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Abstract: Several species of the fungal genus Trichoderma establish biological interactions with various micro- and macro-organisms. Some of these interactions are relevant in ecological terms and in biotechnological applications, such as biocontrol, where Trichoderma could be considered as an invasive species that colonizes a recipient community. The success of this invasion depends on multiple factors, which can be assayed using experimental communities as study models. Therefore, the aim of this work is to develop a species-specific sequence-characterized amplified region (SCAR) marker to monitor the colonization and growth of T. cf. harzianum when it invades experimental communities. For this study, 16 randomly amplified polymorphic DNA (RAPD) primers of 10-mer were used to generate polymorphic patterns, one of which generated a band present only in strains of T. cf. harzianum. This band was cloned, sequenced, and five primers of 20–23 mer were designed. Primer pairs 2F2/2R2 and 2F2/2R3 successfully and specifically amplified fragments of 278 and 448 bp from the T. cf. harzianum BpT10a strain DNA, respectively. Both primer pairs were also tested against the DNA from 14 strains of T. cf. harzianum and several strains of different fungal genera as specificity controls. Only the DNA from the strains of T. cf. harzianum was successfully amplified. Moreover, primer pair 2F2/2R2 was assessed by quantitative real-time polymerase chain reaction (PCR) using fungal DNA mixtures and DNA extracted from fungal experimental communities as templates. T. cf. harzianum was detectable even when as few as 100 copies of the SCAR marker were available or even when its population represented only 0.1% of the whole community.

SCAR分子标记监测菌群中的哈茨木霉

开发具有种属特异性序列特征性扩增区域（SCAR）标记物来监测哈茨木霉在其入侵的试验菌群中的定殖和生长，为哈茨木霉应用于生物防治等生态和生物技术中提供支撑。 多种木霉属真菌能与各种微观和宏观的生物有机体建立相互作用。利用这些相互作用，木霉可做为原生种群的入侵物种而用于生物防治。本文通过使用试验菌群为研究模型，利用随机扩增多态性DNA（RAPD）技术和序列特征性扩增区域（SCAR）标记物来监测菌群中哈茨木霉的生长状态。 利用随机扩增多态性DNA（RAPD）技术，从16个10聚体引物进行多态性筛选，其中1个引物扩增出对应哈茨木霉的条带。对该条带进行克隆测序，并设计5个20-23聚体引物。成功利用引物对2F2/2R2和2F2/2R3278分别特异性地扩增出哈茨木霉BpT10a菌株278 bp和448 bp的DNA片段。同时，用这两个引物对14个哈茨木霉菌株和几种不同的真菌菌株进行特异性对照试验，也只成功扩增出哈茨木霉菌株。此外，使用真菌DNA混合物和试验真菌群的DNA为模板，采用实时聚合酶链式反应（PCR）对引物对2F2/2R2进行评估。当仅使用100份SCAR标记物或哈茨木霉仅占整个菌群的0.1%时，仍能检测出哈茨木霉。 本研究所建立的SCAR分子标记能有效监测菌群中的哈茨木霉的定殖和生长，具有较高特异性、灵敏度和准确度。
哈茨木霉；序列特征性扩增区域（SCAR）；分子标记；试验菌群

[1] Altschul, S.F., Gish, W., Miller, W., 1990. Basic local alignment search tool. J Mol Biol, 215(3):403-410. 

[2] Arnedo-Andrs, M., Gil-Ortega, R., Luis-Arteaga, M., 2002. Development of RAPD and SCAR markers linked to the Pvr4 locus for resistance to PVY in pepper (Capsicum annuum L.). Theor Appl Genet, 105(6-7):1067-1074. 

[3] Atkins, S.D., Clark, I.M., Sosnowska, D., 2003. Detection and quantification of Plectosphaerella cucumerina, a potential biological control agent of potato cyst nematodes, by using conventional PCR, real-time PCR, selective media, and baiting. Appl Environ Microbiol, 69(8):4788-4793. 

[4] Bae, Y.S., Knudsen, G.R., 2001. Influence of a fungus-feeding nematode on growth and biocontrol efficacy of Trichoderma harzianum . Phytopathology, 91(3):301-306. 

[5] Bae, Y.S., Knudsen, G.R., 2005. Soil microbial biomass influence on growth and biocontrol efficacy of Trichoderma harzianum . Biol Control, 32(2):236-242. 

[6] Bautista, R., Crespillo, R., Cnovas, F., 2003. Identification of olive-tree cultivars with SCAR markers. Euphytica, 129(1):33-41. 

[7] Bentez, T., Rincn, A.M., Limn, M.C., 2004. Biocontrol mechanisms of Trichoderma strains. Int Microbiol, 7:249-260. 

[8] Benson, D.A., Cavanaugh, M., Clark, K., 2013. GenBank. Nucl Acids Res, 41(D1):D36-D42. 

[9] Black, J.A., Foarde, K.K., 2007. Comparison of four different methods for extraction of Stachybotrys chartarum spore DNA and verification by real-time PCR. J Microbiol Meth, 70(1):75-81. 

[10] Castillo, P., 2009. Isolation and identification of strains of Trichoderma sp. natives of Chile.  Evaluation of antagonism against sp. Biologist Thesis. Catholic University of Valparaso,Chile :

[11] Cordier, C., Edel-Hermann, V., Martin-Laurent, F., 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(1):60-68. 

[12] del Mar Jimnez-Gasc, M., Jimnez-Diaz, R.M., 2003. Development of a specific PCR-based assay for the identification of Fusarium oxysporum f. sp. ciceris and its pathogenic races 0, 1A, 5, and 6. Phytopathology, 93(2):200-209. 

[13] Druzhinina, I.S., Kubicek, C.P., 2005. Species concepts and biodiversity in Trichoderma and Hypocrea: from aggregate species to species clusters?. J Zhejiang Univ-Sci B, 6(2):100-112. 

[14] Druzhinina, I.S., Kopchinskiy, A.G., Komn, M., 2005. An oligonucleotide barcode for species identification in Trichoderma and Hypocrea . Fung Genet Biol, 42(10):813-828. 

[15] Druzhinina, I.S., Seild-Seiboth, V., Herrera-Estrella, A., 2011.  Trichoderma: the genomics of opportunistic success. Nat Rev Microbiol, 9(10):749-759. 

[16] Feng, X.M., Holmberg, A.I.J., Sundh, I., 2011. Specific SCAR markers and multiplex real-time PCR for quantification of two Trichoderma biocontrol strains in environmental samples. Biocontrol, 56(6):903-913. 

[17] Gams, W., Bissett, J., 1998. Morphology and identification of Trichoderma .  and . Vol. 1. Basic Biology, Taxonomy and Genetics. Taylor & Francis,UK :3-34. 

[18] Green, H., Jensen, D.F., 1995. A tool for monitoring Trichoderma harzianum: II. The use of a GUS transformant for ecological studies in the rhizosphere. Phytopathology, 85(11):1436-1440. 

[19] Grondona, I., Hermosa, R., Tejada, M., 1997. Physiological and biochemical characterization of Trichoderma harzianum, a biological control agent against soilborne fungal plant pathogens. Appl Environ Microbiol, 63(8):3189-3198. 

[20] Hagn, A., Wallisch, S., Radl, V., 2007. A new cultivation independent approach to detect and monitor common Trichoderma species in soils. J Microbiol Meth, 69(1):86-92. 

[21] Harman, G.E., 2006. Overview of mechanisms and uses of Trichoderma spp. Phytopathology, 96(2):190-194. 

[22] Harman, G.E., Howell, C.R., Viterbo, A., 2004.  Trichoderma species—opportunistic, avirulent plant symbionts. Nat Rev Microbiol, 2(1):43-56. 

[23] Hermosa, M.R., Grondona, I., Iturriaga, E.A., 2000. Molecular characterization and identification of biocontrol isolates of Trichoderma spp. Appl Environ Microbiol, 66(5):1890-1898. 

[24] Hoitink, H.A.J., Boehm, M.J., 1999. Biocontrol within the context of soil microbial communities: a substrate-dependent phenomenon. Annu Rev Phytopathol, 37(1):427-446. 

[25] Jessup, C.M., Forde, S.E., Bohannan, B.J.M., 2005. Microbial experimental systems in ecology. Advances in Ecological Research, Elsevier,37:273-307. 

[26] Knudsen, I.M., Jensen, B., Jensen, D.F., 1996. Occurrence of Gliocladium roseum on barley roots in sand and field soil.  Monitoring Antagonistic Fungi Deliberately Released into the Environment. Springer,the Netherlands :33-37. 

[27] Komon-Zelazowska, M., Bissett, J., Zafari, D., 2007. Genetically closely related but phenotypically divergent Trichoderma species cause green mold disease in oyster mushroom farms worldwide. Appl Environ Microbiol, 73(22):7415-7426. 

[28] Koveza, O.V., Kokaeva, Z.G., Gostimsky, S.A., 2001. Creation of a SCAR marker in Pea (Pisum sativum L.) using RAPD analysis. Russ J Genet, 37(4):464-466. 

[29] Kredics, L., Hatvani, L., Naeimi, S., 2014. Biodiversity of the genus Hypocrea/Trichoderma in different habitats. Biotechnology and Biology of , Elsevier,:3-24. 

[30] Kubicek, C.P., Komon-Zelazowska, M., Druzhinina, I.S., 2008. Fungal genus Hypocrea/Trichoderma: from barcodes to biodiversity. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 9(10):753-763. 

[31] Lehmann, P.F., Lin, D., Lasker, B.A., 1992. Genotypic identification and characterization of species and strains within the genus Candida by using random amplified polymorphic DNA. J Clin Microbiol, 30(12):3249-3254. 

[32] Lievens, B., Rep, M., Thomma, B.P.H.J., 2008. Recent developments in the molecular discrimination of formae speciales of Fusarium oxysporum . Pest Manag Sci, 64(8):781-788. 

[33] Martin-Laurent, F., Philippot, L., Hallet, S., 2001. DNA extraction from soils: old bias for new microbial diversity analysis methods. Appl Environ Microbiol, 67(5):2354-2359. 

[34] Massart, S., de Clercq, D., Salmon, M., 2005. Development of real-time PCR using Minor Groove Binding probe to monitor the biological control agent Candida oleophila (strain O). J Microbiol Meth, 60(1):73-82. 

[35] Moore, J.C., Ruiter, P.C., Hunt, H.W., 1996. Microcosms and soil ecology: critical linkages between fields studies and modelling food webs. Ecology, 77(3):694-705. 

[36] Naeimi, S., Kocsub, S., Antal, Z., 2011. Strain-specific SCAR markers for the detection of Trichoderma harzianum AS12-2, a biological control agent against Rhizoctonia solani, the causal agent of rice sheath blight. Acta Biol Hung, 62(1):73-84. 

[37] Paavanen-Huhtala, S., Avikainen, H., Yli-Mattila, T., 2000. Development of strain-specific primers for a strain of Gliocladium catenulatum used in biological control. Eur J Plant Pathol, 106(2):187-198. 

[38] Parasnis, A.S., Gupta, V.S., Tamhankar, S.A., 2000. A highly reliable sex diagnostic PCR assay for mass screening of papaya seedlings. Mol Breed, 6(3):337-344. 

[39] Pasquali, M., Piatti, P., Gullino, M.L., 2006. Development of a real-time polymerase chain reaction for the detection of Fusarium oxysporum f. sp basilici from basil seed and roots. J Phytopathol, 154(10):632-636. 

[40] Pujol, M., Badosa, E., Cabrefiga, J., 2005. Development of a strain-specific quantitative method for monitoring Pseudomonas fluorescens EPS62e, a novel biocontrol agent of fire blight. FEMS Microbiol Lett, 249(2):343-352. 

[41] Rozen, S., Skaletsky, H., 1999. Primer3 on the WWW for general users and for biologist programmers. Bioinformatics Methods and Protocols, Springer,:365-386. 

[42] Rubio, M.B., Hermosa, M.R., Keck, E., 2005. Specific PCR assays for the detection and quantification of DNA from the biocontrol strain Trichoderma harzianum 2413 in soil. Microb Ecol, 49(1):25-33. 

[43] Savazzini, F., Longa, C.M.O., Pertot, I., 2008. Real-time PCR for detection and quantification of the biocontrol agent Trichoderma atroviride strain SC1 in soil. J Microbiol Meth, 73(2):185-194. 

[44] Savazzini, F., Oliveira Longa, C.M., Pertot, I., 2009. Impact of the biocontrol agent Trichoderma atroviride SC1 on soil microbial communities of a vineyard in northern Italy. Soil Biol Biochem, 41(7):1457-1465. 

[45] Schena, L., Finetti-Sialer, M.M., Gallitelli, D., 2002. Molecular detection of strain L47 of Aureobasidium pullulans, a biocontrol agent of postharvest disease. Plant Dis, 86(1):54-60. 

[46] Schuster, A., Schmoll, M., 2010. Biology and biotechnology of Trichoderma . Appl Microbiol Biotechnol, 87(3):787-799. 

[47] van der Putten, W.H., Klironomos, J.N., Wardle, D.A., 2007. Microbial ecology of biological invasions. ISME J, 1(1):28-37. 

[48] Vargas-Gil, S., Pastor, S., March, G.J., 2009. Quantitative isolation of biocontrol agents Trichoderma spp., Gliocladium spp. and actinomycetes from soil with culture media. Microbiol Res, 164(2):196-205. 

[49] Viterbo, A., Haran, S., Friesem, D., 2001. Antifungal activity of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai TM. FEMS Microbiol Lett, 200(2):169-174. 

[50] White, T.J., Bruns, T., Lee, S.J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics.  PCR Protocols: a Guide to Methods and Applications. Academic Press,USA :315-322. 

[51] Williams, J., Clarkson, J.M., Mills, P.R., 2003. A selective medium for quantitative reisolation of Trichoderma harzianum from Agaricus bisporus compost. Appl Environ Microbiol, 69(7):4190-4191. 

[52] Zhang, F., Zhu, Z., Yang, X., 2013.  Trichoderma harzianum T-E5 significantly affects cucumber root exudates and fungal community in the cucumber rhizosphere. Appl Soil Ecol, 72:41-48.