BioControl

, Volume 56, Issue 6, pp 903–913 | Cite as

Specific SCAR markers and multiplex real-time PCR for quantification of two Trichoderma biocontrol strains in environmental samples

  • Xin Mei Feng
  • Anna-Ida Johnsson Holmberg
  • Ingvar Sundh
  • Thomas Ricard
  • Petter Melin
Article

Abstract

Several strains from the genus Trichoderma (Ascomycetes, Hypocreales) are commercially used as biocontrol agents, e.g. in formulations containing the two Trichoderma strains IMI206039 (Hypocrea parapilulifera B.S. Lu, Druzhinina & Samuels) and IMI206040 (T. atroviride P. Karst). To quantify the presence of the two isolates after application, we developed primers for SCAR markers (Sequence-Characterised Amplified Region). In order to quantify both fungal strains simultaneously, we also designed fluorophore-labelled probes distinguishing the two strains, to be used in combination with the SCAR primers. In incubations of two different soils, artificially inoculated and maintained under controlled conditions, the quantification through amplification with the SCAR markers in qPCR and through colony-forming units from plate counting correlated well. Further tests of the markers on samples taken from a golf green treated with a product containing both strains indicated that the two biocontrol strains did not establish, either on the golf green or in the surrounding area.

Keywords

Ascomycetes Golf Hypocrea parapilulifera Hypocreales Trichoderma atroviride Trichoderma polysporum 

References

  1. Abbasi PA, Miller SA, Meulia T, Hoitink HAJ, Kim JM (1999) Precise detection and tracing of Trichoderma hamatum 382 in compost-amended potting mixes by using molecular markers. Appl Environ Microbiol 65:5421–5426PubMedGoogle Scholar
  2. Anonymous (2001) Commission Directive 2001/36/EC concerning the placing of plant protection products on the marketGoogle Scholar
  3. Becker EM, Ball LA, Hintz WE (1999) PCR-based genetic markers for detection and infection frequency analysis of the biocontrol fungus Chondrostereum purpureum on Sitka Alder and trembling aspen. Biol Control 15:71–80CrossRefGoogle Scholar
  4. Benítez T, Rincón AM, Limón MC, Codón AC (2004) Biocontrol mechanisms of Trichoderma strains. Int Microbiol 7:249–260PubMedGoogle Scholar
  5. Castrillo LA, Vandenberg JD, Wraight SP (2003) Strain-specific detection of introduced Beauvaria bassiana in agricultural fields by use of sequence-characterized amplified region markers. J Invertebr Pathol 82:75–83PubMedCrossRefGoogle Scholar
  6. 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 Methods 68:60–68PubMedCrossRefGoogle Scholar
  7. Dauch AL, Watson AK, Jabaji-Hare SH (2003) Detection of the biocontrol agent Colletotrichum coccodes (183088) from the target weed velvetleaf and from soil by strain-specific PCR markers. J Microbiol Methods 55:51–64PubMedCrossRefGoogle Scholar
  8. De Clercq D, Cognet S, Pujol M, Lepoivre P, Jijakli MH (2003) Development of a SCAR marker and a semi-selective medium for specific quantification of Pichia anomala strain K on apple fruit surfaces. Postharvest Biol Technol 29:237–247CrossRefGoogle Scholar
  9. Dodd SL, Hill RA, Stewart A (2004) A duplex-PCR bioassay to detect a Trichoderma virens biocontrol isolate in non-sterile soil. Soil Biol Biochem 36:1955–1965CrossRefGoogle Scholar
  10. Fredricks DN, Smith C, Meier A (2005) Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. J Clin Microbiol 43:5122–5128PubMedCrossRefGoogle Scholar
  11. Ghisalberti EL, Rowland CY (1993) Antifungal metabolites from Trichoderma harzianum. J Nat Prod 56:1799–1804PubMedCrossRefGoogle Scholar
  12. Ginzinger DG (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 30:503–512PubMedCrossRefGoogle Scholar
  13. Grosch R, Schneider JHM, Peth A, Waschke A, Franken P, Kofoet A, Jabaji-Hare SH (2007) Development of a specific PCR assay for the detection of Rhizoctonia solani AG 1-IB using SCAR primers. J Appl Microbiol 102:806–819PubMedCrossRefGoogle Scholar
  14. Haran S, Schickler H, Chet I (1996) Molecular mechanisms of lytic enzymes involved in the biocontrol activity of Trichoderma harzianum. Microbiology 142:2321–2331CrossRefGoogle Scholar
  15. Hermosa MR, Grondona I, Diaz-Minguez JM, Iturriaga EA, 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 38:343–350PubMedCrossRefGoogle Scholar
  16. Holmberg AIJ, Melin P, Levenfors JP, Sundh I (2009) Development and evaluation of SCAR markers for a Pseudomonas brassicacearum strain used in biological control of snow mould. Biol Control 48:181–187CrossRefGoogle Scholar
  17. Howell CR (2003) Mechanisms employed by Trichoderma species in the biological control of plant diseases: The history and evolution of current concepts. Plant Dis 87:4–10CrossRefGoogle Scholar
  18. Ihrmark K, Asmail N, Ubhayasekera W, Melin P, Stenlid J, Karlsson M (2010) Comparative molecular evolution of Trichoderma chitinases in response to mycoparasitic interactions. Evol Bioinform 6:1–26Google Scholar
  19. Lu BS, Druzhinina IS, Fallah P, Chaverri P, Gradinger C, Kubicek CP, Samuels GJ (2004) Hypocrea/Trichoderma species with pachybasium-like conidiophores: teleomorphs for T. minutisporum and T. polysporum and their newly discovered relatives. Mycologia 96:310–342PubMedCrossRefGoogle Scholar
  20. Maddau L, Cabras A, Franceschini A, Linaldeddu BT, Crobu S, Roggio T, Pagnozzi D (2009) Occurrence and characterization of peptaibols from Trichoderma citrinoviride, an endophytic fungus of cork oak, using electrospray ionization quadrupole time-of-flight mass spectrometry. Microbiology 155:3371–3381PubMedCrossRefGoogle Scholar
  21. Paavanen-Huhtala S, Avikainen H, Yli-Mattila T (2000) Development of strain-specific primers for Gliocladium catenulatum strain used in biological control. Eur J Plant Pathol 106:187–198CrossRefGoogle Scholar
  22. Pujol M, Badosa E, Cabrefiga J, Montesinos E (2005) Development of a strain-specific quantitative method for monitoring Pseudomonas fluorescens EPS62e, a novel biocontrol agent of fire blight. FEMS Microbiol Lett 249:343–352PubMedCrossRefGoogle Scholar
  23. Pujol M, Badosa E, Manceau C, Montesinos E (2006) Assessment of the environmental fate of the biological control agent of fire blight, Pseudomonas fluorescens EPS62e, on apple by culture and real-time PCR methods. Appl Environ Microbiol 72:2421–2427PubMedCrossRefGoogle Scholar
  24. Rubio MB, Hermosa MR, 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 49:25–33PubMedCrossRefGoogle Scholar
  25. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  26. Samuels GJ (1996) Trichoderma: a review of biology and systematics of the genus. Mycol Res 100:923–935CrossRefGoogle Scholar
  27. Savazzini F, Longa CMO, Pertot I, Gessler C (2008) Real-time PCR for detection and quantification of the biocontrol agent Trichoderma atroviride strain SC1 in soil. J Microbiol Methods 73:185–194PubMedCrossRefGoogle Scholar
  28. Savazzini F, Longa CMO, Pertot I (2009) Impact of the biocontrol Trichoderma atroviride SC1 on soil microbial communities of a vineyard in northern Italy. Soil Biol Biochem 41:1457–1465CrossRefGoogle Scholar
  29. Thrane C, Lubeck M, Green H, Degefu Y, Allerup S, Thrane U, Jensen DF (1995) A tool for monitoring Trichoderma harzianum. 1. Transformation with the GUS gene by protoplast technology. Phytopathology 85:1428–1435CrossRefGoogle Scholar
  30. Tseng SC, Liu SY, Yang HH, Lo CT, Peng KC (2008) Proteomic study of biocontrol mechanisms of Trichoderma harzianum ETS 323 in response to Rhizoctonia solani. J Agric Food Chem 56:6914–6922PubMedCrossRefGoogle Scholar
  31. Turoczi G, Fekete C, Kerenyi Z, Nagy R, Pomazi A, Hornok L (1996) Biological and molecular characterisation of potential biocontrol strains of Trichoderma. J Basic Microbiol 36:63–72PubMedCrossRefGoogle Scholar
  32. Uchiyama R, Aoki K, Sugimoto H, Taka N, Katayama T, Itonori S, Sugita M, Che FS, Kumagai H, Yamamoto K (2009) Phosphocholine-containing glycosyl inositol-phosphoceramides from Trichoderma viride induce defense responses in cultured rice cells. Biosci Biotechnol Biochem 73:74–78PubMedCrossRefGoogle Scholar
  33. Vargas WA, Djonovic S, Sukno SA, Kenerley CM (2008) Dimerization controls the activity of fungal elicitors that trigger systemic resistance in plants. J Biol Chem 283:19804–19815PubMedCrossRefGoogle Scholar
  34. Vinale F, Flematti G, Sivasithamparam K, Lorito M, Marra R, Skelton BW, Ghisalberti EL (2009) Harzianic acid, an antifungal and plant growth promoting metabolite from Trichoderma harzianum. J Nat Prod 72:2032–2035PubMedCrossRefGoogle Scholar
  35. Weaver M, Vedenyapina E, Kenerley CM (2005) Fitness, persistence, and responsiveness of a genetically engineered strain of Trichoderma virens in soil mesocosms. Appl Soil Ecol 29:125–134CrossRefGoogle Scholar
  36. Williams J, Clarkson JM, Mills PR, Cooper RM (2003) A selective medium for quantitative reisolation of Trichoderma harzianum from Agaricus bisporus compost. Appl Environ Microbiol 69:4190–4191PubMedCrossRefGoogle Scholar

Copyright information

© International Organization for Biological Control (IOBC) 2011

Authors and Affiliations

  • Xin Mei Feng
    • 1
  • Anna-Ida Johnsson Holmberg
    • 1
  • Ingvar Sundh
    • 1
  • Thomas Ricard
    • 2
  • Petter Melin
    • 1
  1. 1.Uppsala BioCenter, Department of MicrobiologySwedish University of Agricultural SciencesUppsalaSweden
  2. 2.BINAB Bio-Innovation ABHelsingborgSweden

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