A New Real-Time PCR Assay for Detecting Fungi in Genus Ceratocystis

Abstract

The genus Ceratocystis contains several significant plant pathogens, causing wilt and canker disease on a wide range of plants species. Currently, there are over 40 known species of Ceratocystis, some of which are becoming increasingly important in agricultural or natural ecosystems. The diagnostics for most Ceratocystis species currently relies on time consuming and labour-intensive culturing approaches. To provide more time efficient and sensitive molecular diagnostic tools for Ceratocystis, a generic Taq-Man real-time PCR assay was developed using the ITS gene. This novel two-probe Taq-man assay amplified DNA from all tested Ceratocystis species. Some non-specific amplification of a few species from closely related genera was observed under certain conditions; however, these false positive detections could be ruled out using the additional PCR primers developed for further sequence based identification of the detected species. The assay was highly sensitive as it detected 0.2 pg/µl of Ceratocystis DNA in water as well as in host DNA matrix. Further validation with artificially inoculated fig stem tissue demonstrated that the assay was also able to effectively detect the pathogen in infected asymptomatic stem tissue. This newly developed real-time PCR assay has practical applications in biosecurity, conservation, and agriculture, enabling to detect Ceratocystis species directly from plant material, to facilitate more sensitive screening of imported plant germplasm, and allow rapid tracking of pathogens in case of disease outbreaks.

Seed viability of Persicaria chinensis (L.) H.Gross in Aotearoa / New Zealand

“…Nucleic acid extraction, PCR and sequencing were undertaken following Dharmaraj et al (2022). A total of 0.3 g of plant tissue from a seedling was ground together with 3 ml of cetyltrimethylammonium bromide (CTAB) lysis buffer prior to incubation.…”