大白菜软腐菌种群组成及优势菌致病型的研究

对黑龙江省成熟大白菜[Brassica pekinensis（Lour．）Rupr．]生产田采集的软腐病菌进行分离、纯化,并根据形态学特征分析了大白菜软腐病菌的种群组成。结果表明,引起黑龙江省秋季大白菜软腐病的主要致病菌是胡萝卜软腐欧文氏菌胡萝卜软腐亚种[Erwinia carotovora（Jones）Bergey et al．subsp、carotovora]（Ecc）;利用20个Ecc菌株的混合菌对来源于不同生态型及不同地区的大白菜品种进行接种,筛选出5个鉴别寄主,以此将20个Ecc菌株划分为5个致病力类型,其中V型为优势致病菌,其分布广且致病力强。     

一株新的胡萝卜软腐欧文氏菌的分离和鉴定

从大白菜软腐组织中分离出一株软腐病细菌BC1,经过形态观察、生理生化特性分析、致病性检测和16S rDNA序列分析,该分离物被鉴定为胡萝卜软腐欧文氏菌胡萝卜亚种（Erwinia carotovora subsp. carotovora, Ecc）的一个新菌株,编号为BC1。这是首次从16S rDNA序列水平上对在我国分布的软腐欧文氏菌进行鉴定。Ecc BC1的16S rDNA序列与其它软腐欧文氏菌株的16S rDNA序列之间同源性达987%～993%,而且在系统发育树中独立于Ecc其它菌株。序列分析结果表明,Ecc BC1具有至少2种不同的16S rDNA序列,它们都在第459位和473位（相对于大肠杆菌16S rDNA序列）发生碱基突变,同一基因中两个突变位点之间彼此互补,处于16S rRNA螺旋H17颈部,而且这两处碱基变异只存在于BC1菌株中。通过与其它软腐欧文氏菌亚种和菌株16S rDNA序列进行比对分析,还进一步鉴定出一些BC1菌株特异的16S rDNA碱基突变位点。本文报道的Ecc BC1两个16S rDNA序列在GenBank中的登录号分别为AY309068和AY309069。     

魔芋软腐病菌分子鉴定与遗传多样性

通过对分离的魔芋软腐病菌株和其它参试菌株的致病性测定、选择性培养基培养性状观察和16S-23S rDNA转录间隔区PCR(ITS-PCR)分析,将测试的33株软腐病菌株主要分为3个组群。第1组群为胡萝卜软腐欧文氏杆菌胡萝卜软腐亚种(Erwinia carotovorasubsp.carotovora,E.c.c.);第2组群为菊欧文氏杆菌(Erwinia chrysanthemi,E.ch.);还有一组未能确定的菌株。利用细菌基因组重复序列通用引物BOX和J3进行Rep-PCR特异性扩增,引起软腐病的菌株E.c.c.和E.ch.(ITS-PCR鉴定)种内的Rep-PCR指纹存在明显的遗传分化,经聚类分析,在0.1水平上把E.c.c.13株区分为5个类群。     

辣椒种质资源抗青枯病的鉴定与评价

采用青枯菌FJC100301菌株对田间辣椒（Capsicum annuum）抗病品种76a和感病品种TW-1分别作了不同温度、不同接种量和不同接种方法的接种试验。结果表明,辣椒青枯病抗性的室内鉴定以接种温度28℃、浸根20 min和3×10^8cfu/mL接种浓度为宜;辣椒种质田间抗青枯病接种鉴定宜选择5月上旬进行,浸根20 min,接种浓度为3×10^8cfu/mL。采用田间抗性接种鉴定的方法,用青枯菌FJC100301菌株对106份辣椒材料进行了抗性鉴定。田间接种后每隔10 d统计病情指数,划分辣椒抗青枯病鉴定分级标准,获得了高抗材料14份、抗病材料8份、中抗材料23份、中感材料23份、感病材料20份、高感材料18份;采用离体叶片接种法对田间筛选得到的高抗和高感纯度较高品种进行抗性分析,结果与田间鉴定一致。     

水稻穗瘟防卫反应相关基因的分离和鉴定

以遗传背景相近、对叶瘟抗性相同但对穗瘟抗性不同的两个水稻株系为材料,利用抑制消减杂交（SSH）技术构建穗瘟抗／感消减cDNA文库,经差异筛选及序列分析,共获得90个独立的差异表达cDNA克隆,根据与它们刚源的基因功能推测,这些克隆可能参与了对病原菌的防卫反应、信号传导和转录等一些重要的生物学过程。利ⅢRT-PCR分析了26个所筛选到的cDNA克隆在抗／感植株接种后的表达,17个基因的表达差异得到验证。对这螳差异表达基因在抗感株系接种后不同时间点的表达谱也进行了RT-PCR的分析。文章首次报道了什关水稻对穗瘟抗性在mRNA水平进行研究,为深入研究水稻对穗瘟抗性的遗传机理打下了基础。     

胡萝卜软腐果胶杆菌CpxP蛋白纯化及抑菌功能鉴定

胡萝卜软腐果胶杆菌是世界十大植物病原菌之一,主要侵染十字花科的经济作物和观赏花卉。文中从胡萝卜软腐果胶杆菌的基因组中克隆1个抗菌基因cpxP(GeneID:29704421),将其构建在原核表达质粒pET-15b上,并转化至大肠杆菌Escherichia coli BL21 (DE3)进行表达,经纯化后进行稳定性和抑菌实验。结果显示,IPTG的诱导终浓度为1mmol/L,实现了蛋白的高效外源表达,纯化后电泳无杂蛋白残留,且该蛋白具有良好的热稳定性和pH稳定性。CpxP蛋白抑菌试验结果显示其对胡萝卜切片的抑菌率可达到44.89%,对马铃薯切片的抑菌率可达到59.41%。为进一步解释其抑菌机理,研究该蛋白的空间结构可为软腐病的防治和新型蛋白农药靶点研究提供新思路。     

珠芽魔芋对细菌性软腐病的抗性鉴定研究

采用魔芋Amorphophallus spp.块茎点种、注射、灌根接种及田间调查等方法,对国内普遍栽种的珠芽红魔芋A. bulbifer、珠芽金魔芋A. muelleri、花魔芋A. konjac和白魔芋A. albus等12个种质材料进行抗软腐病鉴定、比较和评价,以分析珠芽魔芋对抗细菌性软腐病的抗病水平。结果表明,供试材料对软腐病抗性差异较大,珠芽金魔芋种质对细菌性软腐病均有免疫性(I),德宏及临沧珠芽红魔芋种质为高抗病品种(HR);缅甸珠芽红魔芋为抗病品种(R);富源花魔芋、楚雄花魔芋、日本农林2号、鄂魔芋1号、秦魔1号、昭通白魔芋、丽江白魔芋均属易感品种(S),与田间抗性调查情况基本相符。     

EcbCSL101菌株hrpN基因的克隆、表达及HarpinEcbCSL101蛋白的生物学活性

胡萝卜软腐欧文氏菌甜菜亚种(Erwinia carotovora subsp. betavasculorum) EcbCSL101菌株具有很强胞外酶分泌活性, 接种非寄主植物烟草引起过敏反应。Southern blotting结果表明EcbCSL101菌株中含有hrpN 基因。PCR扩增含EcbCSL101完整开放阅读框的DNA片段并克隆到表达载体pET28a(+)中。核苷酸序列分析表明, EcbCSL101菌株的hrpN 基因的ORF为1113 bp, 编码36.65 kD HarpinEcbCSL101蛋白(GenBank, DQ355519),与其它几种软腐欧文氏菌Harpin蛋白有较高的同源性。将含有hrpNEcbCSL101基因的重组质粒转化到大肠杆菌JM109(DE3)中进行表达,纯化后的HarpinEcbCSL101能诱导烟草发生过敏反应。     

EcbCSL101菌株hrpN基因的克隆、表达及HarpinEcbCSL101蛋白的生物学活性

胡萝卜软腐欧文氏茵甜菜亚种(Erwinia carotovora subsp.betavasculorum)EcbCSL101菌株具有很强胞外酶分泌活性,接种非寄主植物烟草引起过敏反应.Southern blotting结果表明EcbCSL101菌株中含有hrpN基因.PCR扩增含EcbCSL101完整开放阅读框的DNA片段并克隆到表达载体pET28a( )中.核苷酸序列分析表明,EcbCSL101菌株的hrpN基因的ORF为1113 bp,编码36.65 kD HarpinEcbCSL101蛋白(GenBank,DQ355519),与其它几种软腐欧文氏菌Harpin蛋白有较高的同源性.将含有hrpNEcbCSL101基因的重组质粒转化到大肠杆菌JM109(DE3)中进行表达,纯化后的HarpinEcbCSL101能诱导烟草发生过敏反应.     

利用KASP标记筛选含rhg1和Rhg4位点的大豆抗病资源

大豆胞囊线虫(SCN,soybean cyst nematode)病是一种世界性大豆病害,培育抗SCN大豆品种是防治SCN的重要措施。本研究利用来自抗SCN主效位点rhg1和Rhg4的2个KASP标记,对487份大豆材料进行筛选,选择含有抗性位点且农艺性状优异的材料;通过室内接种大豆胞囊线虫2号、4号、5号生理小种和新小种X12,进行抗性鉴定验证其抗性水平,为培育抗病品种提供抗源材料。标记筛选结果表明,20份材料含有rhg1和Rhg4这2个主效抗性位点,其中,2份材料仅含有Rhg4位点。表型抗性鉴定结果表明,在接种的22份材料中,有1份材料对3个小种表现中抗,5份材料对2个小种表现抗或中抗。其中,1份材料对2号小种表现抗病、4份表现中抗;2份材料对4号小种表现中抗;4份材料对5号小种表现抗病、14份表现中抗;22份材料对新小种X12均表现出感病或中感。因此,本研究从487份材料中筛选出20份含有2个SCN抗性位点并具优异农艺性状的材料,可通过rhg1和Rhg4位点的累加培育抗病品种。     

Identification of a disease resistance locus in Arabidopsis that is functionally homologous to the RPG1 locus of soybean

A new disease resistance locus in Arabidopsis, RPS3 , was identified using a previously cloned avirulence gene from a non- Arabidopsis pathogen. The avrB avirulence gene from the soybean pathogen Pseudomonas syringae pv. glycinea was transferred into a P. syringae pv. tomato strain that is virulent on Arabidopsis , and conversion to avirulence was assayed on Arabidopsis plants. The avrB gene had avirulence activity on most, but not all, Arabidopsis ecotypes. Of 53 ecotypes examined, 45 were resistant to a P. syringae pv. tomato strain carrying avrB , and eight were susceptible. The inheritance of this resistance was examined using crosses between the resistant ecotype Col-0 and the susceptible ecotype Bla-2. In F₂ plants from this cross, the ratio of resistant:susceptible plants was approximately 3:1, indicating that resistance to P. syringae expressing avrB is determined by a single dominant locus in ecotype Col-0, which we have designated RPS3 . Using RFLP analysis, RPS3 was mapped to chromosome 3, adjacent to markers M583 and G4523, and ≤ 1 cM from another disease resistance locus, RPM1 . In soybean, resistance to P. syringae strains that carry avrB is controlled by the locus RPG1 . Thus, RPG1 and RPS3 both confer avrB -specific disease resistance, suggesting that these genes may be homologs.     

Natural genetic resources of Arabidopsis thaliana reveal a high prevalence and unexpected phenotypic plasticity of RPW8-mediated powdery mildew resistance

Here, an approach based on natural genetic variation was adopted to analyse powdery mildew resistance in Arabidopsis thaliana. Accessions resistant to multiple powdery mildew species were crossed with the susceptible Col-0 ecotype and inheritance of resistance was analysed. Histochemical staining was used to visualize archetypal plant defence responses such as callose deposition, hydrogen peroxide accumulation and host cell death in a subset of these ecotypes. In six accessions, resistance was likely of polygenic origin while 10 accessions exhibited evidence for a single recessively or semi-dominantly inherited resistance locus. Resistance in the latter accessions was mainly manifested at the terminal stage of the fungal life cycle by a failure of abundant conidiophore production. The resistance locus of several of these ecotypes was mapped to a genomic region containing the previously analysed atypical RPW8 powdery mildew resistance genes. Gene silencing revealed that members of the RPW8 locus were responsible for resistance to Golovinomyces orontii in seven accessions. These results suggest that broad-spectrum powdery mildew resistance in A. thaliana is predominantly of polygenic origin or based on RPW8 function. The findings shed new light on the natural variation of inheritance, phenotypic expression and pathogen range of RPW8-conditioned powdery mildew resistance.     

Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.     

Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean.

To develop a model system for molecular genetic analysis of plant-pathogen interactions, we studied the interaction between Arabidopsis thaliana and the bacterial pathogen Pseudomonas syringae pv tomato (Pst). Pst strains were found to be virulent or avirulent on specific Arabidopsis ecotypes, and single ecotypes were resistant to some Pst strains and susceptible to others. In many plant-pathogen interactions, disease resistance is controlled by the simultaneous presence of single plant resistance genes and single pathogen avirulence genes. Therefore, we tested whether avirulence genes in Pst controlled induction of resistance in Arabidopsis. Cosmids that determine avirulence were isolated from Pst genomic libraries, and the Pst avirulence locus avrRpt2 was defined. This allowed us to construct pathogens that differed only by the presence or absence of a single putative avirulence gene. We found that Arabidopsis ecotype Col-0 was susceptible to Pst strain DC3000 but resistant to the same strain carrying avrRpt2, suggesting that a single locus in Col-0 determines resistance. As a first step toward genetically mapping the postulated resistance locus, an ecotype susceptible to infection by DC3000 carrying avrRpt2 was identified. The avrRpt2 locus from Pst was also moved into virulent strains of the soybean pathogen P. syringae pv glycinea to test whether this locus could determine avirulence on soybean. The resulting strains induced a resistant response in a cultivar-specific manner, suggesting that similar resistance mechanisms may function in Arabidopsis and soybean.     

Genetic characterization of five powdery mildew disease resistance loci in Arabidopsis thaliana

This paper reports on six Arabidopsis accessions that show resistance to a wild isolate of the powdery mildew pathogen, Erysiphe cichoracearum . Resistance at 7 days post-inoculation in these accessions was characterized by limited fungal growth and sporadic development of chlorotic or necrotic lesions at inoculation sites. Three accessions, Wa-1, Kas-1 and SI-0, were highly resistant, while the other accessions permitted some fungal growth and conidiation. Papilla formation was a frequent host response; however, cell death appeared to be neither a rapid nor a common response to infection. To determine the genetic basis of resistance, segregation analyses of progeny from crosses between each of the resistant accessions and Columbia ( gl1 ), which is susceptible to the powdery mildew pathogen, were performed. For all accessions except SI-0, resistance was conferred by a single locus. SI-0 was unique in that two unlinked loci controlled the disease reaction phenotype. In accessions Wa-1, Kas-1, Stw-0 and Su-0, powdery mildew resistance was encoded by a semi-dominant allele. However, susceptibility was dominant to resistance in accessions Te-0 and SI-0. Mapping studies revealed that powdery mildew resistances in Kas-1, Wa-1, Te-0, Su-0 and Stw-0 were controlled by five independent loci. This study suggests that the Arabidopsis powdery mildew disease will be a suitable model system in which to investigate powdery mildew diseases.     

Differences in susceptibility of Arabidopsis ecotypes to crown gall disease may result from a deficiency in T-DNA integration.

We show that among ecotypes of Arabidopsis, there is considerable variation in their susceptibility to crown gall disease. Differences in susceptibility are heritable and, in one ecotype, segregate as a single major contributing locus. In several ecotypes, recalcitrance to tumorigenesis results from decreased binding of Agrobacterium to inoculated root explants. The recalcitrance of another ecotype occurs at a late step in T-DNA transfer. Transient expression of a T-DNA-encoded beta-glucuronidase gusA gene is efficient, but the ecotype is deficient in crown gall tumorigenesis, transformation to kanamycin resistance, and stable GUS expression. This ecotype is also more sensitive to gamma radiation than is a susceptible ecotype. DNA gel blot analysis showed that after infection by Agrobacterium, less T-DNA was integrated into the genome of the recalcitrant ecotype than was integrated into the genome of a highly susceptible ecotype.     

Different Mechanisms of Four Aluminum (Al)-Resistant Transgenes for Al Toxicity in Arabidopsis

We have characterized the mechanism of action of four transgenes (AtBCB [Arabidopsis blue copper-binding protein], parB [tobacco (Nicotiana tabacum) glutathione S-transferase], NtPox [tobacco peroxidase], and NtGDI1 [tobacco GDP dissociation inhibitor]) that independently Al resistance on transgenic Arabidopsis. All four transgenic lines showed lower deposition of callose after Al treatment than the Landsberg erecta ecotype of Arabidopsis, confirming that the four genes function to ameliorate Al toxicity. Influx and efflux experiments of Al ions suggested that the AtBCB gene may suppress Al absorption, whereas expression of the NtGDI1 gene promotes a release of Al in the root tip region of Arabidopsis. The total enzyme activities of glutathione S-transferases or peroxidases in transgenic lines carrying either the parB or NtPox genes were significantly higher than in the Landsberg erecta ecotype of Arabidopsis, and these enzyme activities were maintained at higher levels during Al stress. Furthermore, lipid peroxidation caused by Al stress was repressed in these two transgenic lines, suggesting that overexpression of these two genes diminishes oxidative damage caused by Al stress. Al-treated roots of transgenic plants were also stained by 4',6-diamino-2-phenylindole to monitor cell death caused by Al toxicity. The result suggested that cell death is repressed in the NtPox line. Analysis of F(1) hybrids between the four transgenic lines suggests that more resistant transgenic plants can be constructed by combinations of these four genes.     

RCY1, an Arabidopsis thaliana RPP8/HRT family resistance gene,conferring resistance to cucumber mosaic virus requires salicylic acid,ethylene and a novel signal transduction mechanism

The dominant locus, RCY1, in the Arabidopsis thaliana ecotype C24 confers resistance to the yellow strain of cucumber mosaic virus (CMV-Y). The RCY1 locus was mapped to a 150-kb region on chromosome 5. Sequence comparison of this region from C24 and a CMV-Y-susceptible C24 mutant predicts that the RCY1 gene encodes a 104-kDa CC-NBS-LRR-type protein. The RCY1 gene from C24, when expressed in the susceptible ecotype Wassilewskija (Ws), restricted the systemic spread of virus. RCY1 is allelic to the resistance genes RPP8 from the ecotype Landsberg erecta and HRT from the ecotype Dijon-17, which confer resistance to Peronospora parasitica biotype Emco5 and turnip crinkle virus (TCV), respectively. Examination of RCY1 plants defective in salicylic acid (SA), jasmonic acid (JA) and ethylene signaling revealed a requirement for SA and ethylene signaling in mounting a resistance response to CMV-Y. The RCY1 nahG etr1 double mutants exhibited an intermediate level of susceptibility to CMV-Y, compared to the resistant ecotype C24 and the susceptible ecotypes Columbia and Nossen. This suggests that in addition to SA and ethylene, a novel signaling mechanism is associated with the induction of resistance in CMV-Y-infected C24 plants. Moreover, our results suggest that the signaling pathways downstream of the RPP8, HRT, and RCY1 have evolved independently.     

Natural variation in the Arabidopsis response to the avirulence gene hopPsyA uncouples the hypersensitive response from disease resistance

The plant hypersensitive response (HR) is tightly associated with gene-for-gene resistance and has been proposed to function in containing pathogens at the invasion site. This tight association has made it difficult to unequivocally evaluate the importance of HR for plant disease resistance. Here, hopPsyA from Pseudomonas syringae pv. syringae 61 is identified as a new avirulence gene for Arabidopsis that triggers resistance in the absence of macroscopic HR. Resistance to P. syringae pv. tomato DC3000 expressing hopPsyA was EDS1-dependent and NDR1-independent. Intriguingly, several Arabidopsis accessions were resistant to DC3000(hopPsyA) in the absence of HR. This is comparable to the Arabidopsis response to avrRps4, but it is shown that hopPsyA does not signal through RPS4. In a cross between two hopPsyA-resistant accessions that differ in their HR response, the HR segregated as a recessive phenotype regulated by a single locus. This locus, HED1 (HR regulator in EDS1 pathway), is proposed to encode a protein whose activity can cause suppression of the EDS1-dependent HR signaling pathway. HED1-regulated symptomless gene-for-gene resistance responses may explain some cases of Arabidopsis resistance to bacteria that are classified as nonhost resistance.