title: Development of a multiplex Q-PCR to detect Trichoderma harzianum Rifai strain T22 in plant roots.
abstract: The fungal species Trichoderma harzianum is widely used as a biological agent in crop protection. To verify the continued presence of this fungus on plant roots manually inoculated with T. harzianum strain T22, a Q-PCR was designed using specific probes for this particular strain. To develop these molecular diagnostic tools, genome mining was first carried out to retrieve putative new regions by which different strains of T. harzianum could be distinguished. Subsequently, Sanger sequencing of the L-aminoacid oxidase gene (aox1) in T. harzianum was applied to determine the mutations differing between various strains isolated from the Trichoderma collection of Koppert Biological Systems. Based on the sequence information obtained, a set of hydrolysis probes was subsequently developed which discriminated T. harzianum T22 strains varying in only a single nucleotide. Probes designed for two strains uniquely recognized the respective strains in Q-PCR with a detection limit of 12,5ng DNA. Titration assays in which T. harzianum DNA from distinct strains was varied further underscored the specificity of the probes. Lastly, fungal DNA extracted from roots of greenhouse cultured tomato plants was analyzed using the probe-based assay. DNA from T. harzianum strain T22 could readily be identified on roots of greenhouse reared tomato plants inoculated with varying concentrations up to one week after treatment with a detection limit of 3e6 colony forming units of T. harzianum T22. We conclude that the Q-PCR method is a reliable and robust method for assessing the presence and quantity of T. harzianum strain T22 in manually inoculated plant material. Our method provides scope for the development of DNA based strain specific identification of additional strains of Trichoderma and other fungal biological control agents.
authors.affiliation: University of Applied Sciences Leiden, Zernikedreef 11, 2333, CK, Leiden, The Netherlands.
authors.forename: Ivo R
authors.initials: IR
authors.lastname: Horn
authors.affiliation: Koppert Biological Systems, Veilingweg 14, 2651, BE, Berkel en Rodenrijs, The Netherlands.
authors.forename: Menno
authors.initials: M
authors.lastname: van Rijn
authors.affiliation: University of Applied Sciences Leiden, Zernikedreef 11, 2333, CK, Leiden, The Netherlands.
authors.forename: Tom J J
authors.initials: TJ
authors.lastname: Zwetsloot
authors.affiliation: University of Applied Sciences Leiden, Zernikedreef 11, 2333, CK, Leiden, The Netherlands.
authors.forename: Said
authors.initials: S
authors.lastname: Basmagi
authors.affiliation: University of Applied Sciences Leiden, Zernikedreef 11, 2333, CK, Leiden, The Netherlands.
authors.forename: Anita
authors.initials: A
authors.lastname: Dirks-Mulder
authors.affiliation: University of Applied Sciences Leiden, Zernikedreef 11, 2333, CK, Leiden, The Netherlands.
authors.forename: Willem B
authors.initials: WB
authors.lastname: van Leeuwen
authors.affiliation: Koppert Biological Systems, Veilingweg 14, 2651, BE, Berkel en Rodenrijs, The Netherlands.
authors.forename: Willem J
authors.initials: WJ
authors.lastname: Ravensberg
authors.affiliation: University of Applied Sciences Leiden, Zernikedreef 11, 2333, CK, Leiden, The Netherlands; Naturalis Biodiversity Center, Darwinweg 2, 2333, CR Leiden, The Netherlands; Institute Biology Leiden, Leiden University, Sylviusweg 72, 2333, BE, Leiden, The Netherlands. Electronic address: barbara.gravendeel@naturalis.nl.
authors.forename: Barbara
authors.initials: B
authors.lastname: Gravendeel
mesh_terms: D001483:Base Sequence; D004271:DNA, Fungal / Q000032:analysis* / Q000235:genetics; D005800:Genes, Fungal; D018551:Solanum lycopersicum / Q000382:microbiology*; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D016533:Mycological Typing Techniques / Q000191:economics / Q000379:methods*; D018517:Plant Roots / Q000382:microbiology*; D012988:Soil Microbiology; D013559:Symbiosis; D014242:Trichoderma / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D004271:DNA, Fungal
keywords: Biological control agent; Hydrolysis probes; Plant roots; Q-PCR; aox1