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Isoflavones in soybean seed and root exudates are host-specific signal molecules for Phytophthora sojae to recognize its host, soybean. G protein and calcium signaling pathways are involved in the chemotaxis of zoospores in the recognition of isoflavones. The role played by non-host-specific signaling molecules (sugars and amino acids) found in seed and root exudates on zoospore chemotaxis and mycelial growth was studied. The transcriptome of P. sojae responding to aspartic acid (Asp) and glucose (Glc) was analyzed in parellel by the RNA-seq method. We found that the relative in situ concentrations of amino acids and sugars significantly promoted zoospore chemotaxis, as do isoflavones. Transcriptomics showed that both similarities and differences existed in response mechanisms of P. sojae to Asp and Glc. Asp and Glc activated mitogen-activated protein kinase signaling pathways and the phosphatidylinositol signaling system but not the G-protein signaling pathway, which have been reported to be responsible for zoospore chemotaxis. In addition, ubiquitin-mediated proteolysis and ATP binding cassette transporters were also activated by Asp and Glc. Meanwhile, the glutathione signaling pathway uniquely participated in the response of P. sojae to Asp but was not involved in the response process to Glc, which will be evaluated in another study. Our results provide new insights into the molecular mechanisms of zoospore response to Asp and Glc.

Phytophthora sojae does not infect nonhost maize (Zea mays) but infects nonhost common bean (Phaseolus vulgaris) under inoculation. Soybean seed exudates participate in mediating host resistance to P. sojae before infection. This study aims to elucidate the role of seed exudates in mediating the nonhost resistance of maize and common bean to P. sojae before infection. The behaviors of P. sojae zoospores in response to the seed exudates were determined using an assay chamber and a concave slide. The proteomes of P. sojae zoospores in response to the seed exudates were analyzed with the tandem mass tag method. The key proteins were quantitatively verified by parallel reaction monitoring. Maize seed exudates exerted a repellent effect on zoospores of P. sojae. This result explains why zoospores sense repelling signaling molecules in maize seed exudates that weaken and strongly inhibit chemotaxis signals in the phosphatidylinositol signaling pathway and arachidonic acid metabolism pathway. Common bean seed exudates did not exhibit any attraction to the zoospores because the guanine nucleotide-binding protein signaling pathway, which is responsible for transmitting chemotactic signals, had no significant change. The proteins protecting the cell membrane structure were significantly downregulated, and the early apoptosis signal glutathione was enhanced in zoospores responding to common bean seed exudates, which resulted in dissolution of the cysts. Maize and common bean seed exudates mediate part of the nonhost resistance to P. sojae via different mechanisms before infection. The immunity of maize to P. sojae is caused by the repellent effect of maize seed exudates on zoospores. Common bean seed exudates participate in mediating nonhost resistance by dissolving the cysts.

A nine-year survey on pathotypes of Phytophthora sojae from 2007 to 2015 throughout Jilin Province of China was conducted. A total of 219 single-zoospore isolates of P. sojae that came from 250 commercial soybean fields in 25 counties were tested for their virulence phenotypes by using eight soybean differentials representing eight Rps alleles. The investigation showed that Phytophthora root rot (PRR) or P. sojae is mainly distributed in northeast Jilin. A total of 91 pathotypes were identified from 219 isolates, in which 34 matched the virulence formula of 15 known races, and two races (races 28 and 30) were identified in China for the first time. The highest isolation frequency was 6.4% found for race 8, followed by race 3 and race 1, each representing 4.6% and 4.1% of the isolates. The isolation frequencies of the remaining 12 races (races 0, 4, 9, 13, 14, 23, 24, 25, 28, 30, 33, and 44) was less than 2.3%. In addition, nine intermediate reaction types (IRTs) were found (0.5–1.4% isolation frequency). The remaining 67 pathotypes that represented approximately 66% of the isolates were not identified to a race. The isolates each defeated zero to eight Rps genes, and 71.2% of the isolates defeated more than three Rps genes, indicating that virulence of the isolates in Jilin is moderately strong. Virulence frequency of the isolates to eight single Rps genes ranged from 20–77%. More than 57% of the isolates were virulent on Rps1a, Rps1b, Rps1d, Rps6, and Rps7 while less than 30% of the isolates were virulent on Rps1c, Rps1k, and Rps3a, indicating that Rps1c, Rps1k, and Rps3a were more effective than the other five Rps genes in Jilin at reducing infection of soybean by P. sojae.

Differential effects of the seed exudates of susceptible and resistant soybean cultivars Sloan and Williams 82 on the zoospores of soilborne pathogenic Phytophthora sojae are not clear. This study aims to elucidate the role of the seed exudates in the host resistance of soybean against P. sojae during prior to infection. The response behaviors of P. sojae zoospores to the seed exudates were performed in an assay chamber and concave slide, the proteomes of P. sojae zoospores treated with the seed exudates were analyzed by TMT method. The expression of genes encoding key proteins was quantified by qRT-PCR. The zoospores of P. sojae exhibited significant chemotaxis to the both seed exudates and a stronger attracted was found to the seed exudates of Sloan. The seed exudates of Sloan also had a stonger promotion than that of Williams 82 on encystment of zoospores. During the responding process, the key proteins in zoospores related to chemotaxis were upregulated, the upregulation fold of these proteins in responding to Sloan was higher than that of Williams 82. Proteins differentially expressed promote the encystment of zoospores. The changes in expression of these proteins was several fold higher in the zoosprores exposed to the seed exudates of Sloan than that of Williams 82. Evidence of differential changes in protein expression in P. sojae zoospores exposed to the seed exudates of both soybean supports the differential developmental behaviors such as chemotaxis and encystment of the zoospores in responding to the two seed exudates.

Phytophthora root and stem rot of soybean is a destructive disease in many countries caused by the soil-borne pathogen Phytophthora sojae. The interaction between soil-borne pathogens and plant roots before invasion is a focus of interest for revealing host and nonhost resistance mechanisms to soil-borne pathogens. In the present study, soybean cvs. Sloan (susceptible), Williams 82 (resistant) and nonhost common bean cv. Yidianhong were used to determine the effect of roots, root exudates, and isoflavones, amino acids, sugars, and citric acid from the root exudates on the pre-infection behavior (taxis, encystment and cyst germination) of zoospores of P. sojae. The elongation zone of roots of both host soybeans attracted significantly more zoospores than did that of the nonhost bean, and that of the susceptible cultivar attracted significantly more zoospores than did that of the resistant cultivar. Similarly, the host soybean root exudates attracted zoospores and promoted zoospore encystment and cyst germination, but nonhost root exudates had no effect on zoospores. Thus, P. sojae apparently selects its host depending on the root exudates. Of the 26 total components detected from root exudates, the exudates from susceptible and resistant soybeans and nonhost common bean had 12, 17, and 25 components respectively. The differences between the host and nonhost were mainly in the type and concentration of amino acids. Most components were significant chemoattractants of P. sojae zoospores. However, when these attractants were mixed at their relative concentrations in the root exudates, the chemoattraction was significantly reduced compared to that of the sum of each components, and the reduction from strongest to weakest was nonhost common bean, resistant and susceptible soybeans, indicating that these components in the root exudates interact with each other, and the intensity of the interaction relates to the type and concentration of the components. This phenomenon is because some components may share the same receptor, thus causing an obstructive effect. This result may also be a reason that common bean is a nonhost of P. sojae. In addition, the relative local concentrations of citric acid in the root exudates of both nonhost cv. Yidianhong and resistant cv. Williams 82 were repellent to zoospores of P. sojae. Root exudates of susceptible cv. Sloan amended with two concentrations of citric acids especially reduced zoospore chemoattraction, indicating that citric acid in the root exudates also plays an important role in host and nonhost resistance to P. sojae.

Background Recently, a new strain of Cercospora sojina (Race15) has been identified, which has caused the breakdown of resistance in most soybean cultivars in China. Despite this serious yield reduction, little is known about why this strain is more virulent than others. Therefore, we sequenced the Race15 genome and compared it to the Race1 genome sequence, as its virulence is significantly lower. We then re-sequenced 30 isolates of C. sojina from different regions to identifying differential virulence genes using genome-wide association analysis (GWAS). Results The 40.12-Mb Race15 genome encodes 12,607 predicated genes and contains large numbers of gene clusters that have annotations in 11 different common databases. Comparative genomics revealed that although these two genomes had a large number of homologous genes, their genome structures have evolved to introduce 245 specific genes. The most important 5 candidate virulence genes were located on Contig 3 and Contig 1 and were mainly related to the regulation of metabolic mechanisms and the biosynthesis of bioactive metabolites, thereby putatively affecting fungi self-toxicity and reducing host resistance. Our study provides insight into the genomic basis of C. sojina pathogenicity and its infection mechanism, enabling future studies of this disease. Conclusions Via GWAS, we identified five candidate genes using three different methods, and these candidate genes are speculated to be related to metabolic mechanisms and the biosynthesis of bioactive metabolites. Meanwhile, Race15 specific genes may be linked with high virulence. The genes highly prevalent in virulent isolates should also be proposed as candidates, even though they were not found in our SNP analysis. Future work should focus on using a larger sample size to confirm and refine candidate gene identifications and should study the functional roles of these candidates, in order to investigate their potential roles in C. sojina pathogenicity.

Potato virus X (PVX), Potato virus Y (PVY), Potato leafroll virus (PLRV), Potato virus M (PVM), and Potato virus A (PVA) commonly cause mixed infections and major economic losses in potato crops, so a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay has been greatly needed to detect all five viruses simultaneously. Previously published primers were evaluated, and five pairs of highly specific primers were selected for use. The quintuplex RT-PCR assay was optimized for primer concentration, annealing temperature, number of PCR cycles, and extension time. Finally, the quintuplex RT-PCR assay was successfully established. The amplified target fragments of 711 base pairs (bp) for PVX, 447 bp for PVY, 336 bp for PLRV, 520 bp for PVM and 273 bp for PVA, and the viral origins of the PCR products were confirmed by sequencing. This system can be easily customized for simplex, duplex, triplex, tetraplex, and quintuplex RT-PCRs by changing only the primer pairs while keeping the final reaction volume and the concentrations of other reagents constant to detect any combination of PVX, PVY, PLRV, PVM, and PVA viruses. In this way, the detection systems can be customized to the characteristics of viral infections specific to China’s potato-producing regions for excellent detection efficiency and meeting the needs of potato producers. The detection limit of this assay was up to 10−3 dilution in tetraplex and quintuplex RT-PCR, and even higher in simplex, duplex, and triplex RT-PCR. The efficiency of the quintuplex RT-PCR was compared with that of a DAS-enzyme-linked immunosorbent assay to detect the five viruses in 320 field potato samples, 195 in vitro plantlets and pooled potato samples. This new multiplex RT-PCR method is not only effective but also faster, cheaper, more sensitive, and more reliable than DAS-ELISA. It is especially suitable for the testing of in vitro plantlets and pooled samples of postharvest seed tubers.

A total of 395 single-zoospore isolates of Phytophthora sojae that were obtained from 467 soybean fields in Heilongjiang, China from 2011 to 2015 were identified for pathotypes using differential soybean cultivars with Rps1a, Rps1b, Rps1c, Rps1d, Rps1k, Rps2, Rps3a, Rps3b, Rps3c, Rps4, Rps5, Rps6, Rps7, and Rps8 resistance genes. The results showed that P. sojae was widespread in Heilongjiang, but not evenly distributed. A heavy infestation of P. sojae in eastern Heilongjiang coincided with the serious diseases caused by this pathogen in that region. Among 135 pathotypes were identified, 20 isolates matched races of P. sojae based on published race definitions. To our knowledge, this is the first report of races 2, 8, 23, 25, 26, 29, 33, 39, 42, 43, and 48 in Heilongjiang. Race 1, previously considered as the dominant race in Heilongjiang, only comprised 1.52 % of the isolates in the present study. Virulence frequencies of 395 isolates to 14 Rps genes ranged from 17.85 to 82.41 %. Less than 30 % of the isolates were virulent on cultivars containing Rps genes 1k, 1c, or 3a, which indicated that these Rps genes were more effective than other genes in Heilongjiang. All isolates could defeat more than two Rps genes. Ninety-six percent of the isolates were virulent against more than four Rps genes, which indicated that multi-virulence isolates existed in Heilongjiang. All 223 tested isolates were sensitive to metalaxyl, but EC50 for this fungicide against the pathogen increased 18-fold in the last 20 years. The results of the present study suggest that incorporating Rps genes 1c, 1k, and 3a into soybean cultivars and the use of metalaxyl-coated seeds should provide integrated approach to manage Phytophthora root and stem rot of soybean in Heilongjiang, China.

Chemotaxis, encystment, cyst germination, and germ tube growth of Phytophthora sojae zoospores in response to common amino acids and sugars in root exudates (l-glutamate, l-aspartate, l-asparagine, l-alanine, l-serine, l-valine, l-histidine, and l-hydroxyproline; l-glucose, l-sucrose, l-fructose, l-arabinose, and l-galactose were examined. All 8 l-amino acids and 5 l-sugars stimulated zoospore chemotaxis, encystment and cyst germination but did not stimulate subsequent growth of the germ tube in P. sojae. Germ tube growth was significantly retarded in response to each of the test compounds. These findings suggest that chemotaxis, encystment and cyst germination in P. sojae zoospores are receptor-mediated and that the zoospores did not take up and utilize the test amino acids and sugars as nutrients. Different amino acids and sugars affected zoospore chemotaxis to different degrees. Maximum promotion or inhibition occurred at specific concentrations for each compound.

In 2011–2014, ELISA or nucleic acid spot hybridization (NASH) testing for common potato viruses or Potato spindle tuber viroid (PSTVd) was performed on 500 leaf samples collected in potato fields in the northeast provinces Heilongjiang and Inner Mongolia, China. The results revealed that 38.4% (Heilongjiang) and 27.7% (Inner Mongolia) were positive for Potato virus Y (PVY). To unveil the strain composition and population structure of PVY in the region, the multiplex RT-PCR described by Chikh-Ali et al. was performed on all of the ELISA-PVY-positive samples. Of the 158 samples whose PVY strain scenarios could be determined, PVYNTN-NW-SYR-II and PVYN-Wi were the most abundant strains, occurring in 58.9 and 47.5% samples, followed by PVYNTN-NW-SYR-I (31.0%), PVYN:O (19.6%), Eu-PVYNTN (7.6%), NA-PVYN (1.3%), and PVYO (0.6%). In the 84 single-strain-infected samples, PVYN-Wi accounted for 41.7%, PVYNTN-NW-SYR-II for 40.5%, PVYNTN-NW-SYR-I for 14.3%, and PVYN:O and Eu-PVYNTN for 3.6% each. Seven isolates representing PVYNTN-NW-SYR-I (HLJ-6-1 and HLJ-9-4), PVYNTN-NW-SYR-II (INM-W-369-12 and SC-1-1-2), PVYN:O (HLJ-30-2), and PVYN-Wi (HLJ-BDH-2 and HLJ-C-429) were sequenced and analyzed molecularly. Whereas the sequence identities for isolates belonging to the same strain group were >98.5%, they fell for isolates belonging to different strain groups to 92.7–98.1% at the genome level and 96.1–98.4% at the polyprotein level. Interestingly, the exact location of the recombination events varied among isolates within a strain group. Phylogenetic analysis of all 42 full length PVY sequences from China indicated that most clustered to various recombinant groups, despite the fact that the PVY isolates were isolated from at least five host species. Pathological analysis of four isolates representing PVYN:O, PVYN-Wi, PVYNTN-NW-SYR-I, and PVYNTN-NW-SYR-II revealed that the PVYNTN-NW-SYR-II isolate incited the most severe symptoms on potato cultivar Kexin 13, followed by PVYNTN-NW-SYR-I, PVYN:O and PVYN-Wi. The PVYNTN-NW-SYR-I and PVYNTN-NW-SYR-II isolates also caused necrotic ringspots on the tubers of Kexin 13, indicating their ability to induce the potato tuber necrotic ringspot disease in potato.

To estimate inoculum density of Phytophthora sojae in natural soils to predict disease risk and to study the ecology of P. sojae in the soil, we developed two bioassays to quantify zoospores and another for oospores of P. sojae in soil based on inoculation of Mollisol (the most conducive soil type for this disease according to our disease survey) with a known density of zoospores or oospores of enhanced green fluorescent protein (EGFP)-labeled P. sojae Eps597-3 (pathotype 1a3c7). A linear regression equation, \( y_{ 1} = 1.0 9 2 7x_{ 1} + 7. 3 6 5 2 \) (Eq. 1), and an index regression equation, \( {\text{y}}_{ 2} \, \;{ = }\; 1 8 6 2. 9 {\text{e}}^{{0.00 9 5 {\text{x}}_{ 2} }} \) (Eq. 2), were established to quantify the zoospores in the soil, where x1 is the concentration of zoospores counted directly using a light microscope and y1 is the concentration of zoospores added to a soil suspension, x2 is the number of zoospores at the margin of 18 leaf discs, and y2 is the number of zoospores added to 10 mL of a soil suspension. Another linear regression equation, \( y_{ 3} = 3. 3 8 3 6x_{ 3} + 20 7.0 5 \) (Eq. 3), was determined to quantify the oospores in the soil, where x3 is the number of zoosporangia at the margin of 18 leaf discs, and y3 is the number of oospores added to 10 mL of a soil suspension. These three equations provided high reproducibility and accuracy. To verify their applicability, two other isolates with different pathotypes (3a3b678 and 1a1d23a3c578) and two other soil types (Albic luvisols and Aquic cambosols) were tested. Pathotype and soil type did not markedly affect the parameters in the three equations, suggesting that these three equations are accurate enough to serve as universal quantification bioassays of live zoospores and oospores of P. sojae isolates of any pathotype in any soil. Equations (1) and (2) can be used only to estimate zoospores, Eq. (2) for zoospores, and Eq. (3) for oospores of P. sojae in natural or inoculated soil. This method can be finished within 2–3 h for Eq. (1), 4–5 h for Eq. (2), and 7–10 days for Eq. (3).

The F2 population derived from a cross between isolates pRx (Avr1c-Avr1c) and ps1 (avr1c-avr1c) of Phytophthora sojae, fungal agent of soybean stem and root rot, was used to determine the genetic basis of avirulence towards Rps1c gene in soybean. The results indicated that this avirulence is dominant and controlled by a single locus, as expected for a simple gene-for-gene model. Segregation of Avr1c in the F2 progeny of this cross fits a 3:1 ratio. Four of 80 AFLP primers effectively distinguished the avirulent pRx from the virulent ps1. Among the 5 specific markers, band C was amplified from the avirulent pRx by primer set EGC/MAT, then recovered and cloned. This AFLP marker was successfully transfered to a SCAR marker through sequencing, primer design and specific amplication of the DNA of the avirulent pRx. Results of validity and specificity experiments with 50 individuals of the F2 progeny and 50 field isolates demonstrated that this SCAR marker (a 616-bp fragment) can be successfully and specifically amplified from the P. sojae isolates that have Avr1c gene.