Morphological and Molecular Identification of Trichoderma Isolates Used as Biocontrol Agents by DNA Barcoding

Year 2023, Volume: 82 Issue: 1, 59 - 69, 26.06.2023

Yüksel Gezgin , Gülce Güralp Ayşe Berçin Barlas , Rengin Eltem

https://doi.org/10.26650/EurJBiol.2023.1279151

Abstract

Objective: Trichoderma genus are environmentally friendly, targeted biocontrol agents used in organic agriculture. Currently, due to the increasing number of organic farming practices, Trichoderma species form a good market as commercial biocontrol agents. This study aims to make morphological and molecular identification of Trichoderma isolates, which were found to be potential biocontrol agents against plant pathogenic fungi, and to perform phylogenetic diversity analyses of these species using different bioinformatics tools.
Materials and Methods: Two different gene regions (the nuclear ribosomal internal transcribed spacer (ITS) and translation elongation factor 1 (EF) were used for molecular identification of Trichoderma isolates in this study. Polymerase Chain Reaction (PCR) related regions were amplified and sequenced using primers specific to these gene regions. Following molecular identifications based on these two different gene regions, phylogenetic trees were drawn and polymorphic regions in the nucleotide sequences of these genes were determined.
Results: As a result of the study, Trichoderma isolates were determined as T. citrinoviride Bissett and T. atroviride P. Karst. at the species level. This study not only provides essential information about the biodiversity of Trichoderma species, which is a biocontrol agent, but also allows the design of new species-specific primers based on the polymorphic regions of both species.
Conclusion: It will be possible to make fast and low-cost molecular identification independent of sequence analysis by using primers unique to these species in the future.

Keywords

T. citrinoviride, T. atroviride, BLAST, TrichOKEY, TrichoBLAST, targeted biocontrol agents, DNA barcoding

Supporting Institution

Ege University

Project Number

15-MÜH-059

Thanks

This study is supported by Ege University Scientific Research Projects Coordination Unit. Project Number: 15-MÜH-059

References

• 1. Degenkolb T, Grafenhan T, Nirenberg HI, Gams W, Brückner H. Trichoderma brevicompactum complex: rich source of novel and recurrent plant-protective polypeptide antibiotics (peptaibiotics). J Agric Food Chem. 2006; 54(19): 7047-7061. google scholar
• 2. Degenkolb T, Dieckmann R, Nielsen KF, et al. The Trichoderma brevicompactum clade: a separate lineage with new species, new peptaibiotics, and mycotoxins. Mycol Prog. 2008a; 7(3):177-219. google scholar
• 3. Degenkolb T, Von Doehren H, Nielsen KF, Samuels GJ, Brück-ner H. Recent advances and future prospects in peptaibiotics, hydrophobin, and mycotoxin research, and their importance for chemotaxonomy of Trichoderma and Hypocrea. Chem Biodivers. 2008b; 5(5):671-680. google scholar
• 4. Maral D, Sozer S, Gezgin Y, et al. Evaluation of the properties of Trichoderma sp. isolates as a biocontrol agent and biofertilizer. Environ Eng Manag J. 2012; 11(3): S154. google scholar
• 5. Sargin S, Gezgin Y, Eltem R, Vardar FA. Micropropagule pro-duction from Trichoderma harzianum EGE-K38 using solid-state fermentation and a comparative study for drying methods. Turk J Biol. 2013; 37(2):139-146. google scholar
• 6. Gezgin Y, Gül DM, Şenşatar SS, Kara CU, Sargın S, Sukan FV. Evaluation of Trichoderma atroviride and Trichoderma citrinoviride growth profiles and their potentials as biocontrol agent and biofertilizer. Turkish J Biochem. 2020; 45(2):163-175. google scholar
• 7. Sharma A, Arya SK, Singh J, et al. Prospects of chitinase in sustainable farming and modern biotechnology: An update on recent progress and challenges. Biotechnol Genet Eng Rev. 2023; 1-31. google scholar
• 8. Lyubenova A, Rusanova M, Nikolova M, Slavov SB. Plant ex-tracts and Trichoderma spp: possibilities for implementation in agriculture as biopesticides. Biotechnol Biotechnol Equip. 2023; 37(1):159-166. google scholar
• 9. Salem SS. A mini review on green nanotechnology and its devel-opment in biological effects. Arch Microbiol. 2023; 205(4):128. https://doi.org/10.1007/s00203-023-03467-2. google scholar
• 10. Barakat I, Chtaina N, El Kamli T, Grappin P, El Guilli M, Ezzahiri B. Bioactivity of Trichoderma harzianum A peptaibols against Zymoseptoria tritici causal agent of septoria leaf blotch of wheat. J Plant Prot Res. 2023; 63(1):59-67. google scholar
• 11. John NS, Anjanadevi IP, Nath VS, et al. Characterization of Trichoderma isolates against Sclerotium rolfsii, the collar rot pathogen of Amorphophallus-A polyphasic approach. Biol Con-trol. 2015; 90:164-172. google scholar
• 12. Chaverri P, Samuels GJ. Hypocrea/Trichoderma (ascomycota, hypocreales, hypocreaceae): species with green ascospores. Netherlands, N.L.: Centraalbureau voor Schimmelcultures (CBS); 2003: p1-35. google scholar
• 13. Jaklitsch WM. European species of Hypocrea Part I. The green-spored species Stud Mycol. 2009; 63:1-91. google scholar
• 14. Jaklitsch WM. European species of Hypocrea part II: species with hyaline ascospores. Fungal Divers. 2011; 48(1):1-250. google scholar
• 15. Savitha MJ, Sriram S. Morphological and molecular identifica-tion of Trichoderma isolates with biocontrol potential against Phy-tophthora blight in red pepper. Pest Manage Hortic Ecosyst. 2015; 21(2);194-202. google scholar
• 16. Siddiquee S. Morphology-based characterization of Trichoderma species. In: Practical handbook of the biology and molecular diversity of Trichoderma species from tropical regions. Fungal biology. Switzerland, C.H.: Springer International Publishing; 2017:41-73. google scholar
• 17. Dou K, Lu Z, Wu Q. MIST: A multilocus identification system for Trichoderma. Appl Environ Microbiol. 2020; 86(18): e01532-20. https://doi.org/10.1128/AEM.01532-20. google scholar
• 18. Saravanakumar K, Yu C, Dou K, Wang M, Li Y, Chen J. Bio-diversity of Trichoderma community in the tidal flats and wet-land of southeastern China. PLoS One. 2016; 11(12): e0168020. https://doi.org/10.1371/journal.pone.0168020. google scholar
• 19. Srivastava M, Shahid M, Pandey S, et al. Trichoderma genome to genomics: A review. J Data Min Genom Proteom. 2014; 5(162). https://doi.org/10.4172/2153-0602.1000162. google scholar
• 20. Kubicek CP, Steindorff AS, Chenthamara K, et al. Evolution and comparative genomics of the most common Trichoderma species. BMC Genom. 2019; 20:1-24. google scholar
• 21. Kamil D, Prameela Devi T, Choudhary SP, Das A, Kumar A. Genome-mediated methods to unravel the native biogeographi-cal diversity and biosynthetic potential of Trichoderma for plant health. In Fungal diversity, ecology and control management, In: Rajpal VR, Singh I, Navi SS, eds. Fungal diversity, ecology and control management. Fungal biology, Singapore: SG: Springer Nature Singapore, 2022:109-124. google scholar
• 22. Maheshwary NP, Naik BG, Chittaragi A, Morpho-molecular char-acterization, diversity analysis and antagonistic activity of Tricho-derma isolates against predominant soil born pathogens. Indian Phytopathol. 2022; 75(4): 1009-1020. google scholar
• 23. Gal-Hemed I, Atanasova L, Komon-Zelazowska M, Druzhinina IS, Viterbo A, Yarden O. Marine isolates of Trichoderma spp. as potential halotolerant agents of biological control for arid-zone agriculture. J Appl Environ Microbiol. 2011; 77(15):5100-5109. google scholar
• 24. Samuels GJ, Ismaiel A, Mulaw TB, et al., The Longibrachiatum Clade of Trichoderma: a revision with new species. Fungal Divers. 2012; 55(1):77-108. google scholar
• 25. Oskiera M, Szczech M, Bartoszewski G. Molecular identifica-tion of Trichoderma strains collected to develop plant growth-promoting and biocontrol agents. J Hortic Res. 2015; 23(1): 75-86. google scholar
• 26. Kullnig-Gradinger CM, Szakacs G, Kubicek CP. Phylogeny and evolution of the genus Trichoderma: a multigene approach. Mycol Res. 2002; 106(7):757-767. google scholar
• 27. Anees M, Tronsmo A, Edel-Hermann V, Hjeljord LG, Heraud C, Steinberg C. Characterization of field isolates of Trichoderma antagonistic against Rhizoctonia solani. Fungal Biol. 2010; 114(9):691-701. google scholar
• 28. Elad Y, Chet I, Henis Y. A selective medium for improving quan-titative isolation of Trichoderma spp. from soil. Phytoparasitica. 1981; 9:59-67. google scholar
• 29. Howell CR. Selective isolation from soil and separation in vitro of P and Q strains of Trichoderma virens with differential media. Mycologia. 1999; 91(6): 930-934. google scholar
• 30. Gams W, Bissett J. Morphology and identification of Trichoderma and Gliocladium. In: Trichoderma and Gliocladium Volume 1, Basic biology, taxonomy and genetics. Eds. Kubicek CP, Harman GE, Bristol, BS: Taylor and Francis Ltd, Inc; 2002; 3-31. google scholar
• 31. Samuels GJ. Trichoderma: A review of biology and systematics of the genus. Mycol Res. 1996; 100(8):923-935. google scholar
• 32. Jaklitsch WM, Voglmayr H. Biodiversity of Trichoderma (Hypocreaceae) in Southern Europe and Macaronesia. Stud Mycol. 2015; 80:1-87. google scholar
• 33. Liu D, Coloe S, Baird R, Pedersen J. Rapid mini-preparation of fungal DNA for PCR. J Clin Microbiol. 2000; 38(1):471-471. google scholar
• 34. Van den Ende AH, De Hoog GS. Variability and molecular di-agnostics of the neurotropic species Cladophialophora bantiana. Stud Mycol. 1999; 43:151-162. google scholar
• 35. Carbone I, Kohn LM. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia. 1999; 91(3):553-556. google scholar
• 36. Jaklitsch WM, Komon M, Kubicek CP, Druzhinina IS. Hypocrea voglmayrii sp. nov. from the Austrian Alps represents a new phylogenetic clade in Hypocrea/Trichoderma. Mycologia. 2005; 97(6):1365-1378. google scholar
• 37. The Basic Local Alignment Search Tool (BLAST) website, https://blast.ncbi.nlm.nih.gov/Blast.cgi. Accessed May 03, 2023. google scholar
• 38. Druzhinina IS, Kopchinskiy AG, Komon M, Bissett J, Szakacs G, Kubicek CP. An oligonucleotide barcode for species identifi-cation in Trichoderma and Hypocrea. Fungal Genet Biol. 2005; 42(10):813-828. google scholar
• 39. Kopchinskiy A, Komon M, Kubicek CP, Druzhinina IS. Tri-choBLAST: A multilocus database for Trichoderma and Hypocrea identifications. Mycol Res. 2005; 109(6):658-660. google scholar
• 40. Tamura K, Nei M. Estimation of the number of nucleotide sub-stitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol. 1993; 10(3):512-526. google scholar
• 41. Kumar S, Stecher G, LiM, Knyaz C, Tamura K. MEGA X: Molec-ular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018; 35(6):1547-1549. google scholar
• 42. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improv-ing the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994; 22(22):4673-4680. google scholar
• 43. Kuhls K, Lieckfeldt E, Samuels GJ, et al. Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina. Proc Natl Acad Sci. 1996; 93(15):7755-7760. google scholar
• 44. Schoch CL, Seifert KA, Huhndorf S, et al. Nuclear ribosomal in-ternal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci. 2012; 109(16):6241-6246. google scholar
• 45. Ospina-Giraldo MD, Royse DJ, Chen X, Romaine CP. Molecular phylogenetic analyses of biological control strains of Trichoderma harzianum and other biotypes of Trichoderma spp. associated with mushroom green mold. Phytopathology. 1999; 89(4):308-313. google scholar
• 46. Skoneczny D, Oskiera M, Szczech M, Bartoszewski G. Genetic diversity of Trichoderma atroviride strains collected in Poland and identification of loci useful in detection of within-species diversity. Folia Microbiol. 2015; 60(4):297-307. google scholar
• 47. Druzhinina IS, Kubicek CP, Komon-Zelazowska M, Mulaw TB, Bissett J. The Trichoderma harzianum demon: Complex speci-ation history resulting in coexistence of hypothetical biological species, recent agamospecies and numerous relict lineages. BMC Ecol Evol. 2010; 10(1):1-4. google scholar
• 48. Hageskal G, Vrâlstad T, Knutsen AK, Skaar ID. Exploring the species diversity of Trichoderma in Norwegian drinking water systems by DNA barcoding. Mol Ecol Resour. 2008; 8(6):1178-1188. google scholar
• 49. Fahmi AI, Eissa RA, El-Halfawi KA, Hamza HA, Helwa MS. Identification of Trichoderma spp. by DNA barcode and screen-ing for cellulolytic activity. J Microb Biochem Technol. 2016; 8(3):202-209. google scholar
• 50. Saroj DB, Dengeti SN, Aher S, Gupta AK. A rapid, one step molecular identification of Trichoderma citrinoviride and Trichoderma reesei. World J Microbiol Biotechnol. 2015; 31(6):995-999. google scholar