Phytophthora root and stem rot of soybean caused by Phytophthora sojae(P.sojae)is a devastating disease that affects soybean[Glycine max(L.)Merr.]all over the world.S-phase kinase-associated protein 1(SKP1)proteins ar...Phytophthora root and stem rot of soybean caused by Phytophthora sojae(P.sojae)is a devastating disease that affects soybean[Glycine max(L.)Merr.]all over the world.S-phase kinase-associated protein 1(SKP1)proteins are key members of the SKP1/Cullin/F-box protein(SCF)ubiquitin ligase complex and play diverse roles in plant biology.However,the role of SKP1 in soybean against the phytopathogenic oomycete P.sojae remains unclear.In this study,a novel member of the soybean SKP1 gene family,GmSKP1 which was significantly induced by P.sojae,was reported.The expression of GmSKP1 was simultaneously induced by methyl jasmonate(MeJA),salicylic acid(SA)and ethylene(ET),which might suggest an important role for GmSKP1 of plant in responses to hormone treatments.Functional analysis using GmSKP1 overexpression lines showed that GmSKP1 enhanced resistance to P.sojae in transgenic soybean plants.Further analyses showed that GmSKP1 interacted with a homeodomain-leucine zipper protein transcription factor(GmHDL56)and a WRKY transcription factor(GmWRKY31),which could positively regulate responses to P.sojae in soybean.Importantly,several pathogenesis-related(PR)genes were constitutively activated,including GmPR1a,GmPR2,GmPR3,GmPR4,GmPR5a and GmPR10,in GmSKP1-OE soybean plants.Taken together,these results suggested that GmSKP1 enhanced resistance to P.sojae in soybean,possibly by activating the defense-related PR genes.展开更多
The internal transcribed spacer （ITS） region （ITS1, ITS2 and 5.8S rDNA） of the nuclear ribosomal DNA （nrDNA） was amplified via the PCR method in seventeen different isolates of Phytophthora sojae using the commo...The internal transcribed spacer （ITS） region （ITS1, ITS2 and 5.8S rDNA） of the nuclear ribosomal DNA （nrDNA） was amplified via the PCR method in seventeen different isolates of Phytophthora sojae using the common primers of the ITS of fungi. Around 800 bp- 1,000 bp fragments were obtained based on the DL2000 marker and the sequences of the PCR products were tested. Taking isolate USA as outgroup, the phylogenetic tree was constructed by means of maximum parsimony analysis, and the genetic evolution among isolates was analyzed. The results showed that there is a great difference between the base constitution of ITS 1 and ITS2 among various isolates. The seventeen isolates are classified into three groups, and the isolates from the same region belong to the same group, which shows the variation in geography.展开更多
[ Objective] The paper was to screen the antagonistic strain against Phytophthora sojae with biocontrol potential, and provide basis for searching control measures and designing new control strategies against P. sojae...[ Objective] The paper was to screen the antagonistic strain against Phytophthora sojae with biocontrol potential, and provide basis for searching control measures and designing new control strategies against P. sojae. [ Method] The rhizosphere soil of soybean was collected from three different places in Heilongjiang Province, and various soil microorganisms were isolated. Dual culture method was used to screen the microorganism with antagonistic effect against P. sojae. On this basis, the growth inhibition rate of the microorganism with stronger antagonistic effect against P. sojae was determined, and its control effect against P. sojae was also measured. [ Result] A strain of bacterium with relatively good antagonistic effect was isolated from soil, and named as strain B048. Dual test showed that the growth inhibition rate of antagonistic bacterium 11048 against P. sojac reached 97.5%. Antagonistic endurance tests showed that the width of inhibition zone was still 20.0 mm after dual culture with P. sojac for21 d. In potting experiment, the control effect of B048 against P. sojae was 100%. The antagonistic bacterium was primarily identified to be Bacillus pumilus through morphology and 16S rDNA sequence analysis. [Condusion] The antagonistic bacterium B048 had good prospect to be developed as the biocontrol bacterium against P. sojae.展开更多
载体的构建是建立遗传转化体系的基础。以真核表达载体pcDNA3.1(-)/hygro为基本骨架,构建大豆疫霉菌遗传转化载体,通过限制性内切酶酶切、去磷酸化、连接等基因重组技术,将增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP...载体的构建是建立遗传转化体系的基础。以真核表达载体pcDNA3.1(-)/hygro为基本骨架,构建大豆疫霉菌遗传转化载体,通过限制性内切酶酶切、去磷酸化、连接等基因重组技术,将增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)基因和来自莴苣霜霉菌(Bremia lactucae)的启动子(ham34)、终止子重组到真核表达载体pcDNA3.1(-)/hygro中,经大肠杆菌转化后对转化子进行了酶切验证,为大豆疫霉菌遗传转化体系的建立提供载体。展开更多
Phytophthora sojae Kanfman and Gerdemann （P. sojae） is one of the most prevalent pathogens and causes Phytophthora root rot, which limits soybean production worldwide. Development of resistant cultivars is a cost-ef...Phytophthora sojae Kanfman and Gerdemann （P. sojae） is one of the most prevalent pathogens and causes Phytophthora root rot, which limits soybean production worldwide. Development of resistant cultivars is a cost-effective approach to controlling this disease. In this study, 127 soybean germplasm were evaluated for their responses to Phytophthora sojae strain Pm28 using the hypocotyl inoculation technique, and 49 were found resistant to the strain. The hypocotyl of P1, P2, F1, and F2：3 of two crosses of Ludou 4 （resistant）×Youchu 4 （susceptible） and Cangdou 5 （resistant）×Williams （susceptible） were inoculated with Pm28, and were used to analyze the inheritance of resistance. The population derived from the cross of Ludou 4×Youchu 4 was used to map the resistance gene （designated as Rps9） to a linkage group. 932 pairs of SSR primers were used to detect polymorphism, and seven SSR markers were mapped near the resistance gene. The results showed that the resistance to Pm28 in Ludou 4 and Cangdou 5 was controlled by a single dominant gene Rps9, which was located on the molecular linkage group N between the SSR markers Satt631 （7.5 cM） and Sat_186 （4.3 cM）.展开更多
By investigating occurrence of Phytophthora root rot in fields and isolating P.sojae fromdiseased plants and soils, the distribution of P.sojae in China was surveyed. In addition tonortheast region, P.sojae existed in...By investigating occurrence of Phytophthora root rot in fields and isolating P.sojae fromdiseased plants and soils, the distribution of P.sojae in China was surveyed. In addition tonortheast region, P.sojae existed in Huanghe-Huaihe basin and Yangtze basin too. Eighty- threeisolates of P.sojae isolated from different areas were identified on virulence using 13differential soybean cultivars, abundant virulence diversity was found in P.sojae. The greaterdiversity in virulence of P.sojae was in isolates from soil than from plants. And the greatestvirulence diversity of P.sojae was found in Yangtze basin.展开更多
In order to clarify the differential response of Phytophthora sojae to the seed exudates of host soybean and non-host maize and understand the relationship between seed exudates and host selectivity of Phytophthora so...In order to clarify the differential response of Phytophthora sojae to the seed exudates of host soybean and non-host maize and understand the relationship between seed exudates and host selectivity of Phytophthora sojae, non-host maize Suiyu 23 and susceptible host soybean Sloan seed exudates were collected to measure their influence on mycelial growth, formation and germination of oospores, chemotaxis, encystment and germination of zoospores of Phytophthora sojae. The results showed that nonhost maize seed exudates exhibited repellency to zoospores of Phytophthora sojae, it also could significantly inhibited Phytophthora sojae mycelial growth, formation of oospores compared with the control;compared with host soybean, non-host maize seed exudates could significantly inhibited Phytophthora sojae mycelial growth, formation and germination of oospores, germination of cysts, which indicated that the seed exudates was the critical factor to host selectivity of Phytophthora sojae and the maize seed exudates was closely related to its non-host resistance.展开更多
Soybean root and stem rot caused by Phytophthora sojae is a destructive disease worldwide. Using genetic resistance is an important and major component in the integrated pest management of this disease. To understand ...Soybean root and stem rot caused by Phytophthora sojae is a destructive disease worldwide. Using genetic resistance is an important and major component in the integrated pest management of this disease. To understand molecular mechanisms of root and stem rot resistance in soybeans, the gene and protein expression in hypocotyls and stems of variety Suinong 10 carrying resistance genes Rps1a and Rps2 was investigated by using mRNA differential display reverse transcription PCR and two-dimensional electrophoresis at 0, 0.5, 1, 2, and 4 h after inoculation with P. sojae race 1. The results of the comparison of gene and protein expression showed that at least eight differential fragments at the transcriptional level were related to metabolic pathway, phytoalexin, and signal transduction in defense responses. Sequence analyses indicated that these fragments represented cinnamic acid 4-hydroxylase gene, ATP b gene coding ATP synthase b subunit and ubiquitin-conjugating enzyme gene which upregulated at 0.5 h post inoculation, blue copper protein gene and UDP-N-acetyl-a-D-galactosamine gene which upregulated at 2 h post inoculation, TGA-type basic leucine zipper protein TGA1.1 gene, cyclophilin gene, and 14-3-3 protein gene which upregulated at 4 h post inoculation. Three resistance-related proteins, a-subunit and b-subunit of ATP synthase, and cytochrome P450-like protein, were upregulated at 2 h post inoculation. The results suggested that resistance-related multiple proteins and genes were expressed in the recognition between soybean and P. sojae during zoospore germination, penetration and mycelium growth of P. sojae in soybean.展开更多
To elucidate the differential gene expression patterns in soybeans during infection by Phytophthora sojae,a cDNA library for suppression subtractive hybridization （SSH） was constructed with cDNAs from soybean cultiv...To elucidate the differential gene expression patterns in soybeans during infection by Phytophthora sojae,a cDNA library for suppression subtractive hybridization （SSH） was constructed with cDNAs from soybean cultivar Suinong 10 treated with sterile distilled water as the driver and cDNAs from Suinong 10 inoculated with P.sojae as the tester.A total of 2 067 recombinant colonies from the SSH library were randomly picked,amplified,and sequenced.After discarding 312 poor quality expressed sequence tags （EST）,1 755 high quality ESTs were assembled and edited to 1 384 tentatively unique genes （TUG）,in which,586 showed significant homology to known sequences,and 798 had low homology or no match with the known sequences.A cDNA microarray containing 307 singletons from the 586 TUGs and 222 singletons from the 798 TUGs was developed to characterize differentially expressed cDNAs in the SSH library,and eight cDNAs were identified to be up-regulated after microarray analysis and then confirmed by real-time PCR.They were homologous to the protein 10,and were also related to some proteins in disease resistance response,such as pathogen-related protein,phenylalanine ammonia-lyase,isoflavone reductase,WRKY transcription factor 31,major allergen Pru ar 1,and pleiotropic drug resistance protein 12.Most of the up-regulated cDNAs encode enzymes of phytoalexin biosynthesis and pathogenesis-related proteins involved in plant disease resistance.Here,we fist reported the Pru ar 1 in soybeans.The findings of this research have contributed to better understanding of soybean resistance to P.sojae at the molecular level.展开更多
Soil-borne pathogen Phytophthora sojae is an oomycete that causes devastating damage to soybean yield. To mine original resistant genes in soybean is an effective and environmentally-friend approach controlling the di...Soil-borne pathogen Phytophthora sojae is an oomycete that causes devastating damage to soybean yield. To mine original resistant genes in soybean is an effective and environmentally-friend approach controlling the disease. In this study, soybean proteins were extracted from the first trifoliolates infected by predominant P. sojae race 1 and analyzed by twodimensional gel electrophoresis. Nineteen differently-expressed protein spots were detected, and 10 of them were further applied for Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry Assay. One protein containing a dirigent （DIR） domain was identified and belonged to the DIR-b/d family. Therefore, it was named as GmDRR1 （Glycine max Disease Resistance Response 1）. Then, GmDRR1 gene was pathologically confirmed to be involved in the resistant to P. sojae in soybean. GmDRR1-GFP （green fluorescent protein） fusion proteins localized in the cell membrane. qRTPCR results showed GmDRR1 gene expressed differently in P. sojae resistant- and susceptible-soybean cultivars. By the promoter analysis, we found a haplotype H8 was existing in most resistant soybean varieties, while a haplotype H77 was existing in most susceptible soybean varieties. The H77 haplotype had seven SNPs （C to A, G to C, C to A, T to A, T to C, T to C, and T to A） and two single nucleotide insertions. The results supported that the expression difference of GmDRR1 genes between P. sojae resistant- and susceptible-soybean cultivars might depend on the GmDRR1 promoter SNPs. The results suggested that GmDRR1 was a dirigent protein involved in soybean resistant to P. sojae and paved a novel way for investigation of the molecular regulatory mechanism of the defense response to P. sojae in soybean.展开更多
Phytophthora sojae infection severely impairs soybean production. We previously identified a dirigent protein, Gm DRR1(Glycine max Disease Resistant Response 1), that increases soybean resistance to P.sojae. However, ...Phytophthora sojae infection severely impairs soybean production. We previously identified a dirigent protein, Gm DRR1(Glycine max Disease Resistant Response 1), that increases soybean resistance to P.sojae. However, the molecular basis of Gm DRR1 function remained largely uncharacterized. In the present study, analysis of Gm DRR1-RNAi, Gm DRR1-overexpressing, and CRISPR/Cas9-derived Gmdrr1 mutant lines revealed that Gm DRR1 expression significantly restricted P. sojae growth. Combining coimmunoprecipitation with liquid chromatography–tandem mass spectrometry revealed a Gm DRR1-interacting protein, Gm DRR2, which is homologous to Gm DRR1. An E-coniferyl alcohol coupling assay indicated that Gm DRR1 promotes the synthesis of(+)-pinoresinol, which helps to protect plants from P. sojae. The Gm NAC1(Glyma.05 G025500) transcription factor bound to the Gm DRR1 promoter both in vitro and in vivo to upregulate Gm DRR1 expression. Soybean resistance to P. sojae was increased by overexpression of Gm NAC1. Our findings suggest a novel signaling pathway involving a NAC transcription factor that mediates soybean resistance to P. sojae. Specifically, Gm NAC1 directly induces Gm DRR1 expression to increase resistance of soybean plants to P. sojae.展开更多
Soybean Phytophthora root rot (Phytophthora sojae) is a severe disease all over the world. Soybean germplasm from central and southern China for resistance has been evaluated by American researchers on a large scale. ...Soybean Phytophthora root rot (Phytophthora sojae) is a severe disease all over the world. Soybean germplasm from central and southern China for resistance has been evaluated by American researchers on a large scale. P. sojae has been found frequently in northeast of China in recent years, but not systematic evaluation of soybean germplasm for resistance has occurred there. By means of hypocotyl inoculation, 922 cultivars/lines from northeast of China were screened and evaluated for their response to race 1, and 25 of P. sojae. Generally resistance was less frequent in northeast of China than in central and southern China. Five cultivars/lines were identified that confer resistant responses to race 1, 3, 8, 25 and four additional isolates of P. sojae. These cultivars/lines may provide valuable sources of resistance for future breeding programs.展开更多
To estimate the impact of crop rotation on the pathotype and genetic structure of Phythophthora sojae in fields, 372 isolates of P. sojae were obtained from long-term localisation experimental fields in Heilongjiang P...To estimate the impact of crop rotation on the pathotype and genetic structure of Phythophthora sojae in fields, 372 isolates of P. sojae were obtained from long-term localisation experimental fields in Heilongjiang Province of China. The hypocotyl inoculation method was used to characterize the virulence of P. sojae on 13 differential cultivars, and the amplified fragment length polymorphism（AFLP） technique was used to analyze difference in the genetic structure of P. sojae. The results indicated that an abundant diversity of genetic structures and pathotypes of P. sojae, a more uniform distribution of pathotypes and less dominance of pathotypes occurred in corn-soybean and wheat-soybean rotation fields than in a continuous soybean mono-cropping field. These findings suggested that P. sojae did not easily become the dominant race in rotation fields, which maintain disease resistance in soybean varieties. Therefore, the results of this study suggested that Phytophthora stem and root rot of soybeans could be effectively controlled by rotating soybeans with non-host crops of corn and wheat.展开更多
Plants can be infected by multiple pathogens concurrently in natural systems. However,pathogen–pathogen interactions have rarely been studied. In addition to the oomycete Phytophthora sojae, fungi such as Fusarium sp...Plants can be infected by multiple pathogens concurrently in natural systems. However,pathogen–pathogen interactions have rarely been studied. In addition to the oomycete Phytophthora sojae, fungi such as Fusarium spp. also cause soybean root rot. In a 3-year field investigation, we discovered that P. sojae and Fusarium spp. frequently coexisted in diseased soybean roots. Out of 336 P. sojae–soybean–Fusarium combinations,more than 80% aggravated disease. Different Fusarium species all enhanced P. sojae infection when co-inoculated on soybean. Treatment with Fusarium secreted non-proteinaceous metabolites had an effect equal to the direct pathogen coinoculation. By screening a Fusarium graminearum mutant library, we identified Fusarium promoting factor of Phytophthora sojae infection 1(Fpp1),encoding a zinc alcohol dehydrogenase. Fpp1 is functionally conserved in Fusarium and contributes to metabolite-mediated infection promotion, in which vitamin B6(VB6) produced by Fusarium is key. Transcriptional and functional analyses revealed that Fpp1 regulates two VB6 metabolism genes, and VB6 suppresses expression of soybean disease resistance-related genes. These results reveal that co-infection with Fusarium promotes loss of P. sojae resistance in soybean, information that will inform the sustainable use of diseaseresistant crop varieties and provide new strategies to control soybean root rot.展开更多
Leucine-rich repeat receptor-like kinases(LRR-RLKs)are well known to act in plant growth,development,and defense responses.Plant LRR-RLKs locate on cell surface to sense and initiate responsive signals to a variety of...Leucine-rich repeat receptor-like kinases(LRR-RLKs)are well known to act in plant growth,development,and defense responses.Plant LRR-RLKs locate on cell surface to sense and initiate responsive signals to a variety of extracellular stimuli,such as microbe-associated molecular patterns(MAMPs)released from microorganisms.LRR-RLKs are also present in microbes and function in microbial growth and development,but their roles in communicating with hosts are largely unknown.A recent study published in Nature Communications uncovered that a microbial LRR-RLK,PsRLK6,is required for oospore development in the sexual reproduction of Phytophthora sojae,an oomycete pathogen that causes root and stem rot in soybean.Meanwhile,PsRLK6 is recognized as a novel type of MAMP by an unknown plant LRR receptor-like protein and triggers immune responses in soybean,tomato,and Nicotiana benthamiana.The findings reveal dual roles of a pathogen LRR-RLK in determining both life through sexual reproduction and death through triggering plant immunity.展开更多
A strong fibrinolytic activity was demonstrated in the Semen Sojae Praeparatum(SSP), which is a famous traditional Chinese medicine. To study the activities and dynamic changes of fibrinolytic enzyme, standard fibrin ...A strong fibrinolytic activity was demonstrated in the Semen Sojae Praeparatum(SSP), which is a famous traditional Chinese medicine. To study the activities and dynamic changes of fibrinolytic enzyme, standard fibrin plate was used to determine the fibrinolytic activity. For the first time fibrinolytic enzyme was found during the fermentation of SSP and the fibrinolytic activities of samples were shown to increase significantly over time. In the "yellow cladding" stage, the fibrinolytic activity was 619.75 IU/g. On day 6, 12 and 15 of the "secondary fermentation" stage, the fibrinolytic activity was 711.49 IU/g, 866.67 IU/g, 1 022.31 IU/g, respectively. The results indicate that fibrinolytic enzyme was generated during the fermentation of SSP and it displayed increasing activity which peaked at the "secondary fermentation" stage. The fibrinolytic enzyme was found to not only degrades fibrin directly, but also activate plasminogen to do so.展开更多
Plants secrete defense molecules into the extracellular space (the apoplast) to combat attacking microbes. However, the mechanisms by which successful pathogens subvert plant apoplastic immunity remain poorly understo...Plants secrete defense molecules into the extracellular space (the apoplast) to combat attacking microbes. However, the mechanisms by which successful pathogens subvert plant apoplastic immunity remain poorly understood. In this study, we show that PsAvh240, a membrane-localized effector of the soybean pathogen Phytophthora sojae, promotes P. sojae infection in soybean hairy roots. We found that PsAvh240 interacts with the soybean-resistant aspartic protease GmAP1 in planta and suppresses the secretion of GmAP1 into the apoplast. By solving its crystal structure we revealed that PsAvh240 contain six a helices and two WY motifs. The first two a helices of PsAvh240 are responsible for its plasma membrane-localization and are required for PsAvh240's interaction with GmAP1. The second WY motifs of two PsAvh240 molecules form a handshake arrangement resulting in a handshake-like dimer. This dimerization is required for the effector's repression of GmAP1 secretion. Taken together, these data reveal that PsAvh240 localizes at the plasma membrane to interfere with GmAP1 secretion, which represents an effective mechanism by which effector proteins suppress plant apoplastic immunity.展开更多
This paper reported the contents variation analysis ofγ-amino butyric acid(GABA)in Semen sojae praeparatum(SSP)which is a famous traditional Chinese medicine.High performance liquid chromatography(HPLC)was used and G...This paper reported the contents variation analysis ofγ-amino butyric acid(GABA)in Semen sojae praeparatum(SSP)which is a famous traditional Chinese medicine.High performance liquid chromatography(HPLC)was used and GABA was derivatized by online pre-column derivatization with o-phthalaldehyde(OPA).To validate this method,the precision,stability,repeatability and recovery were discussed.In the concentration range from 0.0125 to 0.400 mg/m L,the calibration curve for GABA was linear and the regression equation was obtained with correlation coefficient(R2)of 0.9999.Relatively high levels of GABA exist in SSP and the content changes of GABA at different time points during the fermenting process were detected.At the"yellow cladding"stage,GABA level was very low or even undetectable;the"secondary fermentation"stage witnessed a rapid increase of GABA content to 1.39-5.52 mg/g,which remained stable after 18 days of"secondary fermentation".This study demonstrated that GABA was generated at the"secondary fermentation"stage,revealing the significance and rationality of the"secondary fermentation"stage in the fermenting process of SSP.On the other hand,it suggested the downside of taking soy isoflavones as the only measurement in existing quality assessment and optimization approach for the fermenting process of SSP.展开更多
基金Supported by the NSFC Projects(31971972)the Natural Science Foundation of Heilongjiang Province(ZD2019C001)the Outstanding Talents and Innovative Team of Agricultural Scientific Research。
文摘Phytophthora root and stem rot of soybean caused by Phytophthora sojae(P.sojae)is a devastating disease that affects soybean[Glycine max(L.)Merr.]all over the world.S-phase kinase-associated protein 1(SKP1)proteins are key members of the SKP1/Cullin/F-box protein(SCF)ubiquitin ligase complex and play diverse roles in plant biology.However,the role of SKP1 in soybean against the phytopathogenic oomycete P.sojae remains unclear.In this study,a novel member of the soybean SKP1 gene family,GmSKP1 which was significantly induced by P.sojae,was reported.The expression of GmSKP1 was simultaneously induced by methyl jasmonate(MeJA),salicylic acid(SA)and ethylene(ET),which might suggest an important role for GmSKP1 of plant in responses to hormone treatments.Functional analysis using GmSKP1 overexpression lines showed that GmSKP1 enhanced resistance to P.sojae in transgenic soybean plants.Further analyses showed that GmSKP1 interacted with a homeodomain-leucine zipper protein transcription factor(GmHDL56)and a WRKY transcription factor(GmWRKY31),which could positively regulate responses to P.sojae in soybean.Importantly,several pathogenesis-related(PR)genes were constitutively activated,including GmPR1a,GmPR2,GmPR3,GmPR4,GmPR5a and GmPR10,in GmSKP1-OE soybean plants.Taken together,these results suggested that GmSKP1 enhanced resistance to P.sojae in soybean,possibly by activating the defense-related PR genes.
基金This work was supported by National Natural Science Fundation of China (No.30400285, 30671317), Postdoctoral Grant from Ag-riculture Sciences Academy of Heilongjiang Province (No. LRB06-010), China Postdoctoral Grant, Item for Teachers from Heilongjiang University (No. 140022), Young People’s Science Fund of Heilongjiang Province (No. QC06C012), 973(No. 2004CB117203-4), the Opening Fund of Key Opening Laboratory of Physiology and Ecology of Crop in Cold Terra of Agriculture Ministry "the Cloning and Mapping of cDNA Sequence from Related Gene Resistant to Phytophthora sojae and International Tech-nology Cooperation Item (No. 2005DFA30340).
文摘The internal transcribed spacer （ITS） region （ITS1, ITS2 and 5.8S rDNA） of the nuclear ribosomal DNA （nrDNA） was amplified via the PCR method in seventeen different isolates of Phytophthora sojae using the common primers of the ITS of fungi. Around 800 bp- 1,000 bp fragments were obtained based on the DL2000 marker and the sequences of the PCR products were tested. Taking isolate USA as outgroup, the phylogenetic tree was constructed by means of maximum parsimony analysis, and the genetic evolution among isolates was analyzed. The results showed that there is a great difference between the base constitution of ITS 1 and ITS2 among various isolates. The seventeen isolates are classified into three groups, and the isolates from the same region belong to the same group, which shows the variation in geography.
基金Supported by National Natural Science Foundation of China(30800040)Excellent Youth Science and Technology Fund of Anhui Province(10040606Y04)教育部留学回国人员科研启动基金资助项目~~
文摘[ Objective] The paper was to screen the antagonistic strain against Phytophthora sojae with biocontrol potential, and provide basis for searching control measures and designing new control strategies against P. sojae. [ Method] The rhizosphere soil of soybean was collected from three different places in Heilongjiang Province, and various soil microorganisms were isolated. Dual culture method was used to screen the microorganism with antagonistic effect against P. sojae. On this basis, the growth inhibition rate of the microorganism with stronger antagonistic effect against P. sojae was determined, and its control effect against P. sojae was also measured. [ Result] A strain of bacterium with relatively good antagonistic effect was isolated from soil, and named as strain B048. Dual test showed that the growth inhibition rate of antagonistic bacterium 11048 against P. sojac reached 97.5%. Antagonistic endurance tests showed that the width of inhibition zone was still 20.0 mm after dual culture with P. sojac for21 d. In potting experiment, the control effect of B048 against P. sojae was 100%. The antagonistic bacterium was primarily identified to be Bacillus pumilus through morphology and 16S rDNA sequence analysis. [Condusion] The antagonistic bacterium B048 had good prospect to be developed as the biocontrol bacterium against P. sojae.
文摘载体的构建是建立遗传转化体系的基础。以真核表达载体pcDNA3.1(-)/hygro为基本骨架,构建大豆疫霉菌遗传转化载体,通过限制性内切酶酶切、去磷酸化、连接等基因重组技术,将增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)基因和来自莴苣霜霉菌(Bremia lactucae)的启动子(ham34)、终止子重组到真核表达载体pcDNA3.1(-)/hygro中,经大肠杆菌转化后对转化子进行了酶切验证,为大豆疫霉菌遗传转化体系的建立提供载体。
基金supported by the Earmarked Fund for Modern Agro-Industry Technology Research System, China (nyhyzx07-053)the Program for Changjiang Scholars and Innovative Research Team in University, China (PCSIRT)the Research Fund for the Doctoral Program of Higher Education of China (20090097120023)
文摘Phytophthora sojae Kanfman and Gerdemann （P. sojae） is one of the most prevalent pathogens and causes Phytophthora root rot, which limits soybean production worldwide. Development of resistant cultivars is a cost-effective approach to controlling this disease. In this study, 127 soybean germplasm were evaluated for their responses to Phytophthora sojae strain Pm28 using the hypocotyl inoculation technique, and 49 were found resistant to the strain. The hypocotyl of P1, P2, F1, and F2：3 of two crosses of Ludou 4 （resistant）×Youchu 4 （susceptible） and Cangdou 5 （resistant）×Williams （susceptible） were inoculated with Pm28, and were used to analyze the inheritance of resistance. The population derived from the cross of Ludou 4×Youchu 4 was used to map the resistance gene （designated as Rps9） to a linkage group. 932 pairs of SSR primers were used to detect polymorphism, and seven SSR markers were mapped near the resistance gene. The results showed that the resistance to Pm28 in Ludou 4 and Cangdou 5 was controlled by a single dominant gene Rps9, which was located on the molecular linkage group N between the SSR markers Satt631 （7.5 cM） and Sat_186 （4.3 cM）.
基金supported by the National Natural Science Foundation of China(39970497)Nationa1 Basic Work Program of China(2001BA509B0608).
文摘By investigating occurrence of Phytophthora root rot in fields and isolating P.sojae fromdiseased plants and soils, the distribution of P.sojae in China was surveyed. In addition tonortheast region, P.sojae existed in Huanghe-Huaihe basin and Yangtze basin too. Eighty- threeisolates of P.sojae isolated from different areas were identified on virulence using 13differential soybean cultivars, abundant virulence diversity was found in P.sojae. The greaterdiversity in virulence of P.sojae was in isolates from soil than from plants. And the greatestvirulence diversity of P.sojae was found in Yangtze basin.
基金Supported by the National Natural Science Foundation of China(31670444 31370449)
文摘In order to clarify the differential response of Phytophthora sojae to the seed exudates of host soybean and non-host maize and understand the relationship between seed exudates and host selectivity of Phytophthora sojae, non-host maize Suiyu 23 and susceptible host soybean Sloan seed exudates were collected to measure their influence on mycelial growth, formation and germination of oospores, chemotaxis, encystment and germination of zoospores of Phytophthora sojae. The results showed that nonhost maize seed exudates exhibited repellency to zoospores of Phytophthora sojae, it also could significantly inhibited Phytophthora sojae mycelial growth, formation of oospores compared with the control;compared with host soybean, non-host maize seed exudates could significantly inhibited Phytophthora sojae mycelial growth, formation and germination of oospores, germination of cysts, which indicated that the seed exudates was the critical factor to host selectivity of Phytophthora sojae and the maize seed exudates was closely related to its non-host resistance.
基金supported by the Commonweal Specialized Research Fund of China Agriculture (3-20,201103015)
文摘Soybean root and stem rot caused by Phytophthora sojae is a destructive disease worldwide. Using genetic resistance is an important and major component in the integrated pest management of this disease. To understand molecular mechanisms of root and stem rot resistance in soybeans, the gene and protein expression in hypocotyls and stems of variety Suinong 10 carrying resistance genes Rps1a and Rps2 was investigated by using mRNA differential display reverse transcription PCR and two-dimensional electrophoresis at 0, 0.5, 1, 2, and 4 h after inoculation with P. sojae race 1. The results of the comparison of gene and protein expression showed that at least eight differential fragments at the transcriptional level were related to metabolic pathway, phytoalexin, and signal transduction in defense responses. Sequence analyses indicated that these fragments represented cinnamic acid 4-hydroxylase gene, ATP b gene coding ATP synthase b subunit and ubiquitin-conjugating enzyme gene which upregulated at 0.5 h post inoculation, blue copper protein gene and UDP-N-acetyl-a-D-galactosamine gene which upregulated at 2 h post inoculation, TGA-type basic leucine zipper protein TGA1.1 gene, cyclophilin gene, and 14-3-3 protein gene which upregulated at 4 h post inoculation. Three resistance-related proteins, a-subunit and b-subunit of ATP synthase, and cytochrome P450-like protein, were upregulated at 2 h post inoculation. The results suggested that resistance-related multiple proteins and genes were expressed in the recognition between soybean and P. sojae during zoospore germination, penetration and mycelium growth of P. sojae in soybean.
基金supported by the Program for New Century Excellent Talents in Universities,Ministry of Education,China(NCET-09-164)the National Natural Science Foundation of China(30671317,30971811,31071439,and 31110103001)+1 种基金the Program for New Century Excellent Talents in Universities in Heilongjiang Province,China(NCET-06-007)the Natural Science Foundation of Heilongjiang Province,China(C200814)
文摘To elucidate the differential gene expression patterns in soybeans during infection by Phytophthora sojae,a cDNA library for suppression subtractive hybridization （SSH） was constructed with cDNAs from soybean cultivar Suinong 10 treated with sterile distilled water as the driver and cDNAs from Suinong 10 inoculated with P.sojae as the tester.A total of 2 067 recombinant colonies from the SSH library were randomly picked,amplified,and sequenced.After discarding 312 poor quality expressed sequence tags （EST）,1 755 high quality ESTs were assembled and edited to 1 384 tentatively unique genes （TUG）,in which,586 showed significant homology to known sequences,and 798 had low homology or no match with the known sequences.A cDNA microarray containing 307 singletons from the 586 TUGs and 222 singletons from the 798 TUGs was developed to characterize differentially expressed cDNAs in the SSH library,and eight cDNAs were identified to be up-regulated after microarray analysis and then confirmed by real-time PCR.They were homologous to the protein 10,and were also related to some proteins in disease resistance response,such as pathogen-related protein,phenylalanine ammonia-lyase,isoflavone reductase,WRKY transcription factor 31,major allergen Pru ar 1,and pleiotropic drug resistance protein 12.Most of the up-regulated cDNAs encode enzymes of phytoalexin biosynthesis and pathogenesis-related proteins involved in plant disease resistance.Here,we fist reported the Pru ar 1 in soybeans.The findings of this research have contributed to better understanding of soybean resistance to P.sojae at the molecular level.
基金financially supported by the Academic Skeleton Support Plan of Department of Education of Heilongjiang Province,China (1254G011)the National Natural Science Foundation of China (31271747,31471516,31400074,31401465,31501332)+3 种基金the National High-Tech R&D Program of China (the 863 Program,2013AA102602)the Research Fund for the Doctoral Program of Higher Education of China (20122325120015)the Academic Backbone Project of Northeast Agricultural University,China (15XG02)the Talented Young Project of Northeast Agricultural University,China (518062)
文摘Soil-borne pathogen Phytophthora sojae is an oomycete that causes devastating damage to soybean yield. To mine original resistant genes in soybean is an effective and environmentally-friend approach controlling the disease. In this study, soybean proteins were extracted from the first trifoliolates infected by predominant P. sojae race 1 and analyzed by twodimensional gel electrophoresis. Nineteen differently-expressed protein spots were detected, and 10 of them were further applied for Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry Assay. One protein containing a dirigent （DIR） domain was identified and belonged to the DIR-b/d family. Therefore, it was named as GmDRR1 （Glycine max Disease Resistance Response 1）. Then, GmDRR1 gene was pathologically confirmed to be involved in the resistant to P. sojae in soybean. GmDRR1-GFP （green fluorescent protein） fusion proteins localized in the cell membrane. qRTPCR results showed GmDRR1 gene expressed differently in P. sojae resistant- and susceptible-soybean cultivars. By the promoter analysis, we found a haplotype H8 was existing in most resistant soybean varieties, while a haplotype H77 was existing in most susceptible soybean varieties. The H77 haplotype had seven SNPs （C to A, G to C, C to A, T to A, T to C, T to C, and T to A） and two single nucleotide insertions. The results supported that the expression difference of GmDRR1 genes between P. sojae resistant- and susceptible-soybean cultivars might depend on the GmDRR1 promoter SNPs. The results suggested that GmDRR1 was a dirigent protein involved in soybean resistant to P. sojae and paved a novel way for investigation of the molecular regulatory mechanism of the defense response to P. sojae in soybean.
基金supported by the National Natural Science Foundation of China(U20A2027,32070274,32072014)the Heilongjiang Postdoctoral Science Foundation(LBH-Q16014)。
文摘Phytophthora sojae infection severely impairs soybean production. We previously identified a dirigent protein, Gm DRR1(Glycine max Disease Resistant Response 1), that increases soybean resistance to P.sojae. However, the molecular basis of Gm DRR1 function remained largely uncharacterized. In the present study, analysis of Gm DRR1-RNAi, Gm DRR1-overexpressing, and CRISPR/Cas9-derived Gmdrr1 mutant lines revealed that Gm DRR1 expression significantly restricted P. sojae growth. Combining coimmunoprecipitation with liquid chromatography–tandem mass spectrometry revealed a Gm DRR1-interacting protein, Gm DRR2, which is homologous to Gm DRR1. An E-coniferyl alcohol coupling assay indicated that Gm DRR1 promotes the synthesis of(+)-pinoresinol, which helps to protect plants from P. sojae. The Gm NAC1(Glyma.05 G025500) transcription factor bound to the Gm DRR1 promoter both in vitro and in vivo to upregulate Gm DRR1 expression. Soybean resistance to P. sojae was increased by overexpression of Gm NAC1. Our findings suggest a novel signaling pathway involving a NAC transcription factor that mediates soybean resistance to P. sojae. Specifically, Gm NAC1 directly induces Gm DRR1 expression to increase resistance of soybean plants to P. sojae.
文摘Soybean Phytophthora root rot (Phytophthora sojae) is a severe disease all over the world. Soybean germplasm from central and southern China for resistance has been evaluated by American researchers on a large scale. P. sojae has been found frequently in northeast of China in recent years, but not systematic evaluation of soybean germplasm for resistance has occurred there. By means of hypocotyl inoculation, 922 cultivars/lines from northeast of China were screened and evaluated for their response to race 1, and 25 of P. sojae. Generally resistance was less frequent in northeast of China than in central and southern China. Five cultivars/lines were identified that confer resistant responses to race 1, 3, 8, 25 and four additional isolates of P. sojae. These cultivars/lines may provide valuable sources of resistance for future breeding programs.
基金Supported by the Special Fund for Agro-scientific Research in the Public Interest(201303018)the National Natural Science Foundation of China(31370449)
文摘To estimate the impact of crop rotation on the pathotype and genetic structure of Phythophthora sojae in fields, 372 isolates of P. sojae were obtained from long-term localisation experimental fields in Heilongjiang Province of China. The hypocotyl inoculation method was used to characterize the virulence of P. sojae on 13 differential cultivars, and the amplified fragment length polymorphism（AFLP） technique was used to analyze difference in the genetic structure of P. sojae. The results indicated that an abundant diversity of genetic structures and pathotypes of P. sojae, a more uniform distribution of pathotypes and less dominance of pathotypes occurred in corn-soybean and wheat-soybean rotation fields than in a continuous soybean mono-cropping field. These findings suggested that P. sojae did not easily become the dominant race in rotation fields, which maintain disease resistance in soybean varieties. Therefore, the results of this study suggested that Phytophthora stem and root rot of soybeans could be effectively controlled by rotating soybeans with non-host crops of corn and wheat.
基金supported by grants from the National Natural Science Foundation of China (3217237431721004)the China Agriculture Research System (CARS-004-PS14)。
文摘Plants can be infected by multiple pathogens concurrently in natural systems. However,pathogen–pathogen interactions have rarely been studied. In addition to the oomycete Phytophthora sojae, fungi such as Fusarium spp. also cause soybean root rot. In a 3-year field investigation, we discovered that P. sojae and Fusarium spp. frequently coexisted in diseased soybean roots. Out of 336 P. sojae–soybean–Fusarium combinations,more than 80% aggravated disease. Different Fusarium species all enhanced P. sojae infection when co-inoculated on soybean. Treatment with Fusarium secreted non-proteinaceous metabolites had an effect equal to the direct pathogen coinoculation. By screening a Fusarium graminearum mutant library, we identified Fusarium promoting factor of Phytophthora sojae infection 1(Fpp1),encoding a zinc alcohol dehydrogenase. Fpp1 is functionally conserved in Fusarium and contributes to metabolite-mediated infection promotion, in which vitamin B6(VB6) produced by Fusarium is key. Transcriptional and functional analyses revealed that Fpp1 regulates two VB6 metabolism genes, and VB6 suppresses expression of soybean disease resistance-related genes. These results reveal that co-infection with Fusarium promotes loss of P. sojae resistance in soybean, information that will inform the sustainable use of diseaseresistant crop varieties and provide new strategies to control soybean root rot.
基金supported by grants from the National Natural Science Foundation of China(32372490 to J.F.)the Sichuan Applied Fundamental Research Foundation(2022JDTD0023 to J.F.)the Natural Science Foundation of Sichuan Province(2022NSFSC0174 to H.W.).
文摘Leucine-rich repeat receptor-like kinases(LRR-RLKs)are well known to act in plant growth,development,and defense responses.Plant LRR-RLKs locate on cell surface to sense and initiate responsive signals to a variety of extracellular stimuli,such as microbe-associated molecular patterns(MAMPs)released from microorganisms.LRR-RLKs are also present in microbes and function in microbial growth and development,but their roles in communicating with hosts are largely unknown.A recent study published in Nature Communications uncovered that a microbial LRR-RLK,PsRLK6,is required for oospore development in the sexual reproduction of Phytophthora sojae,an oomycete pathogen that causes root and stem rot in soybean.Meanwhile,PsRLK6 is recognized as a novel type of MAMP by an unknown plant LRR receptor-like protein and triggers immune responses in soybean,tomato,and Nicotiana benthamiana.The findings reveal dual roles of a pathogen LRR-RLK in determining both life through sexual reproduction and death through triggering plant immunity.
基金Supported by the National Natural Science Foundation of China(81660664)the Natural Science Foundation of Jiangxi Province(20192ACBL21032,20192BBGL70051,20192BAB205098,20171BAB21 5061,GJJ150844,GJJ160858)+1 种基金Jiangxi Provincial Health and Family Planning Commission(2017Z016)the Natural Science Foundation of Jiangxi University of Traditional Chinese Medicine(2014BS013).
文摘A strong fibrinolytic activity was demonstrated in the Semen Sojae Praeparatum(SSP), which is a famous traditional Chinese medicine. To study the activities and dynamic changes of fibrinolytic enzyme, standard fibrin plate was used to determine the fibrinolytic activity. For the first time fibrinolytic enzyme was found during the fermentation of SSP and the fibrinolytic activities of samples were shown to increase significantly over time. In the "yellow cladding" stage, the fibrinolytic activity was 619.75 IU/g. On day 6, 12 and 15 of the "secondary fermentation" stage, the fibrinolytic activity was 711.49 IU/g, 866.67 IU/g, 1 022.31 IU/g, respectively. The results indicate that fibrinolytic enzyme was generated during the fermentation of SSP and it displayed increasing activity which peaked at the "secondary fermentation" stage. The fibrinolytic enzyme was found to not only degrades fibrin directly, but also activate plasminogen to do so.
基金supported by grants to Yuanchao Wang from the China National Funds for Innovative Research Groups(31721004)the key program of the National Natural Science Foundation of China(31430073)+2 种基金the Chinese Modern Agricultural Industry Technology System(CARS-004-PS14)the National Key R&D Program of China(SQ2018YFD020042)Research in the W.X.laboratory is supported by the Chinese Thousand Talents Plan and the Chinese Academy of Sciences.B.G.is supported by the Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX18.0662).
文摘Plants secrete defense molecules into the extracellular space (the apoplast) to combat attacking microbes. However, the mechanisms by which successful pathogens subvert plant apoplastic immunity remain poorly understood. In this study, we show that PsAvh240, a membrane-localized effector of the soybean pathogen Phytophthora sojae, promotes P. sojae infection in soybean hairy roots. We found that PsAvh240 interacts with the soybean-resistant aspartic protease GmAP1 in planta and suppresses the secretion of GmAP1 into the apoplast. By solving its crystal structure we revealed that PsAvh240 contain six a helices and two WY motifs. The first two a helices of PsAvh240 are responsible for its plasma membrane-localization and are required for PsAvh240's interaction with GmAP1. The second WY motifs of two PsAvh240 molecules form a handshake arrangement resulting in a handshake-like dimer. This dimerization is required for the effector's repression of GmAP1 secretion. Taken together, these data reveal that PsAvh240 localizes at the plasma membrane to interfere with GmAP1 secretion, which represents an effective mechanism by which effector proteins suppress plant apoplastic immunity.
基金the National Natural Science Foundation of China(81660664,82060709,82060699)the Natural Science Foundation of Jiangxi Province(20192ACBL21032,20192BBGL70051)China Scholarship Council(201908360259)。
文摘This paper reported the contents variation analysis ofγ-amino butyric acid(GABA)in Semen sojae praeparatum(SSP)which is a famous traditional Chinese medicine.High performance liquid chromatography(HPLC)was used and GABA was derivatized by online pre-column derivatization with o-phthalaldehyde(OPA).To validate this method,the precision,stability,repeatability and recovery were discussed.In the concentration range from 0.0125 to 0.400 mg/m L,the calibration curve for GABA was linear and the regression equation was obtained with correlation coefficient(R2)of 0.9999.Relatively high levels of GABA exist in SSP and the content changes of GABA at different time points during the fermenting process were detected.At the"yellow cladding"stage,GABA level was very low or even undetectable;the"secondary fermentation"stage witnessed a rapid increase of GABA content to 1.39-5.52 mg/g,which remained stable after 18 days of"secondary fermentation".This study demonstrated that GABA was generated at the"secondary fermentation"stage,revealing the significance and rationality of the"secondary fermentation"stage in the fermenting process of SSP.On the other hand,it suggested the downside of taking soy isoflavones as the only measurement in existing quality assessment and optimization approach for the fermenting process of SSP.
