Molecular identification of Trichoderma spp. of Indonesia and their antagonistic activities against Fusarium oxysporum f.sp. cubense
Identifikasi Molekuler Trichoderma spp. dan Aktivitas Antagonisnya terhadap Fusarium oxysporum f.sp cubense
Efendi, Vivi Oktavianis
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Trichoderma is a cosmopolitan fungus that widespread in the soil and rotten wood. Various species of Trichoderma have been used, as a producer of enzymes and other metabolites or as a biocontrol agent. Therefore, identification of Trichoderma strains become important. Until now, many identity culture Trichoderma in Indonesia have doubtful validity. Trichoderma concept changed from time to time. Significant efforts in formulating the concept of Trichoderma was first performed by Rifai in 1969 based approach to morphological characters. Trichoderma species are divided into 9 aggregates, namely T. piluliferum, T. polysporum, T. hamatum, T. koningii, T. aureoviride, T. harzianum, T. longibrachiatum, T. pseudokoningii, and T. viride. This concept was later revised by Bissett in 1991 with the introduction of section 5 (section) is section Trichoderma, section Longibrachiatum, section Saturnisporum, section Pachybasium, and section Hypocreanum. Along with the development of methods of DNA extraction and phylogenetic analysis of the fungus, identification of Trichoderma species have been changed from morfohology in to combination of morphology and phylogenetic analysis. The first, phylogenetic analysis approach of Trichoderma by Kindermann in 1998 using DNA sequence analysis of the ITS region to 85 strains of Trichoderma. Then, when the approach of a single gene can not be relied upon to distinguish some species that are morphologically similar, multigene analysis was able to distinguish the species. At this time locus used in phylogenetic analysis and species identification in Trichoderma has been established that, ITS, tef α-1, RPB2, and Endokitinase. Trichoderma research in Indonesia have been carried out, but only a few research that include diversity and taxonomic studies. Trichoderma diversity and taxonomic studies from Indonesia is important because it can serve as a model for other fungal diversity studies. Therefore, this study aims to identify the 27 accession numbers Trichoderma collection of LIPIMC and IPBCC derived from litter and soil in Indonesia through a variety of phylogenetic approaches two genes (ITS and tef α-1). In addition, this research also aimed to characterize the antagonistic activity of Trichoderma spp. against Fusarium oxysporum f.sp. cubense (Foc), causes Fusarium wilt disease in bananas, because of the demands of the community to develop a biocontrol Foc agent. Identification of Trichoderma started early stages by performing morphological characterization. Colonies character observed in 5 days that were incubated at room conditions. After that, the microscopic preparations were made by using the method of Riddle and preparations were observed under a microscope Olympus BX53. Colony characters and microscopic characters are recorded as a material for morphological identification. Molecular phylogenetic analysis was started by growing Trichoderma isolates on media Potato Dextrose Broth (PDB) and incubated at 27˚C for 3-5 days. DNA extraction using PhythopureTM DNA Extraction Kit (GE Healthcare, UK), and then proceed with the ITS region amplification primer pair ITS4 (5'-TC CTCCGCTTATATATGC-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAA GG-3'), and α tef -1 (translation elongation factor-1 α) with primer tef1-fw (5'-GT GAGCGTGGTATCACCATCG-3 ') and tef1-rev (5'-GCCATCCTTGGAGACC AGC-3'). DNA sequencing is done by sending the product to FirstBASE (Malaysia). DNA sequences were analyzed by PCR using the MEGA program (Molecular Evolution and Genetic Analysis) version 5.05. On this analysis, kinship was analyzed by Neighbor Joining method (NJ). Sites that contain gaps partially excluded in the analysis. Support for internal branches obtained by bootstrap analysis with 1000 repition. Single gene and multigene analysis showed that the topology of the phylogenetic tree generated from ITS analysis that similar to the result from phylogenetic tree analysis combination ITS - tef-1α. Based on this multigene analysis, Trichoderma strains LIPIMC collection and IPBCC spread into 3 sections: section Longibrachiatum, section Pachybasium and section Trichoderma. A total of 25 of the 27 accession numbers were identified and spread in 7 species namely T. asperellum, T. atroviride and T. ovalisporum (each 1 number), T. harzianum (11 numbers), T. reesei, and T. virens (each 3 numbers) and T.tawa (5 numbers). Two other accession numbers can not be identified, and will be analyzed further by RPB II gene primers. In the test antagonist to F.oxysporum f.sp. cubense, all the accession numbers have inhibitory effects of direct and indirect (volatile compounds) have variation. Highest inhibition was shown by T. tawa IPBCC 13.1031 is 85.63% with type 3 interaction. In the volatile test, the three best strains namely T. harzianum LIPIMC 0572, T. ovalisporum LIPIMC 0571, and Trichoderma sp. LIPIMC 0570 volatilnya compounds showed antagonist activity with percentage inhibition values, respectively for 45.25%, 45.65%, and 45.38%.