Project description:An extensive survey conducted in the Saïss plain of Morocco during the 2017-2018 growing season revealed that 35 out of 50 apple and pear orchards were infested with a pathogen that causes the decline disease. Morphological and phylogenetic tree analyses using the cox II gene allowed us to identify the pathogen as Phytopythium vexans. Interestingly, no Phytophthora and Pythium species were isolated. The occurrence and prevalence of the disease varied between locations; the most infested locations were Meknes (100%), Imouzzer (83%), and Sefrou (80%). To fulfill Koch's postulate, a greenhouse pathogenicity test was performed on the stem and collar of one-year-old healthy seedlings of apple rootstock M115. Symptoms similar to those observed in the field were reproduced in less than 4 months post-inoculation with root rot disease severity ranging from 70 to 100%. The survey results evidenced that apple rootstocks, soil type, and irrigation procedure may contribute significantly to the occurrence of the disease. The disease was most prevalent in drip water irrigation and sandy-clay soil on wild apple rootstock. Accordingly, a rational drip advanced watering system and good sanitation practices could eliminate water stagnation and help prevent the onset of this disease. It was concluded that Pp. vexans occurrence may be strongly influenced by irrigation mode and type of soil. Therefore, the obtained findings of this study could help to better understand the recurrence of this disease and to develop a reliable integrated strategy for its management.
Project description:Brown root rot caused by Phytopythium vexans is a new destructive root disease on many plants such as Gingko, Citrus, kiwifruit, and ramie. The establishment of loop-mediated isothermal amplification (LAMP) technology for detecting P. vexans can help monitor and control brown root rot quickly, efficiently, and accurately. LAMP technology is known for its simplicity, sensitivity, and speed; and it does not require any specialized equipment - a water bath or a thermoblock is sufficient for isothermal amplifications. LAMP products can be visualized by using hydroxy naphthol blue (HNB) dye or agarose gel electrophoresis. In this study, by searching and comparing the internal transcribed spacer (ITS) sequences of P. vexans and the related species in oomycete genera Pythium, Phytopythium, and Phytophthora, we designed specific primers targeting the ITS gene region of P. vexans. Using HNB dye, we established a LAMP technique for rapid detection of P. vexans by visible color change. In addition, we optimized the protocol to enhance both sensitivity and specificity for P. vexans detection. Under the optimized condition, our protocol based on LAMP technology could detect as low as 24 copies of the P. vexans genomic DNA, which is ∼100 times more sensitive than conventional PCR. This method can successfully detect P. vexans using cell suspensions from P. vexans - infected ramie root tissues.