 August 2023
Available online 2 August 2023
0944-5013/© 2023 Published by Elsevier GmbH.
T.F. Sousa et al. Microbiological Research 275 (2023) 127469

antifungal properties and have been reported as next-generation anti­ PDA, CMD and SNA media and incubated at 25 ◦ C for 72 h, and 30
plasmodial therapeutics (Marik et al., 2019; Gavryushina et al., 2021; structures of conidia, phialide and chlamydospores were measured
Lee et al., 2021). under a microscope (Zeiss). Scanning electronic microscopy (JSM-
The genus Trichoderma was described by Persoon (1794) and many IT500HR) was used to obtain the images of the microstructures, which
revisions of its taxonomy have been carried out in the last decades, was carried out at the Multiuser Center for the Analysis of Biomedical
which have dramatically increased the number of species in the genus Phenomena at the Amazonas State University (CMABio). For this, the
mainly due the incorporation of other barcodes, such as endochitinase microculture was prepared in PDA and incubated at 28 ◦ C for 4 days,
ech42, a beta subunit of DNA-directed RNA polymerase rpb2, and fixed with Karnovsky fixative, and then dehydrated using increasing
translation elongation factor tef1-α genes in molecular identification concentrations of alcohol (30%, 40%, 50%, 70%, 80%, 90%, 100%) for
(Kullnig-Gradinger et al., 2002; Chaverri et al., 2003; Druzhinina et al., 15 min each. The step with absolute alcohol was repeated twice. The
2006; Jaklitsch et al., 2006; Chaverri et al., 2015). Recently, a Tricho­ microculture was dried in a critical point dryer (Leica EM CPD300) and
derma molecular identification guideline was established based on a then subjected to gold plating (DII-29010SCTR Smart Coater).
polyphasic approach, thus preparing the taxonomy of Trichoderma for
the genomic era (Cai and Druzhinina, 2021). According to Index Fun­
gorum (http://www.indexfungorum.org/), more than 500 valid species 2.3. DNA isolation, PCR amplification
have been described for the genus.
Despite the existence of a high number of described species so far, the To obtain the mycelial mass of the isolate INPA 2475, mycelia was
number of complete genomes of Trichoderma available in a public grown on PD medium (20 g/L potato; 20 g/l dextrose) for two days. The
database is still low, with only 87 sequenced genomes in the Joint broth was filtered, and DNA was isolated using the CTAB protocol
Genome Institute JGI (mycocosm.jgi.doe.gov). Complete genomes can (Doyle, 1987). The quantity of the DNA obtained was estimated using
enable the prospection and obtention of new metabolites and enzymes of spectrophotometry (NanoDrop 2000, Thermo Scientific), while the
biotechnological interest (Gupta et al., 2016; Sun et al., 2019; Shenouda integrity was verified via electrophoresis in a 0.8% (w/v) agarose gel.
and Cox, 2021; Zhu et al., 2021). The use of genome mining for pre­ Each PCR reaction was performed in a 25 µL final volume, containing
dicting molecules also can accelerate new drug discovery via the 2.5 µL of 10X Easytaq® buffer, 10 nM dNTP, 1 U Easytaq® DNA poly­
synthetic-bioinformatic natural product discovery approach (Chu et al., merase, 0.5 pM of each primer, 1 µL of total DNA (50 ng) and autoclaved
2016; Chu et al., 2019; Chu et al., 2020). In the near future, the whole distilled water to achieve the final volume. The amplification reactions
genome sequencing of the type species will allow a global comparison of of the three loci (ITS, tef1-α and rpb2) were as follows: initial denatur­
the genome, with analysis of hundreds or thousands of genes, which will ation of DNA at 95 ◦ C for 3 min, 35 cycles with denaturation at 95 ◦ C for
help to resolve cryptic taxonomic species and ambiguous identification 45 s, annealing step at 55 ◦ C for 45 s, followed by extension at 72 ◦ C for
in Trichoderma via phylogenomic analysis. 60 s. The final extension was performed at 72 ◦ C for 5 min. PCR products
Species of Trichoderma sections (sensu Viride clade) included Tri­ were resolved on agarose gel 1.5% (w/v) stained with ethidium bro­
choderma species of the extinct clades Rufa and Pachybasium A and mide, photo documented using a molecular imaging system (Loccus
encompasses Trichoderma koningii aggregate species such as Biotecnologic L-Pix. Chemi), and amplicons were compared with 1 kb
T. ovalisporum, T. caribbaeum, T. dorotheae, T. dingleyae, T. koningiopsis, plus marker (Invitrogen, catalog number: 10787018). The pair of
T. petersenii and T. taiwanense, T. texanum, T. rogersonii, T. austrokoningii, primers EF-1αF (ATGGGTAAGGARGACAAGAC) and EF-1αR (GGARG­
and T. stilbohypoxyli (Samuels et al., 2006; Montoya et al., 2016; Cai TACCAGTSATCATGTT) were used to amplify the partial sequence of the
et al., 2021). This clade is characterized by pustules containing aggre­ translation elongation factor gene (O’Donnell et al., 1998; Carbone and
gate formations of conidiophores and has applications in agriculture as a Kohn, 1999), The ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4
biocontrol agent (Samuels et al., 2006; John et al., 2010; Ayele et al., (TCCTCCGCTTATTGATATG) were used for amplification of internal
2021; Poveda, 2021; Rush et al., 2021). transcribed spacer (White et al., 1990) and the fRPB2–5 f (GAY­
In this study, we isolated a new fungal species of Trichoderma from GAYMGWGATCAYTTYGG) and fRPB2–7cr (CCCATRGCTbTGTYYY)
Scleronema micranthum (Malvaceae), a native plant to the Amazon were used for amplifying the partial sequence of the second largest
rainforest that is used in forestry. This new species has been extensively subunit of RNA polymerase II (Liu et al., 1999).
characterized based on morphological characters such as the length of Prior to sequencing, the PCR products were cleaned with ExoSAP-IT
the conidia, phialides and chlamydospores, sequence analysis of the ITS, (Applied Biosystems, product code: 15819906). For this, 5 µL of PCR
tef-1α and rpb2 loci, mycoparasite activity, production and activity of products were mixed with 2 µL of EXOSAP and incubated at 37 ◦ C for 15
volatile organic compounds (VOCs) against different phytopathogens, min, followed by the ExoSAP-IT inactivation step at 80 ◦ C for 15 min.
secondary metabolite annotation and comparative genomic analysis. Then, the cleaned PCR products were submitted to sequencing using the
BigDye terminator protocol using a 10 µL volume reaction containing, 2
2. Materials and methods µL of ultrapure water, 1.5 µL of 5X BigDye buffer, 0.5 µL of BigDye
Terminator v3.1 (Thermo Fisher), 1 µL of each primer and 5 µL of
2.1. Strain origin cleaned PCR products. The thermocycling conditions consisted of a
denaturation at 96 ◦ C for 60 s, followed by 35 cycles at 96 ◦ C for 15 s,
The examined strain INPA 2475 was obtained from the National 50 ◦ C for 15 s and 60 ◦ C for 4 min. Reactions were resolved using
Institute for Amazonian Research (INPA) and was isolated in 2004 as an capillary electrophoresis on sequencing instrument (3500 Genetic
endophyte isolated from the sapwood of Scleronema micranthum in Analyzer, Thermo Fisher).
Manaus, Amazonas state, Brazil.

2.2. Morphological characterization 2.4. Genome sequencing

Macromorphology was performed in four different culture media: The complete genome was sequenced using the 150 bp paired-end
PDA (200 g/L potato; 20 g/L dextrose; 15 g/L agar), MEA (30 g/mL malt Illumina platform. The “de novo” assembly was performed using the
extract; 5 g/mL peptone; 15 g/L agar), CMD (50 g/L cornmeal; 15 g/L SPAdes (Prjibelski et al., 2020) assembler, and resulted in 36426151 bp
agar; 20 g/L) and SNA (1 g/L KH2PO4, 1 g/L KNO3, 0.5 g/L MgSO4, 0.5 with 103X coverage and 109 scaffolds with an N50 of 1650868 bp. The
g/L KCl, 0.2 g/L glucose, 0.2 g/L sucrose, 20 g/L agar) under incubation assembled genome was deposited in the Genbank under access number
at 25 ◦ C for 72 h. For the micromorphology, the culture was prepared in JARPVV000000000.

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2.5. Phylogenetic analysis 2.8. Antifungal activity of the VOCs

Consensus sequences were obtained based on alignment of forward To analyze the production of volatile organic compounds (VOCs) and
and reverse sequences using DNA baser assembly software (http:// its mycelial growth inhibition effect, the sandwich plate technique was
www.dnabaser.com/). The new sequences obtained were deposited in used (Ebadzadsahrai et al., 2020), in which the bases of petri dishes of
GenBank under accession numbers OQ221157 (ITS) OQ241434 (tef1-α) the same size containing PDA medium were inoculated with mycelial
and OQ241435 (rpb2). The ITS sequences were used for identification of discs, one dish with the isolate INPA 2475 and in the other a mycelial
the genera as per the recommendations in the Trichoderma guidelines disk of the phytopathogen so that the isolate INPA 2475 was at the
(Cai et al., 2021), and phylogenetic identification of the INPA 2475 bottom and the phytopathogen on top. These dishes were sealed with
isolate was conducted by an initial proximity screening using tef1-α parafilm and incubated at 28 ◦ C for 7 days. A dish with the phyto­
sequences of 103 Trichoderma species belonging to clade 5 obtained in pathogen paired with PDA medium without the presence of the isolate
the Trichokey database in the section Trichoderma taxonomy (trichokey. INPA 2475 was used as a positive control. The inhibition calculation was
com/index.php/trichoderma-taxonomy-2020). The sequences of closely performed using the same formula that was used in dual-culture assay.
related species were selected for concatenated analysis. The final dataset
resulted in 46 Trichoderma spp. sequences (22 = closely related species 2.9. Efficacy of INPA 2475 VOCs in the control of fruit anthracnose in
of clade 5; 23 = species belongs to clade 1) in which Trichoderma Capsicum chinense Jaq. caused by Colletotrichum scovillei
undulatun was used as an outgroup.
The sequences of tef1-α and rpb2 were individually aligned using the A 200 mL borosilicate graduated glass reagent bottle containing
MAFFT tool in the UGENE software (Okonechnikov et al., 2012). 50 mL of PDA medium was inoculated with mycelium discs of the isolate
Alignments were plotted in IQ-TREE 2 (Minh et al., 2020) and a INPA 2475 and incubated for two days at 28 ◦ C. After this period, the
phylogenetic analysis using maximum likelihood (mL) was performed pepper fruits were soaked with a concentrated spore solution of
with the concatenated tef1-α and rpb2 sequences. Bayesian inference (BI) C. scoville (108 spores/mL), wrapped in gauze, which was secured with
was performed via CIPRES (www.phylo.org). the lid of the bottle, which was sealed with parafilm. The bottle was
The ML analysis included 1000 replicates (bootstrap) using all the incubated at 28 ◦ C and evaluated after seven days. For the control, the
sites, with the best model selected by IQ-TREE. Bayesian inference (BI) same proceedings were used though with the exception of the inocula­
was based on the model adopted in PAUP * 4 and Mrmodeltest2 v2 tion of Trichoderma INPA2475. The experiment was conducted in
(Nylander 2004). All sites in the loci were considered; the analysis was triplicate.
performed for ten million generations, with the first 25% of the trees
discarded and burned using the MrBayes v 3.7 tool available from 2.10. Statistical analysis
CIPRES (https://www.phylo.org/). Posterior probability (PP) and tree
topology were visualized via Figtree v 1.1.2 (Rambaut 2009). The The data of for the nine plant pathogens were first tested for
consensus tree of the ML and BI analyses was generated manually from normality of residuals (Shapiro-Wilk test, P ≥ 0.05) using the R package
the topology obtained using the iTol platform (https://itol.embl.de/) in stats v4.1.3 (R Core Team, 2022) and for homogeneity of variance
BI analysis with the posterior probability values, plus the bootstrap (Levene’s test, P ≥ 0.05) using the R package car v3.0–12. Then, the
values generated by the maximum likelihood analysis, using the Corel­ data was submitted to analysis of variance (ANOVA), followed by a
Draw 2020 editing package. Tukey’s HSD test (P ≤ 0.05) for all possible pairwise comparisons of the
plant pathogens tested, using the R package agricolae v1.3–5 (Mendi­
2.6. Phytopathogens used in dual culture and VOC assays buru, 2021).

The phytopathogens used in this work belong to the microbiological 2.11. Comparative genomic analysis
collection of the Laboratory of Phytopathology at National Institute for
Amazonian Research (INPA) and the Laboratory of Molecular Biology at The analyses of orthologous genes were carried out using the genome
Embrapa Amazônia Ocidental. The species and collection code are: of INPA 2475 and four closely related species with genome sequences
Corynespora cassiicola INPA 2671, Colletotrichum scovillei INPA 2910, available. These sequences were submitted to protein prediction using
Colletotrichum sp. INPA 2973, Rhizoctonia sp. INPA 2942, Alternaria sp. the ASAP pipeline (Brandes et al., 2016) and the FASTA protein data
SMPT 22, Colletotrichum siamense CPAA Coll 2 N, Fusarium decemcellulare generated were plotted in Orthovenn2 (orthovenn2.bioinfotoolkits.net).
CPAA 307 and Fusarium sp. MCT 10621. The phytopathogen Colleto­ The INPA 2475 genome also was submitted to the fungiSMASH platform
trichum sp. INPA 2931 was used only in dual culture assay. The origin (https://fungismash.secondarymetabolites.org/) for identification of
and host of these isolates are listed in Table S2. biosynthetic gene clusters.
The putative protein sequences of T. agriamazonicum and of four
2.7. Dual culture assay other closely related species (T. viride, T. atroviride, T. koningiopsis and
T. koningii) were submitted to the dbCAN2 Meta server (https://bcb.unl.
The antagonist potential of the isolate INPA 2475 towards nine plant edu/dbCAN2/blast.php) in order to perform the CAZy family annotation
pathogens was tested using the dual culture method. Discs with mycelia using the HMMER tool. The heatmap of the main cell wall degradation
(5 mm) of the isolate INPA 2475 and of the phytopathogens were enzymes was plotted using the R package “pheatmap” v1.0.12 (https://
inoculated in Petri dishes (90 mm) containing PDA culture medium in CRAN.Rproject.org/package=pheatmap).
opposite directions at a 50 mm from the edge. Dishes containing only the
phytopathogen were used as a positive control. Inoculums were incu­ 2.12. VOC identification via GC-MS analysis
bated at 28 ◦ C and evaluated after 7, 14 and 21 days. The percentage of
mycelial growth inhibition (MGI) of phytopathogens was calculated For the VOC assays, Kitassato-type bottles (500 mL) were used to
according to the formula: Inhibition% : GC−GCGT x100, in which GC repre­ perform the different treatments: 1) only the pepper sample (Capsicum
sents the mean diameter of the control and GT represents the mean chinense); 2) pepper sample inoculated with Colletotrichum scoville; 3)
diameter of the treatment. PDA culture medium (25 mL) with INPA 2475; and 4) PDA culture
medium (25 mL) with INPA 2475 in the presence of the pepper sample
inoculated with C. scoville. The vials were sealed with a polypropylene
cap (top-opening) and GC septa (side-opening) and kept at 28 ◦ C for

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T.F. Sousa et al. Microbiological Research 275 (2023) 127469

seven days. On the seventh day, the flask was heated at 45 ◦ C for 30 min; chromatograms and was expressed as a relative percentage. Compounds
then, the SPME fiber was inserted via the side opening of the Kitassato were identified based on similarities with data from the NIST library,
bottles and the divinylbenzene-carboxen-polydimethylsiloxane fiber elution order and retention indexes (RI). A mixture of linear alkanes
(DVB/CAR/PDMS, 50/30 µm, Supelco) was exposed to the volatile (C7–C30) was analyzed with the samples to calculate the RI according to
organic compounds (VOCs) for 60 min at the same temperature. Sub­ the Van Den Dool–Kratz equation (Van, Kratz, 1963).
sequently, the injections were performed manually in a gas chromato­
graph coupled to a mass spectrometer (GC-MS) (QP2010 plus,
Shimadzu). 2.13. HPLC/MS-MS analysis
Following extraction, the fiber assembly was subjected to desorption
for 300 s at 270 ◦ C. Chromatographic separation was performed in a The separation was carried out on a high-performance liquid chro­
Rtx-5 (Restek) fused silica capillary column (30 m × 0.25 mm i.d., matograph (HPLC) (Thermo-Ultimate 3000, Thermo Scientific) in a C18
0.25 µm film thickness) using helium as the carrier gas (1 mL/min). The Kinetex Coreshell column (100 ×4.6 mm and 2.6 µm particle size) at 50
following temperature program was used: initial temperature of 50 ◦ C, ºC. A volume of 5 µL of the sample solution was injected in this analysis.
ramped at 5 ◦ C /min to 300 ◦ C, then held for 10 min. The injector, A flow rate of 0.4 mL/min was applied to the chromatographic method,
GC–MS interface, and MS source temperatures were 270 ◦ C, 280 ◦ C and in which the mobile phases comprised water (A) and acetonitrile (B),
250 ◦ C, respectively. The quadrupole was operated in the full scan mode both containing 0.1% formic acid and pumped in a gradient as follows:
with an m/z range of 50–550. The proportion of each compound was 0–2 min 10% B, 2–12 min 10–95% B, 12–15 min 95% B, 15–16 min
estimated by dividing its mean area by the total identified mean area of 10% B, 16–19 min 10% B.
The HPLC was connected in-line to a mass spectrometer (Orbitrap ID-

Fig. 1. Phylogram generated by concatenated analysis of tef1-α and rpb2 sequences of Trichoderma agriamazonicum and closely related species of clade 5 and Tri­
choderma belong to clade 1. The numbers indicate the support of the branches (mL/PP). The new species is marked in dark blue. Trichoderma undulatum (clade 8) was
used as an outgroup.

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T.F. Sousa et al. Microbiological Research 275 (2023) 127469

X™ Tribrid, Thermo Scientific). Acquisitions were carried in positive Typification: Mycobank: MB 848125 Type:—BRAZIL. Amazonas, in
electrospray ionization with a capillary voltage of + 3500 V and tem­ the tissue of Scleronema micranthum, 2004, type: INPA 2475. The type is
perature of 263 ºC. The search for masses was done in the interval of m/z deposited in the National Research Institute of Amazon Research under
100–1500 (70 K resolution at m/z 200), automatic gain control (AGC) collection code: INPA 2475. Sequences from this strain have been
was set to accumulate 3 × 106 ions with a maximum injection time of deposited in GenBank with accession numbers: ITS= OQ221157, tef1-
100 ms. Tandem MS analysis was performed at a 17.5 K resolution, AGC α = OQ241434, rpb2 = OQ241435.
set to 105 in 50 ms (maximum) and isolation window of m/z 3.0. Macromorphology: Colonies at 25 ◦ C and growing for 72 h in PDA
medium present a white mycelium of a cottony appearance with low
3. Results production of conidia and conidiophores and a sparse mycelium in the
center; no pigment or exudate production was observed; colony radius
3.1. Phylogenetic analysis after 72 h on PDA: 55.4 − 56.3 mm (Fig. 3A). Colonies at 25 ◦ C and
growing for 72 h in CMD medium present a white to hyaline sparse
The phylogeny using 103 tef1-α sequences of Trichoderma spp. belong mycelium with low production of pustules in the hyphae contact zone,
to clade 5 reveals an unambiguous position of isolated INPA 2475 with low production of conidia and conidiophores; no pigments or exudates
high bootstrap support, indicating a new species and presents proximity are observed in CMD media; colony radius after 72 h on CMD:
with T. ovalisporum species (Fig. S1). Based on concatenated alignment 43.8–44 mm; Colonies at 25 ◦ C and growing for 72 h in MEA medium
of tef1-α and rpb2 sequences of the most closely related species, we found present a white mycelium of a cottony appearance with sparse mycelium
1166 characters including gaps (tef1-α: 607; rpb2: 559). The best-fit in the border and low production of conidia and conidiophores; no
evolution model for BI analysis adopted by PAUP* 4 was HKY+I+G pigments or exudates are observed in MEA media; colony radius after
for tef1-α and SYM+G for rpb2. For the mL analysis, the best-fit evolution 72 h on MEA: 48.1–48.8 mm. Colonies at 25 ◦ C and growing for 72 h in
model was HKY+F+I+G4 for tef1-α and TNe+G4 for rpb2. The concat­ SNA medium present a white sparse mycelium with abundant pustules,
enated analysis showed the formation of a sister clade with T. texanum conidia and conidiophores production, forming a concentric ring; no
species, thus indicating a new taxon (Fig. 1). The phylogeny using un­ pigment or exudate production was observed; colony radius after 72 h
linked loci supports the new species proposition via distinct clade for­ on SNA: 25.4–25.8 mm (Fig. 3A).
mations in the phylograms (Figs. S1 and S2). Micromorphology in PDA: Conidiophores present smooth-walls
By investigating the nucleotide differences between the isolate INPA containing 2–3 lageniform phialides measuring 5.27–8.9 µm
2475 and the closely related T. texanum and T. ovalisporum, we found (M=7.16 µm) × 1.9–3.3 µm (M=2.27 µm) (Fig. 3B,C,F) with the pres­
differences in 39 nucleotide positions for T. texanum (tef1-α: 31 and rpb2: ence of greenish-yellow, globose conidia measuring 2.96–4.10 µm
8) and, in 37 nucleotides, differences were found for T. ovalisporum (tef1- (M=3.47 µm) × 2.82–3.64 µm (M=3.21 µm) (Fig. 3G). Presence of ter­
α: 28 and rpb2: 9). By extending the comparison for T. koningiopsis, the minal chlamydospores with 4–5.37 µm (M=4.99 µm) × 4.36–4.92 µm
differences were as high as 52 nucleotide positions (tef1-α: 42 and rpb2: (M=4.65 µm) (Fig. 3D,E) and intercalary chlamydospores with
10). Moreover, the isolate INPA 2475 of T. agriamazonicum displayed 5.94–6.3 µm (M=6.1 µm) x 3.2–3.82 µm (M=3.5 µm) (Fig. 3H).
single nucleotide polymorphisms (SNP) in 20 nucleotides (tef1-α: 15 and Micromorphology in SNA: Conidiophores present smooth-walls
rpb2: 5) in relation to T. ovalisporum, T. texanum and T. koningiopsis containing 2–3 lageniform phialides measuring 5.1–8.6 µm
(Fig. 2). Taken together, the morphological characteristics and molec­ (M=7.12 µm) × 1.7–3.2 µm (M=2.19 µm) with the presence of
ular data support the proposal of INPA 2475 as a new fungal species greenish-yellow, globose conidia measuring 2.97–3.98 µm
herein named Trichoderma agriamazonicum. (M=3.46 µm) × 2.67–3.47 µm (M=3.13 µm). Presence of terminal
chlamydospores with 5.44–9.04 µm (M=6.93 µm) × 5.32–8.38 µm
3.2. Taxonomy (M=6.74 µm) and intercalary chlamydospores with 7–8.8 µm
(M=7.64 µm) × 3.8–4.72 µm (M=4.73 µm).
Trichoderma agriamazonicum T.F. Sousa & G.F. Silva, sp. nov. Micromorphology in CMD: Conidiophores present smooth-walls

Fig. 2. Polymorphism identified in tef1-α and rpb2 genes in Trichoderma agriamazonicum sp. nov. and closely related species; Asterisk indicates the single nucleotide
polymorphism (SNP) present only in T. agriamazonicum.

5
T.F. Sousa et al. Microbiological Research 275 (2023) 127469

12.75% and 35% of MGI respectively was observed on the 7th day;
nonetheless, on the 14th day, the MGI increased to 53.43% and 69.26%
(Fig. 4C). The phytopathogen Alternaria sp. SMPT22 presents an MGI of
66.66%.
The interaction between Colletotrichum siamense CPAA Coll 2 N and
T. agriamazonicum was better characterized using SEM and the myco­
parasitism status has been confirmed. In the contact zone in the dual
culture assay, the hyphae of T. agriamazonicum constrict the hyphae of
C. siamense (Fig. 4AB).

3.4. Effect of volatile organic compounds (VOCs) on the mycelial growth

of phytopathogens

The growth of all the phytopathogens was affected by the VOCs of

T. agriamazonicum. The main inhibition was observed for Corynespora
cassiicola INPA 2671 (54%), while lesser inhibition was observed in
Fusarium spp. (17% and 26%) (Fig. 5AB). Colletotrichum spp., Alternaria
sp. SMPT22 and Rhizoctonia sp. INPA 2942 present > 40% MGI. Among
the Colletotrichum species, C. scoville INPA 2910, which causes fruit rot in
Capsicum chinense, has the greatest inhibition (50%) (Fig. 5AB).

3.5. Effect of VOCs in the control of fruit rot caused by Colletotrichum

scovillei

Based on the results of the mycelial growth inhibition by the VOCs of

T. agriamazonicum, an in vivo assay using Capsicum chinense fruits was
conducted to evaluate the effects of the VOCs in the control of fruit rot
caused by C. scovillei INPA 2910. On the 7th day after inoculation of the
phytopathogen, no symptoms were observed in the treatment with
T. agriamazonicum, while the control (not inoculated with INPA2475) in
this same period presented a completely rotten fruit with abundant
spores on the surface of the fruit (Fig. 6).

3.6. VOC characterization using GC-MS

Fig. 3. a Colony morphology of Trichoderma agriamazonicum sp. nov. in PDA, To identify the volatile organic compounds produced by Trichoderma
MEA, CMD and SNA media. b, c, e Conidiophores. d Chlamydospore. f
agriamazonicum for the control of anthracnose fruit rot in Capsicum
Greenish-yellow conidia (red arrow) and chlamydospores of cotton-blue color.
chinense caused by Colletotrichum scovillei, we conducted a GC/MS-MS
g Intercalary chlamydospore (yellow arrow).
analysis. The VOCs that are produced by T. agriamazonicum during the
interaction with C. chinense and Colletotrichum scovillei were 3-octanone,
containing 2–3 lageniform phialides measuring 5.09–9.16 µm
2-octen-1-ol, α-copaene and benzeneacetil acid. The other VOCs iden­
(M=7.11 µm) × 2.11–4.05 µm (M=3.12 µm) with the presence of
tified in T. agriamazonicum are listed in Table 1.
greenish-yellow, globose conidia measuring 3.07–4.05 µm
(M=3.44 µm) × 2.93–4.19 µm (M=3.60 µm). Presence of terminal
3.7. HPLC-MS/MS analysis
chlamydospores with 6.82–9.38 µm (M=8.23 µm) × 4.89–7.26 µm
(M=5.79 µm).
The investigation of the secondary metabolites was carried out
Sexual stage: Not observed.
manually via the analysis of accurate mass, considering the mass error as
Distribution: Brazil, Amazonas state.
5 ppm; fragmentation profile and specific annotations in the databases
Etymology: Refers to potential for controlling phytopathogens of
are used (Fig. S5, Table S1). The process of chemical dereplication was
agricultural interest and location where the type was isolated.
conducted using Dereplicator+ and METABOLOMICS-SNETS-V2 on the
Notes: Differs from Trichoderma texanum by the absence of the yellow
Global Natural Products Social Molecular Networking (GNPS) site.
mycelium pigment in PDA, MEA and SNA media, low pustule formation
The interpretation of manual spectra resulted in the identification of
in 72 h in PDA, conidia color, smaller phialide size and presence of
two compounds with m/z 754.5 and 1350.8. The molecules annotated in
terminal chlamydospores; Differs from Trichoderma ovalisporum by its
the GNPS were hypocrenone C, 5-hydroxy-de-O-methyllasiodiplodin
low pustule, conidia and conidiophores production.
and trikoningin KBII. The MS/MS spectra of the m/z 153.0 ion corrob­
orate the annotation of hypocrenone C by the presence of signs of loss of
3.3. Mycelial growth inhibition of phytopathogens formic acid, ethylene, H2 and CO (Ding et al., 2015). The MS/MS spectra
of the m/z 295.1 ion, on the other hand, presents a greater number of
To examine its ability to biocontrol phytopathogens, peaks from which losses of H2O, CO and losses of more complex struc­
T. agriamazonicum was evaluated via a dual culture assay. Mycelial tures are observed (Yang et al., 2000).
growth inhibition (MGI) > 50% was observed for all phytopathogens at The MS spectra of the m/z 1052.7 [M+H]+ ion, supposedly from
the end of the 21st day. The highest MGI percentage was for Corynespora lipopeptaibol trikoningin KBII (Oc-Iva-Gly-Val-Aib-Gly-Gly-Val-Aib-
cassiicola INPA 2671 (80.74%) (Fig. 4C, Fig. S3). In contrast, the lowest Gly-Ile-Leuol), showed signs of acylium ions resulting from the
percentage was observed for Rhizoctonia sp. INPA 2942 (59.63%). In sequential fragmentation of the peptide chain, starting from the ami­
general, the Colletotrichum species present > 67.04% of MGI (Fig. 4C). In noalcohol leucinol of the C-terminal portion (Fig. S8). The m/z 382.2 ion
Fusarium decemcellulare CPAA307 and Fusarium sp. CPAA 10621, only encompassed the N-terminal portion of this peptide, which is related to

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T.F. Sousa et al. Microbiological Research 275 (2023) 127469

Fig. 4. Mycoparasitism of Tricoderma agriamazonicum INPA 2475. a Dual culture dish of T. agriamazonicum and Colletotrichum siamense CPAA Coll 2 N; b Scanning
electron microscopy of contact zone between T. agriamazonicum and C. siamense. Black arrows show T. agriamazonicum constricting the hyphae of C. siamense; c
Mycelial growth inhibition (MGI) by Trichoderma agrimazonicum INPA 2475 against nine phytopathogens at 7, 14 and 21 days of inhibition.

an acetylated Iva-Gly-Val fragment with an octanoyl group (Auvin-Gu­ losses with an exception in the C-terminal ion 169.1, which corresponds
ette et al., 1993). to Gly-Vxxol. The annotated 7-res peptaibols (Ac-Pro-Gly-Glu-Leu-Aib-
A short lipopeptaibol, consisting of seven amino acid residues not Gly-Lxxol and Ac-Pro-Gly-Glu-Leu-Aib-Gly-Vxxol) are unprecedented in
reported in the literature, could be annotated from the MS spectrum of the literature and are putative new peptide sequences (Fig. S6-S7).
the m/z 754.5 [M+H]+ ion. From the interpretation of the fragmenta­ Additionally, the MS spectrum of the m/z 1350.8 [M+H]+ ion
tion pattern of this substance, it was possible to propose the sequence showed characteristic loss peaks of the aminoalcohol leucinol or iso­
Oc-Vxx-Gly-Vxx-Aib-Gly-Lxx-Lxxol (where Vxx can be valine or isova­ leucinol plus seven signals, which correspond to the residue Aib-Gly-
line, Lxx leucine or isoleucine and Lxxol leucinol or isoleucinol) (Fig S9). Gly-Vxx-Aib-Gly-Lxx-Lxxol for fragment m/z 680.4 (Fig. S10). Addi­
In addition to lipopeptaibol, two new 7-res peptaibol sequences were tional information on the MS spectrum of the [M+ 2 H]+ adduct allowed
also detected at m/z 684.4 and 698.40 (Fig S6-S7). In the fragmentation us to infer the N-terminal portion as Oc-Vxx-Gly-Vxx-Aib-Gly-Gly-Vxx,
of m/z 698.40, the C-terminal showed loss of 174.10 u and indicated the forming a 15-res lipopeptaibol (Oc-Vxx-Gly-Vxx-Aib-Gly-Gly-Vxx-Aib-
amino acids Gly-Lxxol. The other mass losses included m/z 129.04 (Glu), Gly-Gly-Vxx-Aib-Gly-Lxx-Lxxol) whose sequence has not yet been re­
m/z 113.09 (Leu), m/z 85.05 (Aib) and the N-terminal ion at m/z 197.1 ported in the literature. The data for the fragmentation and mass error in
represents the Ac-Pro-Gly. The ion m/z 684.4 presents the same mass ppm are available in Table S1.

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T.F. Sousa et al. Microbiological Research 275 (2023) 127469

Fig. 5. a Effect of volatile organic compounds (VOCs) on mycelial growth of phytopathogens after seven days. b Percentage of mycelial growth inhibition.

3.8. Comparative genomics using orthologous protein analysis (GO:006810) and 5% are for establishment of localization
(GO:0051234) (Fig. 7).
The OrthoVenn analysis of T. agriamazonicum resulted in 10,173
predicted proteins and 9799 clusters, of which 7350 are shared among 3.9. CAZymes of Trichoderma agriamazonicum INPA 2475
T. viride, T. atroviride, T. koningiopsis and T. koningii, which are the closest
species that have available genomic sequences. Based on the genome of T. agriamazonicum, the CAZymes were pre­
T. koningiopsis was the species that most shares orthologous proteins dicted, classified and compared with other related Trichoderma spp.
(9363) with T. agriamazonicum. Only two clusters are unique to (Fig. 8). In total, 198 CAZymes were predicted in T. agriamazonicum,
T. agriamazonicum; nonetheless, these are hypothetical proteins. 182 while T. viride have 223, T. atroviride 203, T. koningiopsis 181 and
orthologous clusters are shared only between T. agriamazonicum and T. koningii 151. In the dendrogram based on the composition of
T. koningiopsis, 150 between T. atroviride, 23 between T. viride and 9 CAZymes, T. agriamazonicum has significant similarity with T. atroviride.
between T. koningii. CAZyme licheninase (lichenin BGLU GH12) was predicted only in
Of the 182 clusters shared between T. koningii and T. agriamazonicum. The licheninase is responsible for the hydrolysis of
T. agriamazonicum, according to the molecular function, 46% are related lichenin present in many lichen species and cleaves the bonds (1→4)-
to oxidoreductase activity (GO:0016491), 12% are predicted for trans­ β-D-glucosidic in β-D-glucans containing (1→3)- and (1→4)-bonds. The
ferase activity (GO:0016740), 12% are for ion binding (GO:0043167), majority of the enzymes found were related to lignin and chitin degra­
9% are for hydrolase activity (GO:0016787), 3% are for nucleotide dation with a high number of copies of genes that encode the enzymes
binding (GO:0000166), 3% are for nucleoside binding (GO:0001882); lignin glucooligosaccharide oxidase AA7 and chitinase GH18 (Fig. 8).
3% have an unknown function (GO:0003674), 3% are for mono­
oxygenase activity (GO:0004497), 3% are for transporter activity
3.10. Biosynthetic gene cluster potential
(GO:0005215) and 3% are for other ligands (GO:0005488). According to
the biological processes, 20% have an unknown function (GO:0008150),
T. agriamazonicum has 33 biosynthetic gene clusters (BGC), of which
18% participate in metabolic processes (GO:0008152), 14% are related
14 are for polyketide production (PKS), 3 non-ribosomal peptide syn­
to cellular metabolic processes (GO:0044237), 9% are for nitrogen
thetases (NRPS), 8 non-ribosomal peptide synthetase-like (NRPS-like), 6
compound metabolic process (GO:0006807), 7% are for primary meta­
terpenes and 2 hybrids (PKS-NRPS). Only 2 BGCs present 100% simi­
bolic process (GO:0044238), 6% are for cellular process (GO:0009987),
larity with pathways that have already been characterized, such as for
6% are for macromolecule metabolic process (GO:0043170), 5% are for
production of NRPS siderophore dimethylcoprogen and T1-PKS naph­
organic acid metabolic processes (GO:006082), 5% are for transport
thopyrone. Another 4 BGCs present < 40% similarity with BGCs for

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Fig. 6. Effect of the volatile organic compounds of Trichoderma agriamazonicum on the Capsicum chinense fruit rot caused by Colletotricum scoville INPA 2910 after
seven days.

T. agriamazonicum using only tef1-α sequence data reveals a non-

Table 1
ambiguous position and presents proximity with T. ovalisporum while
Volatile organic compounds identified using GC/MS-MS in Trichoderma agria­
rpb2 data shows T. texanum as a sister clade.
mazonicum INPA 2475 cultivated in PDA and during the interaction in Capsicum
chinense fruit inoculated with Colletotrichum scovillei INPA 2910. The low number of SNPs in the rpb2 gene compared to tef1-α (Fig. 2)
T + Cc+P = Trichoderma agriamazonicum INPA 2475 + Capsicum chinense and reduces the phylogenetic resolution power for this clade, and conse­
Colletotrichum scovillei. quently reduces PP and bootstrap support as observed in Fig. 1. None­
theless, the morphological comparisons among related species in the
Compounds RT Formula Trichoderma Interaction:
agriamazonicum T þ Cc þ P tef1-α, rpb2 and concatenated analysis confirms the status of
T. agriamazonicum as a new fungal species that has greater conidia and
1-Octen-3-ol 10.5 C8H16 - +
3-Octanone 10.8 C8H16 + +
phialide length and poor conidia production.
2-Octen-1-ol 13.6 C8H16 + + T. agriamazonicum presents significant inhibition of mycelial growth
6-Amyl- 24.8 C10H14O2 + - in all phytopathogens tested, and this activity can be related to the
α-pyrone mycoparasite activity that was confirmed in Colletotrichum siamense by
Furan-2-pentil 10.9 C9H14O -
constriction of the hyphal structures. The mycoparasite activity of the
+
α-Terpinene 11.8 C10H16 + -
1-Octanol 13.7 C18H18O + - genus Trichoderma is an ancestral trait and is a complex process that
2-Heptyfuran 17.5 C11H18O + - involves enzymes, signaling, and production of metabolites (Mukherjee
α - Copaene 24.5 C15H24 + + et al., 2022; Atanasova et al., 2018; Tamandegani et al., 2020). The
Benzeneacetil 19.0 C10H12O2 + +
mycoparasitism traits have also been associated with plant disease
acid
suppression (Mukherjee et al., 2022).
In addition to mycoparasitic activity, T. agriamazonicum also
production of tryptoquialanine, fusaric acid, squalestatin S1 and neu­ inhibited the mycelial growth of phytopathogens through its production
rosporin A. In contrast, 27 BGCs do not present any similarity with BGCs of VOCs (Fig. 5AB, Table 1), in which compounds were identified with
already characterized in the MIBiG repository, thus showing the unex­ previously reported antifungal and fungistatic activity (Salwan et al.,
ploited biosynthetic potential of T. agriamazonicum. In addition, 2019). The 6-amyl-α-pyrone, a lactone with a characteristic aroma of
T. agriamazonicum presents a greater number of PKS when compared to coconut, has been characterized as one of the main volatile constituents
the other five closely related Trichoderma species that have genome se­ produced by many Trichoderma species, and has antifungal properties
quences available in public databases (Fig. S3). (Kalyani et al., 2000). Likewise, volatiles such as 1-octen-3-ol and
3-octanone, exhibit fungistatic and fungicidal effects (Nemčovič et al.,
4. Discussion 2008). The production of VOCs by Trichoderma is associated with
various biological processes such conidia germination, signaling and
In this study, we added a new member to the Trichoderma genus, defense (Salwan et al., 2019).
which is named here as Trichoderma agriamazonicum. This new species In the inhibition of mycelial growth by VOCs and mycoparasitism,
was isolated as an endophyte of a native Amazonian plant Scleronema T. agriamazonicum showed potential for controlling Colletotrichum spp.
micranthum and, to our knowledge, this is the first study to report a We selected C. scovillei isolated from Capsicum chinense fruit rot for post-
fungus associated with this species. The phylogenetic position of harvest control assays due the great inhibition by the VOCs of

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T.F. Sousa et al. Microbiological Research 275 (2023) 127469

Fig. 7. a Venn diagram of Trichoderma agriamazonicum INPA 2475 and related species; b Protein cluster number; c Number of elements: specific (1) and proteins
shared by 2, 3, 4, and 5 species in the Venn diagram.

T. agriamazonicum and the results show that this new species has po­ ribosomal peptide synthase (Fig. S8-S10). Two 7-mer peptaibols (m/z
tential to be used post-harvest. This work reports for the first time the 684.3 and m/z 698.4) are also not found in the literature and appear not
VOCs for controlling anthracnose fruit rot in Capsicum chinense caused to have the same biosynthetic origin as the lipopeptaibols (Fig. S6-S7).
by C. scovillei. The occurrence of a 7-mer peptaibol is rare in Trichoderma and, to date,
The use of volatile organic compounds produced by species of Tri­ have only been reported trichodecenin I (Oancea et al., 2008) and tri­
choderma has already been reported for Carica papaya in the control of chopolyns I and II (Fujita et al., 1981).
fruit rot caused by Colletotrichum gleosporioides (Valenzuela et al., 2015), Other secondary metabolites annotated in this work include hypo­
for Citrus sinensis in the control of green mold caused by Penicillium crenone C and 5-hydroxy-de-O-methyllasiodiplodin. The hypocrenone C
digitatum (Ferreira et al., 2020), for Musa acuminata in the control of was isolated from a marine strain of the fungus Hypocrea koningii PF04
crown rot caused by Colletotrichum musae (Sangeetha et al., 2009), in the (Ding et al., 2015), and 5-hydroxy-de-O-methyllasiodiplodin was ob­
control of fruit rot in Cucumis melo caused by Fusarium incarnatum tained from the endophytic fungus Lasiodiplodia theobromae IFO21059
(Intana et al., 2021), for anthracnose in Capsicum frutescens caused by (Yang et al., 2000) and from some Trichoderma species (Zhang et al.,
Colletotrichum gleosporioides (Ruangwong et al., 2021). Moreover, VOCs 2017). Regarding its biological potential, 5-hydrox­
can be applied in plant growth promotion (Lee et al., 2016; You et al., y-de-O-methyllasiodiplodin has had potato microtuber inducing activity
2022). (Yang et al., 2000), antibiotic activity (Yang et al., 2006) and cytotoxic
We reported that T. agriamozonicum is an unconventional 7-mer activity reported (Buayairaksa et al., 2011).
peptaibol and lipopeptaibol producer (S5-S10). The lipopeptaibol tri­ The biosynthetic gene clusters of T. agriamazonicum are mainly
koningin KBII annotated in T. agriamazonicum has antibiotic activity directed to polyketide production; nonetheless, the majority of these
against Staphylococcus aureus, as reported by Auvin-Guette (1993). The represent non-characterized BGCs. The biosynthetic potential of Tri­
lipopeptaibols at m/z 754.5 and m/z 1350.8 were not found in the choderma has gained attention recently, and analysis with different
literature and these compounds are structurally similar in the C-terminal Trichoderma genomes show low numbers of shared BGC inter-species,
and the N-terminal portions, but differ in the amount of amino acid thus indicating the vast biosynthetic repertoire for the genus (She­
residues, thus suggesting the module-skipping mechanism by the non- nouda et al., 2021). Among the Trichoderma species with whole

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T.F. Sousa et al. Microbiological Research 275 (2023) 127469

genomes, T. agriamazonicum presents a higher number of orthologous

protein shared with T. koningiopsis, while in the CAZyme composition,
T. agriamazonicum is more closely related to Trichoderma atroviride IMI
206040, which are two well-known biocontrol strains.
With the current scenario of deforestation in the Amazon and the
crisis generated by climate change, the use of sustainable agricultural
practices urgently needs to be implemented. The exploration of
Amazonian biodiversity for the discovery of new species of Trichoderma
can help in not only increasing the productivity of the world’s main
commodities, but also in reducing the use of pesticides, while at the
same time keeping the Amazon rainforest standing.

5. Conclusion

In this paper, we described a new fungal species Trichoderma agria­

mazonicum and demonstrated its biotechnological potential in the con­
trol of phytopathogens by means of mycoparasitism activity and
production of volatile organic compounds (VOCs). The VOCs of
T. agriamazonicum are able to control the anthracnose fruit rot caused by
Colletotricum scovillei in Capsicum chinense, and three VOCs (6-amyl-
α-pyrone, 1-octen-3-ol and 3-octanone) were identified as a main con­
stituent and as being responsible for the fungistatic and fungicidal ac­
tivity. The peptaibol annotation reveals that the new species is an
unconventional producer of 7-mer peptaibol and lipopeptaibol and has
not been described in the literature so far. Genomic analysis indicated
the vast metabolite repertoire via the presence of non-characterized
BGCs. Finally, the CAZyme comparison shows a specific licheninase
(GH12) only found in Trichoderma agriamazonicum.

Funding

This work was funded by Coordenação de Aperfeiçoamento de Pes­

soal de Nível Superior (CAPES-grant number 88881.200469/2018–01
Procad AmazonMicro and Grant Nº 88887.510218–2020–00 CAPES-
Amazônia-Legal) and CAPES—Finance code 001. We also would like to
thank Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) for the productivity scholarship grant provided to H.H.F.K. (No.
305942/2020–4). G.F.S would like to thank the Fundação de Amparo à
Pesquisa do Estado do Amazonas (FAPEAM) for the productivity
scholarship grant provided (call 013/2022). The authors H.H.F.K, G.F.S
and R.E.H, also acknowledge FAPEAM for the funding via the projects
PROSPAM (call 008/2021), AMAZONAS ESTRATÉGICO (call 004/
2018), ÁREAS PRIORITÁRIAS (call 010/2021) and POSGRAD 2022/
2023 (call 005/2022).

CRediT authorship contribution statement

Gilvan Ferreira da Silva: Conceptualization, Methodology, Data

analysis, Writing − original draft preparation, Writing − review &
editing. Thiago Fernandes Sousa: Conceptualization, Methodology,
Data analysis, Writing − original draft preparation, Writing − review &
editing. Rogério Eiji Hanada: Conceptualization, Methodology, Data
analysis, Writing − original draft preparation, Writing − review &
editing. Ingride Jarline Santos da Silva: Bench experiments, Data
curation. Bruna Nayara Pantoja Vieira Reça: Bench experiments, Data
curation. Gleucinei Santos Castro: Bench experiments, Data curation.
Fernanda Fátima Caniato: Statistical analysis. Hector Henrique Fer­
reira Koolen: Chemical analysis. Giovana Anceski Bataglion: Chem­
ical analysis. Moysés Batista de Araújo Júnior: Chemical analysis.
Michel Eduardo Beleza Yamagishi: Bioinformatics analysis.

Declaration of Competing Interest

Fig. 8. CAZyme composition in Trichoderma agriamazonicum-related species.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

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T.F. Sousa et al. Microbiological Research 275 (2023) 127469

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