Abstract
Early and accurate detection of the causal pathogen Phytophthora sojae is crucial for effective prevention and control of root and stem rot and seedling damping-off of soybean. In the present study, a novel isothermal amplification assay was developed for detecting P. sojae. This 25 min assay included a two-step approach. First, a pair of novel primers, PSYPT-F and PSYPT-R were used to amplify a specific fragment of the Ypt1 gene of P. sojae in a 20 min recombinase polymerase amplification (RPA) step. Second, lateral flow dipsticks (LFD) were used to detect and visualize RPA amplicons of P. sojae within 5 min. This RPA-LFD assay was specific to P. sojae. It yielded negative detection results against 24 other Phytophthora, one Globisporangium, and 14 fungal species. It was also found to be sensitive, detecting as low as 10 pg of P. sojae genomic DNA in a 50-μL reaction. Furthermore, P. sojae was detected from artificially inoculated hypocotyls of soybean seedlings using this novel assay. In a comparative evaluation using 130 soybean rhizosphere samples, this novel assay consistently detected P. sojae in 55.4% of samples, higher than other three methods, including loop-mediated isothermal amplification (54.6%), conventional PCR (46.9%), and leaf-disc baiting (38.5–40.0%). Results in this study indicated that this rapid, specific, and sensitive RPA-LFD assay has potentially significant applications to diagnosing Phytophthora root and stem rot and damp-off of soybean, especially under time- and resource-limited conditions.
Highlights
Phytophthora sojae is one of the most devastating pathogens of soybean crops (Glycine max), causing damping-off on seedlings and root and stem rot on older plants
Test lines were visible on dipsticks using genomic DNAs (gDNAs) of P. sojae isolates
Test lines were visible on dipsticks correlating with 100, 10, 1, 0.1, or 0.01 ng of P. sojae gDNA template used per each recombinase polymerase amplification (RPA) reaction
Summary
Phytophthora sojae is one of the most devastating pathogens of soybean crops (Glycine max), causing damping-off on seedlings and root and stem rot on older plants. After assessing its potential risks to agricultural and economic security, the Ministry of Agriculture of the People’s Republic of China identified P. sojae as a quarantine pest in 20071, whereas it was discovered in Jilin and Heilongjiang Provinces in 1989 (Su and Shen, 1993). Spread of this pathogen has been accelerated by China’s increasing international and interprovincial trade and transportation of soybean seeds and plants (Cui et al, 2010; Wu et al, 2017). The pathogen has been found in the Inner Mongolia Autonomous Region, Xinjiang Uygur Autonomous Region, Huanghe-Huaihe River Basin and Yangtze River Basin (Chen and Wang, 2017), as well as Jilin, Heilongjiang (Su and Shen, 1993), Fujian (Cui et al, 2010; Wu et al, 2017), and Anhui (Dai Y.L. et al, 2015) Provinces
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