In order to identify a specific marker for T. harzianum AS12-2, a strain capable of controlling rice sheath blight caused by Rhizoctonia solani, UP-PCR was performed using five universal primers (UP) both separately and in pairwise combinations. The application of two UP primers resulted in the amplification of unique fragments from the genomic DNA of T. harzianum AS12-2, clearly distinguishing it from other Trichoderma strains. The unique fragments had no significant sequence homology with any other known sequence available in databases. Based on the sequences of the unique fragments, 14 oligonucleotide primers were designed. Two primer sets amplified a fragment of expected size from the DNA of strain T. harzianum AS12-2 but not from any other examined strains belonging to T. harzianum, to other Trichoderma species assayed, or to other common fungi present in paddy fields of Mazandaran province, Iran. In conclusion, SCAR (sequence characterized amplified regions) markers were successfully identified and rapid, reliable tools were provided for the detection of an effective biocontrol Trichoderma strain, which can facilitate studies of its population dynamics and establishment after release into the natural environment.
Abbasi, P. A., Miller, S. A., Meulia, T., Hoitink, H. A. J., Kim, J. (1999) Precise detection and tracing of Trichoderma hamatum 382 in compost-amended potting mixes by using molecular markers. Appl. Environ. Microbiol. 65, 5421–5426.
Kim J. , 'Precise detection and tracing of Trichoderma hamatum 382 in compost-amended potting mixes by using molecular markers ' (1999 ) 65 Appl. Environ. Microbiol. : 5421 -5426 .
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410.
Lipman D. J. , 'Basic local alignment search tool ' (1990 ) 215 J. Mol. Biol. : 403 -410 .
Avis, T. J., Hamelin, R. C., Bélanger, R. R. (2001) Approaches to molecular characterization of fungal biocontrol agents: some case studies. Can. J. Plant Pathol. 23, 8–12.
Bélanger R. R. , 'Approaches to molecular characterization of fungal biocontrol agents: some case studies ' (2001 ) 23 Can. J. Plant Pathol. : 8 -12 .
Bae, Y., Knudsen, G. R. (2000) Cotransformation of Trichoderma harzianum with beta glucuronidase and green fluorescent protein genes provides a useful tool for monitoring fungal growth and activity in natural soils. Appl. Environ. Microbiol. 66, 810–815.
Knudsen G. R. , 'Cotransformation of Trichoderma harzianum with beta glucuronidase and green fluorescent protein genes provides a useful tool for monitoring fungal growth and activity in natural soils ' (2000 ) 66 Appl. Environ. Microbiol. : 810 -815 .
Bulat, S. A., Mironenko, N. V., Lapteva, M. N., Strelchenko, P. P. (1994) Polymerase chain reaction with universal primers (UP-PCR) and its application to plant genome analysis. In: Adams, R. P., Miller, J. S., Goldenberg, E. M., Adams, J. E. (eds) Conservation of Plant Genes II. Utilization of Ancient and Modern DNA. Missouri Botanical Garden, St. Louis, USA, pp, 113–129.
Strelchenko P. P. , '', in Conservation of Plant Genes II. Utilization of Ancient and Modern DNA , (1994 ) -.
Bulat, S. A., Lübeck, M., Mironenko, N. V., Jensen, D. F., Lübeck, P. S. (1998) UP-PCR analysis and ITS1 ribotyping of Trichoderma and Gliocladium fungi. Mycol. Res. 102, 933–943.
Lübeck P. S. , 'UP-PCR analysis and ITS1 ribotyping of Trichoderma and Gliocladium fungi ' (1998 ) 102 Mycol. Res. : 933 -943 .
Cordier, C., Edel-Hermann, V., Martin-Laurent, F., Blal, B., Steinberg, C., Alabouvette, C. (2007) SCAR-based real-time PCR to identify a biocontrol strain (T1) of Trichoderma atroviride and study its population dynamics in soils. J. Microbiol. Meth. 68, 60–68.
Alabouvette C. , 'SCAR-based real-time PCR to identify a biocontrol strain (T1) of Trichoderma atroviride and study its population dynamics in soils ' (2007 ) 68 J. Microbiol. Meth. : 60 -68 .
Cumagun, C. J. R., Hockenhull, J., Lübeck, M. (2000) Characterization of Trichoderma isolates from Philippine rice fields by UP-PCR and rDNA-ITS1 analysis: Identification of UP-PCR markers. J. Phytopathol. 148, 109–115.
Lübeck M. , 'Characterization of Trichoderma isolates from Philippine rice fields by UP-PCR and rDNA-ITS1 analysis: Identification of UP-PCR markers ' (2000 ) 148 J. Phytopathol. : 109 -115 .
Hermosa, M. R., Grondona, I., Diaz-Minguez, J. M., Iturriaga, E. A., Monte, E. (2001) Development of a strain-specific SCAR marker for the detection of Trichoderma atroviride 11, a biological control agent against soilborne fungal plant pathogens. Curr. Genet. 38, 343–350.
Monte E. , 'Development of a strain-specific SCAR marker for the detection of Trichoderma atroviride 11, a biological control agent against soilborne fungal plant pathogens ' (2001 ) 38 Curr. Genet. : 343 -350 .
Lo, C. T., Nelson, E. B., Harman, G. E. (1996) Biological control of turfgrass diseases with a rhizosphere competent strain of Trichoderma harzianum. Plant Dis. 80, 736–741.
Harman G. E. , 'Biological control of turfgrass diseases with a rhizosphere competent strain of Trichoderma harzianum ' (1996 ) 80 Plant Dis. : 736 -741 .
Lübeck, P. S., Alekhina, I. A., Lübeck, M., Bulat, S. A. (1998) UP-PCR genotyping and rDNA analysis of Ascochyta pisi Lib. J. Phytopathol. 146, 51–55.
Bulat S. A. , 'UP-PCR genotyping and rDNA analysis of Ascochyta pisi Lib ' (1998 ) 146 J. Phytopathol. : 51 -55 .
Lübeck, M., Alekhina, I. A., Lübeck, P. S., Jensen, D. F., Bulat, S. A. (1999) Delineation of Trichoderma harzianum Rifai into two different genetic entities by a highly robust fingerprinting method, UP-PCR. Mycol. Res. 103, 289–298.
Bulat S. A. , 'Delineation of Trichoderma harzianum Rifai into two different genetic entities by a highly robust fingerprinting method, UP-PCR ' (1999 ) 103 Mycol. Res. : 289 -298 .
Lübeck, M., Bulat, S. A., Alekhina, I. A., Lieckfeldt, E. (2004) Delineation of species within the Trichoderma viride/atroviride/koningii complex by UP-PCR cross-blot hybridization. FEMS Microbiol. Lett. 237, 255–260.
Lieckfeldt E. , 'Delineation of species within the Trichoderma viride/atroviride/koningii complex by UP-PCR cross-blot hybridization ' (2004 ) 237 FEMS Microbiol. Lett. : 255 -260 .
Lübeck, M., Jensen, D. F. (2002) Monitoring Trichoderma strains using dilution plating and UP-PCR fingerprinting in glasshouses of nursery gardens that apply commercial biocontrol agents. Biocontrol Sci. Technol. 12, 371–380.
Jensen D. F. , 'Monitoring Trichoderma strains using dilution plating and UP-PCR fingerprinting in glasshouses of nursery gardens that apply commercial biocontrol agents ' (2002 ) 12 Biocontrol Sci. Technol. : 371 -380 .
Lübeck, M., Poulsen, S. B., Lübeck, P. S., Jensen, D. F., Thrane, U. (2000) Identification of Trichoderma strains from buildings materials by ITS1 ribotyping, UP-PCR fingerprinting and UP-PCR cross-hybridization. FEMS Microbiol. Lett. 185, 129–134.
Thrane U. , 'Identification of Trichoderma strains from buildings materials by ITS1 ribotyping, UP-PCR fingerprinting and UP-PCR cross-hybridization ' (2000 ) 185 FEMS Microbiol. Lett. : 129 -134 .
Miller, S. A. (1996) Detecting propagules of plant pathogenic fungi. In: De Boer, S. H. (ed.) Advances in Plant Pathology: Pathogen Indexing Technique. Academic Press, London, pp. 73–102.
Miller S. A. , '', in Advances in Plant Pathology: Pathogen Indexing Technique , (1996 ) -.
Naeimi, S. (2010) Development of a Specific Molecular Marker for Detection of the Most Effective Trichoderma Strain Against Thanatephorus cucumeris, the Causal Agent of Rice Sheath Blight Disease. PhD thesis, Faculty of Agriculture, University of Tehran, Karaj, Iran, 106 pp.
Naeimi S. , '', in Development of a Specific Molecular Marker for Detection of the Most Effective Trichoderma Strain Against Thanatephorus cucumeris, the Causal Agent of Rice Sheath Blight Disease , (2010 ) -.
Naeimi, S., Okhovvat, S. M., Javan-Nikkhah, M., Kredics, L., Khosravi, V. (2010) Frequency and distribution of Trichoderma spp. in the rice fields of Mazandaran province, Iran. Iran. J. Plant Prot. Sci. 40, 79–91.
Khosravi V. , 'Frequency and distribution of Trichoderma spp. in the rice fields of Mazandaran province, Iran ' (2010 ) 40 Iran. J. Plant Prot. Sci. : 79 -91 .
Paran, I., Michelmore, R. W. (1993) Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theoret. Appl. Genet. 85, 985–993.
Michelmore R. W. , 'Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce ' (1993 ) 85 Theoret. Appl. Genet. : 985 -993 .
Rubio, M. B., Hermosa, M. R., Keck, E., Monte, E. (2005) Specific PCR assays for the detection and quantification of DNA from the biocontrol strain Trichoderma harzianum 2413 in soil. Microb. Ecol. 49, 25–33.
Monte E. , 'Specific PCR assays for the detection and quantification of DNA from the biocontrol strain Trichoderma harzianum 2413 in soil ' (2005 ) 49 Microb. Ecol. : 25 -33 .
Savazzini, F., Longa, C. M. O., Pertot, I., Gessler, C. (2008) Real-time PCR for detection and quantification of the biocontrol agent Trichoderma atroviride strain SCI in soil. J. Microbiol. Meth. 73, 185–194.
Gessler C. , 'Real-time PCR for detection and quantification of the biocontrol agent Trichoderma atroviride strain SCI in soil ' (2008 ) 73 J. Microbiol. Meth. : 185 -194 .
Zimand, G., Valinsky, L., Elad, Y., Chet, I., Manualis, S. (1994) Use of RAPD procedure for the identification of Trichoderma strains. Mycol. Res. 98, 531–534.
Manualis S. , 'Use of RAPD procedure for the identification of Trichoderma strains ' (1994 ) 98 Mycol. Res. : 531 -534 .