A New TaqMan Real-Time PCR Assay for Detecting the Blueberry Pathogen Monilinia vaccinii-corymbosi.

Monilinia vaccinii-corymbosi (Mvc) is an important fungal pathogen of blueberry, causing mummy berry disease. While the symptoms of the advanced stages of the disease can be obvious, diagnosing its early stages can be challenging. To facilitate fast and sensitive screening of asymptomatic or latently infected plant material for Mvc, we developed a specific TaqMan real-time PCR assay targeting the internal transcribed spacer (ITS) region. The assay was shown to be specific to Mvc and did not cross react with any of the other tested Monilinia species or other blueberry pathogens. Using the multicopy ITS region ensured high analytical sensitivity, enabling very low concentrations of Mvc DNA (0.1 pg) to be detected both in water and host DNA matrix. Comparable results were obtained in interlaboratory testing, showing that the assay is robust, and can be effectively used in other laboratories. Assay sensitivity was also confirmed on infected plant tissue, showing that it is effective in detecting the pathogen in infected asymptomatic stem tissue, as well as infected tissue that was mixed with healthy tissue at a ratio of 1:10 by weight. The assay was duplexed with a plant internal control (cytochrome oxidase gene) for simultaneous amplification of the pathogen and plant internal control in a single reaction. This new diagnostic tool can be used for sensitive and rapid screening of blueberry plants for the presence of Mvc in many different settings, e.g., for breeding programs, research, or biosecurity diagnostics.

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

The genus Ceratocystis contains several significant plant pathogens, causing wilt and canker disease on a wide range of plant species. There are >40 known species of Ceratocystis, some of which are becoming increasingly important in agricultural or natural ecosystems. The diagnostic procedures for most Ceratocystis species rely on time-consuming and labor-intensive culturing approaches. To provide more time-efficient and sensitive molecular diagnostic tools for Ceratocystis, a generic TaqMan real-time PCR assay was developed using the ITS gene. This novel two-probe TaqMan assay amplified DNA from all tested Ceratocystis species. Some nonspecific 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 found to be 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; it will enable the detection of Ceratocystis species directly from plant material to facilitate more sensitive screening of imported plant germplasm, and allow rapid tracking of pathogens in the case of disease outbreaks.

Construction of a kiwifruit yeast two-hybrid cDNA library to identify host targets of the Pseudomonas syringae pv. actinidiae effector AvrPto5.

OBJECTIVE: Bacterial canker is a destructive disease of kiwifruit caused by the Gram-negative bacterium Pseudomonas syringae pv. actinidiae (Psa). To understand the disease-causing mechanism of Psa, a kiwifruit yeast two-hybrid cDNA library was constructed to identify putative host targets of the Psa Type Three Secreted Effector AvrPto5. RESULTS: In this study, we used the Mate & Plate™ yeast two-hybrid library method for constructing a kiwifruit cDNA library from messenger RNA of young leaves. The constructed library consisted of 2.15 × 106 independent clones with an average insert size of 1.52 kb. The screening of the kiwifruit yeast two-hybrid cDNA library with Psa AvrPto5 revealed the interaction of a V-type proton ATPase subunit-H, a proline rich-protein and heavy metal-associated isoprenylated plant protein 26. Among these, heavy metal-associated isoprenylated plant protein 26 showed a positive interaction with Psa AvrPto5 as both prey and bait.