The transcriptome of the oomycete plant pathogen Phytophthora sojae was profiled at ten different developmental and infection stages based on a 3′-tag digital gene-expression protocol. More than 90 million clean sequence tags were generated and compared with the P. sojae genome and its 19,027 predicted genes. A total of 14,969 genes were detected, of which 10,044 were deemed reliable because they mapped to unambiguous tags. A comparison of the whole-library genes' expression patterns suggested four groups: i) mycelia and zoosporangia, ii) zoospores and cysts, iii) germinating cysts, and iv) five infection site libraries (IF1.5 to IF24h). The libraries from the different groups showed major transitional shifts in gene expression. From the ten libraries, 722 gene expression–pattern clusters were obtained and the top 16 clusters, containing more than half of the genes, comprised enriched genes with different functions including protein localization, triphosphate metabolism, signaling process, and noncoding RNA metabolism. An evaluation of the average expression level of 30 pathogenesis-related gene families revealed that most were infection induced but with diverse expression patterns and levels. A web-based server named the Phytophthora Transcriptional Database has been established.

Phytophthora spp. are widespread eukaryotic phytopathogens and often extremely harmful. Phytophthora can infect many types of plants important to agriculture and forestry and thus cause large economic losses. Perhaps due to inappropriate recognition of Phytophthora as a common pathogen in history, research on the biological control of Phytophthora is limited.

Necrosis- and ethylene-inducing-like proteins (NLP) are widely distributed in eukaryotic and prokaryotic plant pathogens and are considered to be important virulence factors. We identified, in total, 70 potential Phytophthora sojae NLP genes but 37 were designated as pseudogenes. Sequence alignment of the remaining 33 NLP delineated six groups. Three of these groups include proteins with an intact heptapeptide (Gly-His-Arg-His-Asp-Trp-Glu) motif, which is important for necrosis-inducing activity, whereas the motif is not conserved in the other groups. In total, 19 representative NLP genes were assessed for necrosis-inducing activity by heterologous expression in Nicotiana benthamiana. Surprisingly, only eight genes triggered cell death. The expression of the NLP genes in P. sojae was examined, distinguishing 20 expressed and 13 nonexpressed NLP genes. Real-time reverse-transcriptase polymerase chain reaction results indicate that most NLP are highly expressed during cyst germination and infection stages. Amino acid substitution ratios (Ka/Ks) of 33 NLP sequences from four different P. sojae strains resulted in identification of positive selection sites in a distinct NLP group. Overall, our study indicates that expansion and pseudogenization of the P. sojae NLP family results from an ongoing birth-and-death process, and that varying patterns of expression, necrosis-inducing activity, and positive selection suggest that NLP have diversified in function.

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.

Phytophthora sojae is a devastating pathogen that causes soybean Phytophthora root rot. This study reports the development of a loop-mediated isother

Phytophthora cinnamomi is an ecologically and agriculturally significant plant pathogen. Early and accurate detection of P. cinnamomi is paramount to disease prevention and management. In this study, a loop-mediated isothermal amplification (LAMP) assay utilizing a new target gene Pcinn100006 identified from genomic sequence data was developed and evaluated for the detection of P. cinnamomi. This Pcinn100006 LAMP assay was found highly specific to P. cinnamomi. All 10 tested isolates of P. cinnamomi yielded positive results, whereas 50 isolates belonging to 16 other Phytophthora species, Globisporangium ultimum, and 14 fungal species lacked detection. This assay was 10 times more sensitive (100 pg in a 25-µl reaction mixture) than a conventional PCR assay (2 ng in a 50-µl reaction mixture) for detecting the genomic DNA of P. cinnamomi. In addition, it detected P. cinnamomi from artificially inoculated leaves of Cedrus deodara. Moreover, detection rates of P. cinnamomi using environmental DNAs extracted from 13 naturally infested rhizosphere samples were 100% in the Pcinn100006 LAMP assay versus 46% in the conventional PCR assay. Considering its higher accuracy and shorter time span, this Pcinn100006 LAMP assay is a promising diagnostic tool to replace conventional PCR-based and culture-dependent assays for screening of P. cinnamomi in regions at risk of infection or contamination.