Biological control is a promising and sustainable strategy to reduce damage caused by agricultural pests and the use of chemical fungicides. Fungal strains of the genus Clonostachys are studied as biocontrol agent of fungi and nematodes. However, the presence of this fungus in the soils of Misiones remains unexplored. Traditional fungal identification is generally carried out by morphological characterization in Petri dishes, and by observing their reproductive structures under the microscope. In general, with this methodology it is possible to identify to the genus level, however determining up to the species level is usually very complicated in some genera and many times ambiguities are achieved. In this context, molecular data emerges as an important tool to complement morphological information and thus achieve a correct fungal identification. The objective of this work was to molecularly identify with ITS markers a strain of the mycoparasitic fungus Clonostachys HEP30. The nucleic acids were isolated for molecular corroboration. From the extracted genetic material, the ITS1-5,8S-ITS2 region was amplified and sequenced. Once the region of interest was obtained, the information obtained was compared with that existing in the databases, using the Blast (Basic Local Alignment Search Tool) of the NCBI (National Center for Biotechnology Information) and the fungal barcoding database and then phylogenetic analysis was done. The molecular identification and phylogenetic analysis allowed us to classify the fungal isolate Clonotachys HEP 30 with high percentage of identity as a member of Clonostachys pityrodes species.

Keywords: identification, mycoparasitic, ITS, fungi, biocontrol

Biological control is a promising and sustainable strategy to reduce damage caused by agricultural pests and the use of chemical fungicides. Fungal mycoparasites include many living microorganisms that can act against another fungus to obtain different nutrients from the host to live.¹ Species from the Clonostachys genus (Ascomycota, Bionectriaceae) are common soil inhabitants and plant decomposers, and also some species are referred as endophytes commonly found in tropical and subtropical regions.^2,3 The Trichoderma genus is the best studied biocontrol agent (direct and indirect) of many microbial phytopathogens (fungi and bacteria).^3–8 However, nowadays some members of the Clonostachys genus are also referred as unspecific destructive mycoparasites, including against some microorganisms referred as important plant pathogens.^3,4,9,10 Traditional fungal identification is generally carried out by morphological characterization in Petri dishes, and by observing their reproductive structures under the microscope. In general, with this methodology it is possible to identify to the genus level, however determining up to the species level is usually very complicated in some genera and many times ambiguities are achieved. Molecular identification often is recommended as a valuable tool since many fungal isolates could not be differentiated easily and adequately by traditional cultural and morphological methods.^3,11,12 So, the objective of this work was to molecularly identify a strain of the mycoparasitic fungus Clonostachys HEP30 with ITS markers.

The Clonostachys HEP 30 strain was isolate in soil samples from non-anthropic environments from the Capital department of Misiones province, Argentina. The Clonostachys HEP 30 strain was stored at 4°C under the accession number LBM247 in the Biotechnological Fungal Strain Culture Collection of the Misiones Biotechnology Institute.

Molecular identification

For DNA extraction and amplification the mycelia of the Clonostachys HEP 30 strain was grown in the dark in flask with malt extract broth with a concentration of 1.27% (w/v) (ME - Britania SA) at 28±1°C for 5 days. The extraction of genomic DNA was performed with standard protocols.^11–13 The obtained DNA was resuspended in 30 µL of sterile distilled free of DNAse water (Biopak®). Also, the obtained DNA was further examined by electrophoresis agarose gels at standard concentration of in 1% (w/v - InBio) and stained with a solution of Gel Red (Biotium, 10,000 X). A portion of the ITS region (ITS1-5.8S-ITS2) of approximately 550 bp was amplified by the polymerase chain reaction (PCR). This PCR amplifications were done in a standard 20µL reaction mixture composed of 1X PCR Buffer, 200 μM of dNTP mix, 2.5mM MgCl₂, 10 pmol of each of the amplification primers described below, 0.5 u of Taq polymerase (InBio), and approximately 1µg of genomic DNA.^11–13 The universal fungal primers used were the ITS1 F- (5'-TCCgTAggTgAACCTgCgg-3') and ITS4 R- (5'-TCCTCCgCTTATTgATATgC-3') (White et al., 1990).¹⁴ A stadard amplification protocol was used; it included an initial denaturation at 94°C for 4 min, followed by 35 PCR amplification iterations of 94°C for 40 s, 53°C for 40 s and 72ºC for 40 s. Also a final PCR-extension step of 72°C for 10 min was included.¹¹ The amplified fragment was further examined by electrophoresis agarose gels at a concentration of 2% (w/v - InBio) and stained with a solution of Gel Red (Biotium, 10,000X). Both strands of the amplicón (PCR product) were further sequenced by Macrogen Korea for the phylogenetic studies.¹²

Phylogenetic analysis

The ITS1-5.8S-ITS2 nucleotide sequence of Clonostachys HEP 30 isolate was compared against different nucleotide sequences deposited in the GenBank and Fungal barcoding databases for the molecular species identification. Twenty-two ITS sequences were retrieved from the GenBank and Fungal barcoding databases. All these sequences represented species within the Clonostachys genus (Table 1). Nucleotide sequences retrieved in this study consisted of about 600 bp corresponding to the complete ITS and partial 18S and 28S regions.¹⁵ A sequence of Trametes versicolor (NR_154494.1) was used as an outgroup to root the Clonostachys phylogenetic tree. All DNA sequences selected were aligned using the Clustal W program.¹⁶ The analyses used were based on a distance-based method (Neighbor joining, NJ) and the MEGA 6.0 package¹⁷ was used for the analyses. Also, for supporting the specific clades represented in the tree obtained bootstrap analyses of 1,000 replicates were carried out. The nucleotide divergences were estimated using Kimura’s two-parameter method.

For molecular identification the genomic DNA of Clonostachys HEP 30 strain was extracted and amplified using a pair of primers, ITS 1 and ITS 4. The ITS sequence obtained had 563 bp after sequencing and contig construction. The ITS1-5.8S-ITS2 nucleotide sequence obtained of Clonostachys HEP 30 strain was deposited in GenBank-NCBI database under the accession number MH048667. The comparison and molecular analysis of Clonostachys HEP 30 sequence with the selected sequences in both genetic databases allowed us to state this sequence as belonging to Clonostachys pityrodes species. Our sequence obtained high percentages of identity of 99% with the MN637804 accession number from NCBI database, and with a sequence belonging to Bionectria pityrodes species (anamorph of C. pityrodes) with the CBS126394 accession number from Fungal barcoding database. The ITS1-5.8S-ITS2 phylogenetic trees obtained by the NJ method revealed that our fungal isolate under study belongs to e monophyletic clade of C. pityrodes species (92% bootstrap) (Figure 1). Also, our phylogenetic analyses revealed close positioning of C. pityrodes with Clonostachys candelabrum in a closely related group, separated of the others species of Clonostachys genus.

Figure 1 ITS1-5.8S-ITS2 phylogenetic trees obtained by the NJ method of Clonostachys HEP 30.

The identification of species belonging to this genus is notoriously difficult by traditional morphological methods. Nowadays the molecular identification methods are commonly referred to do not replace the morphological traditional identification of fungi; conversely, they complement the morphological methods.¹² It is proposed that the effective and safe application of any biocontrol agents (liquid or solid) depend on environmental conditions and target species, but also in an accurate and reliable identification of the biocontrol agents in time and space.³

The molecular identification and phylogenetic analysis allowed us to classify the fungal isolate Clonotachys HEP 30 with high percentage of identity as a member of Clonostachys pityrodes species.

None.

The authors declare that there is no conflict of interest.

1. Sun ZB, Li SD, Ren Q, et al. Biology and applications of Clonostachys rosea. Journal of applied microbiology. 2020;129(3):486–495.
2. Schroers HJ. A monograph of Bionectria (Ascomycota, Hypocreales, Bionectriaceae) and its Clonostachys anamorphs. Stud Mycol. 2001;46:1–214.
3. Alvindia DG, Hirooka Y. Identification of Clonostachys and Trichoderma spp. from banana fruit surfaces by cultural, morphological and molecular methods. Mycology. 2011;2(2):109–115.
4. Elad Y, Chet I, Katan J. Trichoderma harzianum: A biocontrol agent effective against Sclerotium rolfsii and Rhizoctonia solani. Phytopathology. 1980;70(2):119–121.
5. Papavizas GC. Trichoderma and Gliocladium: biology, ecology, and potential for biocontrol. Annual review of phytopathology. 1985;23(1):23–54.
6. Howell CR. Cotton seedling preemergence damping-off incited by Rhizopus oryzae and Pythium spp. and its biological control with Trichoderma spp. Phytopathology. 2002;92(2):177–180.
7. Hermosa MR, Grondona I, Iturriaga ET, et al. Molecular characterization and identification of biocontrol isolates of Trichoderma spp. Applied and Environmental Microbiology. 2000;66(5):1890–1898.
8. Samuels GJ. Trichoderma: systematics, the sexual state, and ecology. Phytopathology. 2006;96(2):195–206.
9. Jensen DF, Knudsen IM, Mamarabadi M, et al. Development of a biocontrol agent for plant disease control with special emphasis on the near commercial fungal antagonist Clonostachys rosea strain ‘IK726’. Australasian Plant Pathology. 2007;36(2):95–101.
10. Abreu LM, Moreira GM, Ferreira D, et al. Diversity of Clonostachys species assessed by molecular phylogenetics and MALDI-TOF mass spectrometry. Fungal biology. 2014;118(12):1004–1012.
11. Bich GA, Castrillo ML, Villalba LL, et al. Isolation of the symbiotic fungus of Acromyrmex pubescens and phylogeny of Leucoagaricus gongylophorus from leaf-cutting ants. Saudi Journal of Biological Sciences. 2017;24(4):851–856.
12. Castrillo ML, Bich GA, Amerio NS, et al. Assessment of cellulase complex secretory capacity of Trichoderma strains and morphological and molecular identification of the isolate with the highest enzymatic secretion capacity: Cellulase complex secretory capacity of Trichoderma strains. Journal of microbiology, biotechnology and food sciences. 2021;10(5):e1357–e1357.
13. Castrillo ML, Fonseca MI, Bich GA, et al. Taxonomy and phylogenetic analysis of Aspergillus section nigri isolated from yerba mate in Misiones (Argentina). BAG. Journal of basic and applied genetics. 2012;23(2).
14. White TJ, Bruns T, Lee SJWT, et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications. 1990;18(1):315–322.
15. Castrillo ML, Bich GA, Zapata PD, et al. Biocontrol of Leucoagaricus gongylophorus of leaf-cutting ants with the mycoparasitic agent Trichoderma koningiopsis. 2016.
16. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic acids research. 1994;22(22):4673–4680.
17. Tamura K, Stecher G, Peterson D, et al. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular biology and evolution. 2013;30(12):2725–2729.