Quantification of Trichoderma afroharzianum, Trichoderma harzianum and Trichoderma gamsii inoculants in soil, the wheat rhizosphere and in planta suppression of the crown rot pathogen Fusarium pseudograminearum.

AIMS: Develop quantitative assays (qPCR) to determine the detection threshold limits, colonisation and persistence of Trichoderma gamsii, T. afroharzianum and T. harzianum inoculants in cropping soils, the wheat rhizosphere and their in planta suppressive efficacy against the crown rot pathogen Fusarium pseudograminearum. METHODS AND RESULTS: Trichoderma qPCR primers were designed from the internal transcribed spacer region of 5.8S rDNA and from sequences of DNA fragments diagnostic for each inoculant genotype. The minimum detection thresholds (MDT) of qPCR assays varied between 1 x 10(3) (log 3) and 8 x 10(4) (log 4.9) conidia (genome) equivalents g(-1) of soil for multi- and single- copy target sequences, respectively and were independent of soil type. There was a strong correlation (r > 0.974) between culture-dependent and culture-independent (qPCR) quantification methods. In wheat bioassays, Trichoderma inoculants colonised rhizosphere soils and wheat roots at 56-112 days post-emergence to a depth of 20 cm but were more abundant (P<0.001) at 0-10 cm root depth, means ranging from 2 x 10(2) (log 2.3) to 4 x 10(5) (log 5.6) conidia equivalents g(-1) of rhizosphere soil or root tissue. Inoculants reduced (P<0.001) F. pseudograminearum biomass in wheat crown and root tissue by up to 5,754 -fold and increased (P=0.008) plant biomass, relative to the pathogen control. CONCLUSIONS: The qPCR assays provided sensitive and accurate assessment of wheat root and rhizosphere soil colonisation of Trichoderma inoculants. Strains persisted through to grain maturity at levels shown to significantly suppress F. pseudograminearum in planta. SIGNIFICANCE AND IMPACT OF THE STUDY: The qPCR assays developed here were used to determine the wheat rhizosphere dynamics of T. harzianum, T. afroharzianum and T. gamsii inoculants and their suppressive efficacy against F. pseudograminearum in planta. These assays can be applied to monitor inoculant dynamics in suppressing crown rot and other wheat root diseases in the field.