Quantification of the biocontrol agent Trichoderma harzianum with real-time TaqMan PCR and its potential extrapolation to the hyphal biomass.
The species of the genus Trichoderma are used successfully as biocontrol agents against a wide range of phytopathogenic fungi. Among them, Trichoderma harzianum is especially effective. However, to develop more effective fungal biocontrol strategies in organic substrates and soil, tools for monitoring the control agents are required. Real-time PCR is potentially an effective tool for the quantification of fungi in environmental samples. The aim of this study consisted of the development and application of a real-time PCR-based method to the quantification of T. harzianum, and the extrapolation of these data to fungal biomass values. A set of primers and a TaqMan probe for the ITS region of the fungal genome were designed and tested, and amplification was correlated to biomass measurements obtained with optical microscopy and image analysis, of the hyphal length of the mycelium of the colony. A correlation of 0.76 between ITS copies and biomass was obtained. The extrapolation of the quantity of ITS copies, calculated based on real-time PCR data, into quantities of fungal biomass provides potentially a more accurate value of the quantity of soil fungi.
López-Mondéjar
Rubén
R
Department of Soil Water Conservation and Organic Waste Management, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), P.O. Box 164, 30100 Espinardo, Murcia, Spain. rlmondejar@cebas.csic.es
Antón
Anabel
A
Raidl
Stefan
S
Ros
Margarita
M
Pascual
José Antonio
JA
D001483:Base Sequence; D018533:Biomass; D015169:Colony Count, Microbial / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D025301:Hyphae / Q000254:growth & development; D008969:Molecular Sequence Data; D010572:Pest Control, Biological / Q000379:methods*; D011156:Population Density; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D019914:Taq Polymerase; D014242:Trichoderma / Q000235:genetics* / Q000254:growth & development*
D017931:DNA Primers; D021903:DNA, Ribosomal Spacer; D019914:Taq Polymerase