Development of real-time PCR systems based on SYBR® Green I and TaqMan® technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus

Real-time PCR assays based on SYBR®
Green I and TaqMan® technologies were developed
for in planta detection and quantification of Phoma
tracheiphila, the mitosporic fungus causing ‘mal secco’
disease on citrus. Primers and a hybridization probe were
designed on the basis of the internal transcribed spacer
(ITS) region of the nuclear rRNA genes. The real-time
PCR assays were compared with a classic isolation method
in two separate experiments carried out on 6 and
24 month-old sour orange seedlings, artificially inoculated
with a conidial suspension of the pathogen. Both
technologies made it possible to follow the progression of
infection by P. tracheiphila, enabling detection and
quantification of the target fungus prior to the development
of symptoms. The detection limit was 10 copies of
the cloned target sequence and 15 pg of genomic DNA
extracted from fungal spores. The values of the cycle
threshold (Ct) were linearly correlated with the concentration
of the target DNA, indicating that the method is
suitable as a qualitative and quantitative assay. The
presence of non-target fungal DNA had no effect on the
specificity of the assay, but resulted in a 10-fold
reduction of sensitivity. Total inhibition of the reaction
occurred when conidia of the target pathogen were
mixed with an organic soil substrate before extracting
DNA using the standard protocol, while an alternative
purification kit resulted in a significant decrease in
sensitivity. Compared to classic methods, real-time PCR
proved faster and easier to perform and showed a higher
sensitivity. These results suggest that real-time PCR,
based on both chemistries, has a great potential for early
diagnosis of ‘mal secco’ disease and for quantitative estimation
of fungal growth within host tissue.