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Races of Phytophthora sojae in Iran  [PDF]
Abbas Mohammadi,Azizollah Alizadeh,Mansore Mirabolfathey,Nasrin Nooras Mofrad
Pakistan Journal of Biological Sciences , 2008,
Abstract: Phytophthora root and stem rot of soybean is a destructive disease of soybean in Iran. Races 1 and 3 of pathogen have already been reported from two major growing regions of the crop, Lorestan and Golestan provinces. In a survey during 2004-2005, 142 isolates of P. sojae were recovered from infected plants and naturally infested soil samples using selective media and soybean leaf baiting technique. The majority of tested isolates (110 isolates) belonged to race one of P. sojae and 32 isolates belonged to race 3. ITS region of 23 isolates were amplified with specific primers Ps1 and Ps2. Sequences of this regions were similar to other gene banks sequences except two isolates from China. This survey showed low diversity in Iranian population of P. sojae.
Molecular Detection of Phytophthora sojae

LIU Chun-Lai,YANG Ming-Xiu,WEN Jing-Zhi,

微生物学通报 , 2007,
Abstract: An oligonucleotide primer pair was designed and synthesized after comparison and homological analysis of rDNA ITS sequences among Phytophthora sojae,its related Phytophthora species,and allied fungal and bacterial species from GenBank.PCR amplifications were carried out for 140 isolates including Phytophthora sojae.It showed that only isolates of Phytophthora sojae can be amplified and a special fragment of 288bp were produced by the primers.These primers were used to detect Phytophthora sojae in pure culture,inoculated diseased soybean plants,and inoculated soil samples.The detection protocol has good sensitivity to diseased tissues.
大豆疫霉Phytophthora sojae卵孢子在黑龙江省土壤中的越冬存活率
Overwintering survival rate of Phytophthora sojae oospores in soils in Heilongjiang Province

陈秋明,肖彩霞,孙欠欠,文景芝,Chen Qiuming,Xiao Caixia,Sun Qianqian,Wen Jingzhi
- , 2015, DOI: 10.13802/j.cnki.zwbhxb.2015.01.011
Abstract: 为探究大豆疫霉 Phytophthora sojae卵孢子在黑龙江省土壤中的越冬存活率及其与所处土壤深度和媒介的相关性,以增强型绿色荧光蛋白为报告基因,将培养基及病残体中的大豆疫霉卵孢子分别接种到试验田框栽土壤表层下不同深处,检测其卵孢子的越冬存活率,同时在框栽中定量播种不含任何已知抗疫霉根腐病基因的大豆品种Sloan(rps),苗期调查其发病率。结果表明,大豆疫霉卵孢子在黑龙江省土壤中的适生性较强,可在5~15 cm深度土壤中安全越冬,越冬存活率高达81.67%~96.33%。卵孢子越冬存活率与其所处的越冬媒介关系不大,而与土壤深度有关。在5~15 cm范围内,随着土壤深度的增加,卵孢子越冬存活率增加。处于深层土壤中的卵孢子更容易打破休眠,进入萌发前的萌动状态。各处理间卵孢子越冬存活率的显著性差异并未在发病率上表现出来,说明除了土壤深度外,还有其它因素影响发病率。
In order to investigate the associations between the survival rate of overwintering oospores of Phytophthora sojae and the soil depth and media in which the oospores were in Heilongjiang Province. The oospores in the media and diseased soybean tissues were inoculated at different depths below the soil surface in experimental fields under natural conditions. Some samples were collected randomly to measure the survival rate of oospores under a fluorescent microscope. Meanwhile, the seeds of susceptible soybean cultivar Sloan containing no R gene (rps) to Phytophthora root and stem rot were planted quantificationally in the frames to measure the incidence at seedling stage. The results showed that the oospores of P. sojae could overwinter with a survival rate of 81.67%-96.33%, indicating that they were adapted to the soil environment in Heilongjiang Province. The survival rate of overwintering oospores was related to the soil depth where oospores located, and had little relationship with the media. With the depth increasing, the survival rate of overwintering oospores increased. Oospores that located in deep soils were more likely to break dormancy to germinate. The significant differences in the survival rate of overwintering oospores between different treatments did not express in the seedling incidence, indicating that, in addition to the soil depth, there were other factors influencing the incidence of this disease.
Proteomics study of changes in soybean lines resistant and sensitive to Phytophthora sojae
YuMei Zhang, JinMing Zhao, Yang Xiang, XiaoChun Bian, QiaoMei Zuo, Qi Shen, JunYi Gai, Han Xing
Proteome Science , 2011, DOI: 10.1186/1477-5956-9-52
Abstract: In the present study, 46 differentially expressed proteins were identified in soybean hypocotyls infected with P. sojae, using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization tandem time of flight (MALDI-TOF/TOF). The expression levels of 26 proteins were significantly affected at various time points in the tolerant soybean line, Yudou25, (12 up-regulated and 14 down-regulated). In contrast, in the sensitive soybean line, NG6255, only 20 proteins were significantly affected (11 up-regulated and 9 down-regulated). Among these proteins, 26% were related to energy regulation, 15% to protein destination and storage, 11% to defense against disease, 11% to metabolism, 9% to protein synthesis, 4% to secondary metabolism, and 24% were of unknown function.Our study provides important information on the use of proteomic methods for studying protein regulation during plant-oomycete interactions.Soybean is one of the main sources of edible vegetable oil and high-protein livestock feed [1]. Phytophthora root and stem rot of soybeans, caused by the facultative pathogen Phytophthora sojae, is a serious disease. Each year it causes soybean damage estimated at one to two billion US dollars worldwide [2]. Breeding resistant cultivars is considered the most practical means of controlling this disease.Two types of resistance to phytophthora root rot in soybeans have been described, partial and race-specific resistance. Partial resistance limits the spread of lesions in infected tissues. Race-specific resistance is monogenic and confers immunity or near immunity on the plant through a hypersensitive response. The different physiological pathotypes are governed by an Rps gene [3,4]. Currently, only 14 Rps genes at eight loci (Rps1 to Rps8) are known to confer soybean resistance to P. sojae and these have been designated and mapped to four molecular linkages: F, G, J, and N [5-8]. However, single Rps genes remain effective for 8-15 years [9]. The continuous
土壤环境对大豆疫霉Phytophthora sojae卵孢子萌动的影响
Effects of soil environment on activation of oospores of Phytophthora sojae

所冰,崔人方,田苗,文景芝,Suo Bing,Cui Renfang,Tian Miao,Wen Jingzhi
- , 2015, DOI: 10.13802/j.cnki.zwbhxb.2015.03.013
Abstract: 为明确土壤环境对大豆疫霉Phytophthora sojae卵孢子萌动的影响,将增强型绿色荧光蛋白标记的大豆疫霉卵孢子以2 500个卵孢子/g干土的比例接种于菌黑土中,荧光显微镜下计数卵孢子的萌动率以明确其最适宜的土壤温度和含水量;在此基础上,从5种类型土壤和5种轮作体系土壤中筛选适宜卵孢子萌动的土壤环境.结果表明,25 ℃土壤温度和30%土壤含水量最适合大豆疫霉卵孢子萌动,萌动率为95.78%;黑土和盐碱土分别是最适合和最不适合卵孢子萌动的土壤类型,萌动率分别为94.94%和14.67%;卵孢子萌动率与土壤有机质含量、pH 值和Ca2+含量间明显相关性.大豆连作田和玉米连作田土壤适合卵孢子萌动,萌动率为96.33%和95.00%,小麦连作田土壤不适合卵孢子萌动,萌动率仅为39.33%.
In the present study, the oospores marked by the enhanced green fluorescent protein (EGFP) were inoculated at a certain proportion to the Mollisols through a sterile Eppendorf tube to determine the most suitable soil temperature and water content by calculating the activation rate of oospores under the fluorescent microscope. The same method was used to measure the oospore activation rate in five types of soils and five types of crop rotation soils under the most suitable soil temperature and soil water content to determine the most suitable soil environment. The results showed that the soil temperature of 25 ℃ and the soil water content of 30% were the most suitable for oospore activation, with an activation rate of 95.78%. Mollisols and Halosols were the most suitable and unsuitable soil types, respectively, for the activation of oospores, with an activation rate of 94.94% and 14.67%, respectively. There was no correlation between the activation rate of oospores and pH value, organic matter and Ca2+ content of the soil. Also, the soil from soybean mono-cropping field and corn mono-cropping field were more suitable for the activation of oospores than that from wheat mono-cropping field, with an activation rate of 96.33%, 95.00% and 39.33%, respectively.
Dissection of two soybean QTL conferring partial resistance to Phytophthora sojae through sequence and gene expression analysis  [cached]
Wang Hehe,Wijeratne Asela,Wijeratne Saranga,Lee Sungwoo
BMC Genomics , 2012, DOI: 10.1186/1471-2164-13-428
Abstract: Background Phytophthora sojae is the primary pathogen of soybeans that are grown on poorly drained soils. Race-specific resistance to P. sojae in soybean is gene-for-gene, although in many areas of the US and worldwide there are populations that have adapted to the most commonly deployed resistance to P. sojae ( Rps) genes. Hence, this system has received increased attention towards identifying mechanisms and molecular markers associated with partial resistance to this pathogen. Several quantitative trait loci (QTL) have been identified in the soybean cultivar ‘Conrad’ that contributes to the expression of partial resistance to multiple P. sojae isolates. Results In this study, two of the Conrad QTL on chromosome 19 were dissected through sequence and expression analysis of genes in both resistant (Conrad) and susceptible (‘Sloan’) genotypes. There were 1025 single nucleotide polymorphisms (SNPs) in 87 of 153 genes sequenced from Conrad and Sloan. There were 304 SNPs in 54 genes sequenced from Conrad compared to those from both Sloan and Williams 82, of which 11 genes had SNPs unique to Conrad. Eleven of 19 genes in these regions analyzed with qRT-PCR had significant differences in fold change of transcript abundance in response to infection with P. sojae in lines with QTL haplotype from the resistant parent compared to those with the susceptible parent haplotype. From these, 8 of the 11 genes had SNPs in the upstream, untranslated region, exon, intron, and/or downstream region. These 11 candidate genes encode proteins potentially involved in signal transduction, hormone-mediated pathways, plant cell structural modification, ubiquitination, and basal resistance. Conclusions These findings may indicate a complex defense network with multiple mechanisms underlying these two soybean QTL conferring resistance to P. sojae. SNP markers derived from these candidate genes can contribute to fine mapping of QTL and marker assisted breeding for resistance to P. sojae.
The Phytophthora sojae Avirulence Locus Avr3c Encodes a Multi-Copy RXLR Effector with Sequence Polymorphisms among Pathogen Strains  [PDF]
Suomeng Dong, Dinah Qutob, Jennifer Tedman-Jones, Kuflom Kuflu, Yuanchao Wang, Brett M. Tyler, Mark Gijzen
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005556
Abstract: Root and stem rot disease of soybean is caused by the oomycete Phytophthora sojae. The avirulence (Avr) genes of P. sojae control race-cultivar compatibility. In this study, we identify the P. sojae Avr3c gene and show that it encodes a predicted RXLR effector protein of 220 amino acids. Sequence and transcriptional data were compared for predicted RXLR effectors occurring in the vicinity of Avr4/6, as genetic linkage of Avr3c and Avr4/6 was previously suggested. Mapping of DNA markers in a F2 population was performed to determine whether selected RXLR effector genes co-segregate with the Avr3c phenotype. The results pointed to one RXLR candidate gene as likely to encode Avr3c. This was verified by testing selected genes by a co-bombardment assay on soybean plants with Rps3c, thus demonstrating functionality and confirming the identity of Avr3c. The Avr3c gene together with eight other predicted genes are part of a repetitive segment of 33.7 kb. Three near-identical copies of this segment occur in a tandem array. In P. sojae strain P6497, two identical copies of Avr3c occur within the repeated segments whereas the third copy of this RXLR effector has diverged in sequence. The Avr3c gene is expressed during the early stages of infection in all P. sojae strains examined. Virulent alleles of Avr3c that differ in amino acid sequence were identified in other strains of P. sojae. Gain of virulence was acquired through mutation and subsequent sequence exchanges between the two copies of Avr3c. The results illustrate the importance of segmental duplications and RXLR effector evolution in the control of race-cultivar compatibility in the P. sojae and soybean interaction.
Identification and Functional Characterization of the Soybean GmaPPO12 Promoter Conferring Phytophthora sojae Induced Expression  [PDF]
Chunyue Chai, Yanling Lin, Danyu Shen, Yuren Wu, Hongjuan Li, Daolong Dou
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0067670
Abstract: Identification of pathogen-inducible promoters largely lags behind cloning of the genes for disease resistance. Here, we cloned the soybean GmaPPO12 gene and found that it was rapidly and strongly induced by Phytophthora sojae infection. Computational analysis revealed that its promoter contained many known cis-elements, including several defense related transcriptional factor-binding boxes. We showed that the promoter could mediate induction of GUS expression upon infection in both transient expression assays in Nicotiana benthamiana and stable transgenic soybean hairy roots. Importantly, we demonstrated that pathogen-induced expression of the GmaPPO12 promoter was higher than that of the soybean GmaPR1a promoter. A progressive 5’ and 3’ deletion analysis revealed two fragments that were essential for promoter activity. Thus, the cloned promoter could be used in transgenic plants to enhance resistance to phytophthora pathogens, and the identified fragment could serve as a candidate to produce synthetic pathogen-induced promoters.
Sequence Variants of the Phytophthora sojae RXLR Effector Avr3a/5 Are Differentially Recognized by Rps3a and Rps5 in Soybean  [PDF]
Suomeng Dong, Dan Yu, Linkai Cui, Dinah Qutob, Jennifer Tedman-Jones, Shiv D. Kale, Brett M. Tyler, Yuanchao Wang, Mark Gijzen
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0020172
Abstract: The perception of Phytophthora sojae avirulence (Avr) gene products by corresponding soybean resistance (Rps) gene products causes effector triggered immunity. Past studies have shown that the Avr3a and Avr5 genes of P. sojae are genetically linked, and the Avr3a gene encoding a secreted RXLR effector protein was recently identified. We now provide evidence that Avr3a and Avr5 are allelic. Genetic mapping data from F2 progeny indicates that Avr3a and Avr5 co-segregate, and haplotype analysis of P. sojae strain collections reveal sequence and transcriptional polymorphisms that are consistent with a single genetic locus encoding Avr3a/5. Transformation of P. sojae and transient expression in soybean were performed to test how Avr3a/5 alleles interact with soybean Rps3a and Rps5. Over-expression of Avr3a/5 in a P. sojae strain that is normally virulent on Rps3a and Rps5 results in avirulence to Rps3a and Rps5; whereas silencing of Avr3a/5 causes gain of virulence in a P. sojae strain that is normally avirulent on Rps3a and Rps5 soybean lines. Transient expression and co-bombardment with a reporter gene confirms that Avr3a/5 triggers cell death in Rps5 soybean leaves in an appropriate allele-specific manner. Sequence analysis of the Avr3a/5 gene identifies crucial residues in the effector domain that distinguish recognition by Rps3a and Rps5.
A Myb Transcription Factor of Phytophthora sojae, Regulated by MAP Kinase PsSAK1, Is Required for Zoospore Development  [PDF]
Meng Zhang, Jing Lu, Kai Tao, Wenwu Ye, Aining Li, Xiaoyun Liu, Liang Kong, Suomeng Dong, Xiaobo Zheng, Yuanchao Wang
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0040246
Abstract: PsSAK1, a mitogen-activated protein (MAP) kinase from Phytophthora sojae, plays an important role in host infection and zoospore viability. However, the downstream mechanism of PsSAK1 remains unclear. In this study, the 3'-tag digital gene expression (DGE) profiling method was applied to sequence the global transcriptional sequence of PsSAK1-silenced mutants during the cysts stage and 1.5 h after inoculation onto susceptible soybean leaf tissues. Compared with the gene expression levels of the recipient P. sojae strain, several candidates of Myb family were differentially expressed (up or down) in response to the loss of PsSAK1, including of a R2R3-type Myb transcription factor, PsMYB1. qRT-PCR indicated that the transcriptional level of PsMYB1 decreased due to PsSAK1 silencing. The transcriptional level of PsMYB1 increased during sporulating hyphae, in germinated cysts, and early infection. Silencing of PsMYB1 results in three phenotypes: a) no cleavage of the cytoplasm into uninucleate zoospores or release of normal zoospores, b) direct germination of sporangia, and c) afunction in zoospore-mediated plant infection. Our data indicate that the PsMYB1 transcription factor functions downstream of MAP kinase PsSAK1 and is required for zoospore development of P. sojae.
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