RESUMO
Phytophthora sojae is a devastating plant pathogen that causes soybean Phytophthora root rot worldwide. Early on-site and accurate detection of the causal pathogen is critical for successful management. In this study, we have developed a novel and specific one-pot RPA/PCR-CRISPR/Cas12 assay for on-site detection (Cas-OPRAD) of Phytophthora root rot (P. sojae). Compared to the traditional RPA/PCR detection methods, the Cas-OPRAD assay has significant detection performance. The Cas-OPRAD platform has excellent specificity to distinguish 33 P. sojae from closely related oomycetes or fungal species. The PCR-Cas12a assay had a consistent detection limit of 100 pg. µL-1, while the RPA-Cas12a assay achieved a detection limit of 10 pg. µL-1. Furthermore, the Cas-OPRAD assay was equipped with a lateral flow assay for on-site diagnosis and enabled the visual detection of P. sojae on the infected field soybean samples. This assay provides a simple, efficient, rapid (<1 h), and visual detection platform for diagnosing Phytophthora root rot based on the one-pot CRISPR/Cas12a assay. Our work provides important methods for early and accurate on-site detection of Phytophthora root rot in the field or customs fields.
RESUMO
Chinese water bamboo (Dracaena sanderiana) is a popular houseplant due to its ability to survive in various indoor conditions. In October 2020, pronounced leaf blight symptoms with approximately 50% disease incidence were observed on water bamboo in the 35-ha field of Wenchang county (19°50'45'' N, 110°21'38'' E), Hainan, China. The diseased leaves showed pale green with yellowish lesions, dried and shrivelled tips. As the disease progressed, upward-spreading necrosis led to stem weakness, and then the plants wilted and died within a few weeks (Fig. 1A,B). Ten symptomatic leaves were collected, and leaf pieces (5 mm × 5 mm) from the edge of the lesion were cut, disinfected for 30 s using 75% ethanol, and rinsed five times in d.d. H2O, and placed on V8 media amended with 10 mg/L pimaricin, 150 mg/L ampicillin, and 16 mg/L rifampicin (Guo et al., 2012). Eight isolates of Phytophthora recovered from 10 leaves were characterized morphologically. Colony morphology showed slightly radiate to stellate patterns, with cottony aerial mycelium (Fig.1D). Hyphae were smooth and uniform, branching at nearly right angles into stout (Fig.1E). Mycelial swellings were observed in plate culture (Fig.1F). Chlamydospores were abundant and spherical (Fig.1G). Sporangia were produced externally through sympodial development of the sporangiophore immediately below a sporangium (Fig.1H, I). The sporangia were 29.3-63.9 µm (mean, 48.5 µm) in length and 19.3-46.8 µm (mean, 33.5 µm, n=50) in width. No sexual organs were observed in the 8 isolates. The morphological characteristics of the isolates were consistent with the description of P. palmivora (Erwin and Ribeiro, 1996). The internal transcribed spacer (ITS) region of rDNA, the translation elongation factor 1α gene (TEF1), and ß-Tubulin gene (TUB) sequences of the 8 isolates were amplified using the primers ITS1/ITS4 (Cooke et al., 2000), ELONGF1/ELONGR1 and TUBUF2/TUBUR1 (Kroon, et al., 2004), respectively, followed by sequencing analysis. The sequences of representative isolate HK-5 for the ITS, TEF1 and TUB were deposited in GenBank with the accession numbers of OK349485, OK349488 and OK349487. BLAST results showed that the ITS (MG865559, identity=735/735; 100%), TEF1(MH359047, identity=682/684; 99.7%) and TUB (MH493992, identity=467/468; 99.8%) sequences were all highly similar with a sequence of P. palmivora strain CPHST BL105. Phylogenetic analysis using the combined ITS, TEF1, and TUB sequences showed that the isolate HK-5 were grouped with the P. palmivora with good bootstrap support (Fig.2). Based on morphological and molecular identification, the pathogens were identified as P. palmivora. The pathogenicity tests showed that all whole plants (1-year-old) sprayed with the 5 mL of zoospore suspension (1×106 zoospores mL-1) initially presented with discoloured spots on their leaves after 4 days of inoculation, and the symptoms gradually progressed from spots to leaf blight (Fig. 1C). Each isolate was applied onto 10 plants and control plants were sprayed with d.d.H2O. The results of the pathogenicity test exhibited typical symptoms as observed in the field. No significant differences of virulence were observed among the 8 isolates, and the control plants remained symptomless. P. palmivora was re-isolated from the leaves of inoculated plants. This is the first report of P. palmivora on water bamboo in China, and appropriate measures must be undertaken to control this agent in this region.