| Phytophthora root rot caused by Phytophthora sojae Kaufinann & Gerdemann is one of the most destructive diseases of soybean. P. sojae is spread by oospores that persist in the soil carried with transported soybeans. A rapid and accurate method for the specific detection of P. sojae in soil is essential to control the spread of Phytophthora root rot. The internal transcribed spacer regions of the ribosomal DNA (rDNA) of eight P. sojae isolates from different areas were amplified using the polymerase chain reaction (PCR) with the universal primers DC6 and ITS4. The sequences of the resulting PCR products were aligned with published sequences of 50 other Phytophthora species, and a region specific to P. sojae was used to design the specific PCR primers, PS1 and PS2. More than 201 isolates representing 14 species of Phytophthora and at least 36 other species of fungi were used to test the specificity of the primers. PCR amplification with primers PS1 and PS2 results in the amplification of a product of approximately 330 bp, exclusively from isolates of P. sojae. Tests with P. sojae total genomic DNA determined that the sensitivity of primers PS1 and PS2 is about 1 fg. This PCR assay, combined with a simple soil screening method developed in this work, allowed the detection of P. sojae in soil within one day, with a detection sensitivity of 2 oospores in 20 g of soil. In addition, combined with a rapid NaOH lysis technique, PCR with the PS primers could be used to detect P. sojae from diseased soybean tissue and soybean residues. The PS-primer-based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in soil and infected soybean tissue. Detection and quantification of genomic DNA from the plant pathogen Phytophthora sojae was achieved from soil substrate. Specific primers targeting a 330-bp fragment from the ITS of P. sojae were used in real-time polymerase chain reaction (PCR) assays conjugated with the fluorescent SYBR? Green I dye. Standard curves showed a linear relation (r~2= 0.993) between log values of fungal genomic DNA of pathogen and real-time PCR threshold cycles and were quantitative over 5 orders of magnitude. Real-time PCR assays were applied to suspension and sterile and non-sterile soil substrate inoculated with known oospore numbers of P. sojae. Detection and genomic DNA quantification was obtainedfrom the different treatments, while no amplicon was detected from non-seeded non-sterile soil samples, confirming the absence of cross-reactivity with the soil microflora DNA. A significant difference was obtained among the amounts of genomic DNA of oospores present in suspension and seeded soil substrate. The DNA extraction protocol and real-time PCR quantification assay can be adaptable to detect and quantify genomic DNA from the soilborn P. sojae.In this work, two inhibitor of the Ca2t/calmodulin signalling pathway were tested to verify the role of this pathway in the zoospores germination process of Phytophthora sojae. Chelator EGTA and calcium channel blocker verapamil were able to inhibit the germination of zoospores. Many roundlets were observed in the zoospores inhibited completely and some zoospores' memberane dissolved. The calmodulin gene of P. sojae was cloned by reverse transcriptase and polymerase chain reaction. The DNA sequence of calmodulin gene of P. sojae comprises about 763 nucleotides including introns. The cDNA sequence is composed of 523 nucleotides and predicted amino acid is 54-91% identical to calmodulin genes of other species of fungi. In the compatible interaction between soybean and P. sojae, cam of P. sojae downregulated in 24h and the expression reached a minimum at 24h. At 48h, the expressive level increased to the level of control. Relative quantitative RT-PCR was used to detect the change of calmodulin expression treated with H2O2. The result suggested that calmodulin gene of P. sojae is not essential for oxidative stress responses.
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