西南地区水稻细菌性条斑病菌致病力的分化

以水稻品种IRBB4、IRBB5、IRBB14、IRBB18、IRBB21和IR24为鉴别品种,在孕穗期,采用针刺接种的方法,将水稻条斑病菌接种在水稻剑叶上,根据病菌与品种互作反应和亲和性的类型差异,将供试的75株条斑病菌菌株区分为13个小种群.大多数水稻条斑病菌株在鉴别品种上的互作反应表现为弱互作模式.云南、贵州、四川3省菌株小种组成的比较表明:以小种C9为目前3省的共同优势种群;云南省小种组成多样,所有的小种均有分布,贵州小种类型有6、9、11、13,四川小种类型有3、4、6、7、9、11、12、13,小种的分布受地理区域的影响.     

Xoryp_08180 of Xanthomonas oryzae pv. oryzicola,Encoding a Hypothetical Protein,is Regulated by HrpG and HrpX and Required for Full Virulence in Rice

Xanthomonas oryzae pv. oryzicola (Xoc) causes a destructive bacterial leaf streak disease in rice. Some of the gene products annotated as hypothetical proteins in the genome of Xoc may contribute to its virulence in rice. A mutant, Mxoc1679, screened from our previous Tn5-tagged mutant library for Xoc strain RS105, showed reduced virulence in rice. In this mutant, a gene named as Xoryp_08180 was disrupted by Tn5 insertion. Xoryp_08180 encodes a 1 306-aa hypothetical protein which is highly conserved in Xanthomonas spp. Non-polar mutation of Xoryp_08180 in RS105 strain led to a significant reduction in bacterial virulence and growth in rice, a delayed hypersensitive response (HR) in non-host tobacco, and a decrease in extracellular protease activity. The deficiencies above were restored to wild-type level in the complementary strain by expressing Xoryp_08180 in trans. In addition, the expression of Xoryp_08180 was repressed in hrpG and hrpX mutants in planta but not in a nutrient-rich condition. These results suggested that Xoryp_08180 is a virulence factor required for extracellular protease production, HR induction and full virulence of Xoc.     

利用PCR技术专化性检测水稻细菌性条斑病菌

 设计水稻细菌性条斑病菌的专化性引物,并建立相应的PCR检测体系,分别对31株水稻细菌性条斑病菌和15株水稻白叶枯病菌及其它相关菌株进行了测试。结果表明,建立的PCR检测体系可专化性检测水稻细菌性条斑病菌,而水稻白叶枯病菌和其它菌株均没有扩增信号。检测灵敏度可以达到20个细菌菌体,从自然发病和人工接种发病的水稻种子成功地检测出条斑病菌。实现了对水稻细菌性条斑病菌的快速和专化性检测。     

水稻细菌性条斑病菌LAMP快速检测方法的建立

以SYBR Green I荧光染料为指示剂,针对水稻细菌性条斑病菌糖基转移酶,设计了4条普通LAMP引物,并在此基础上设计了2条LAMP环引物,建立了水稻细菌性条斑病菌的环介导等温扩增可视化快速检测方法。结果表明:水稻细菌性条斑病菌呈荧光的阳性反应,近缘菌株则呈橙色的阴性反应;检测灵敏度DNA为100 fg/μL,菌悬液为3×103 cfu/m L。LAMP技术的建立为现场检疫和大规模检测提供了新的方法。     

西甜瓜果斑病菌avrRxo1同源基因激发玉米Rxo1抗性产生的研究

生物信息学分析发现西(甜)瓜果斑病菌(Acidovorax citrulli,Ac)基因组中存在水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)avrRxo1的同源基因avrRxo1_(Ac)。为了揭示avrRxo1_(Ac)是否能够激发玉米R基因Rxo1介导的过敏反应(Hypersensitive response,HR),本研究克隆了avrRxo1_(Ac)基因;氨基酸同源性分析结果显示:AvrRxo1_(Ac)与AvrRxo1的同源性为53.16%。将avrRxo1_(Ac)置于Xoc的avrRxo1突变体中,发现其不能恢复该突变体在含有Rxo1玉米上激发HR反应的能力;借助农杆菌瞬时表达体系发现AvrRxo1A,也不能在Rxo1玉米上激发HR反应。这些结果表明avrRxo1_(Ac)-Rxo1互作不表现为基因-对-基因关系,暗示Rxo1抗病基因转入西甜瓜中,将不能起到抗西(甜)瓜细菌性果斑病的作用。     

江西省细菌性条斑病菌的致病型划分和水稻抗性资源的鉴定

为探明江西省水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)的致病力分化水平和品种资源的抗病性,本研究利用IRBB5、IRBB14、IR24、IRBB4、IRBB21和金刚30等6个鉴别品种,将分离自江西6个稻区的129株Xoc菌株划分为C1~C9 9个致病型;发现仅C5对鉴别品种IRBB4、C7对鉴别品种IRBB21表现为强互作关系;不同稻区的Xoc优势种群呈现多样性。孕穗期抗性测定结果显示:124个籼稻品种中,对C2型菌株5-16表现为中抗的品种仅占8.06%,对C6型菌株08-3-2表现为抗的品种占14.5%;252个东乡野生稻单株中,1株单株(东113)对C2型菌株表现高抗,14个单株表现抗性。东乡野生稻苗期注射接种结果显示,苗期抗病性与孕穗期抗性呈显著相关性,但苗期抗病性普遍低于孕穗期抗性,仅东113表现为中抗。但东乡野生稻对于Xoc的抗病性明显强于栽培稻,从野生稻中寻找抗性资源将是培育抗病品种的理想途径之一。     

Strategy of Echinochloa oryzicola Vasing. for survival in flooded rice

Echinochloa oryzicola Vasing. (= Echinochloa phyllopogon Stapf ex Kessenko) is an obligate weed with an elaborated survival strategy in the flooded rice of Japan. In this review various adaptive characters of the weed, which comprise the survival strategy, are discussed through the life cycle. The weed is distributed only in flooded rice. Seeds (spikelets) buried in the soil exhibit annual cycles between dormant and non‐dormant state, and non‐dormant seeds recurrently appear in spring when rice growers start to prepare seedling beds and fields for rice transplanting. The non‐dormant seeds have unique characters metabolically adapted to submerged conditions to germinate and grow by the anaerobic respiration through alcohol fermentation. The weed has seemingly perfect mimicry of the rice plants throughout its development from seedling to heading, by which the weed escapes from manual weeding. In a rice paddy, the weed starts heading coincidentally with the rice plants at the period when the growers are reluctant to walk in the rice paddy to weed. Irrespective of plant height of the rice cultivar, the weed develops a few upper leaves above the rice canopy during the heading period of rice. This phenotypic plasticity of E. oryzicola in plant height is one of the characters conferring its competitive aggressiveness in flooded rice. When weeding is begun again after heading, the dormant weed seeds escape weeding by shattering and join the soil seedbank. The dormant seeds express the gene of an enzyme catalyzing ATP synthesis through the mitochondrial oxidative phosphorylation more abundantly, and have larger oxygen absorption and enzyme activity of the aerobic respiration than the non‐dormant seeds, suggesting that the dormant seeds maintain viability by the conventional aerobic respiration in the paddy soil drained from rice harvesting in fall to the next early spring. The various adaptive characters comprising the survival strategy of E. oryzicola in flooded rice consist of those inherited from the wild progenitor and those selected by the crop cultivation pressure. It is suggested that both the mimicry of the weed and the heading coincident with the rice plants have been acquired by the large selection pressure of frequent weeding, which has been done over the past hundred years. However, today, the manual weeding is substituted with herbicides, which cannot detect the mimicry and heading photoperiodic sensitivity. As a result, the dominant species of Echinochloa weeds in flooded rice is changing from E. oryzicola to Echinochloa crus‐galli var. crus‐galli that has neither mimicry nor photoperiodic sensitivity synchronizing to that of rice, but is more competitive against rice.     

水稻条斑病细菌hrp调节基因hrpGXooc和hrpXooc的克隆和序列分析

 Xanthomonas oryzae pv. oryzicola基因文库的hrp基因克隆pUHRS138携39.3-kb大小的hrp基因片段。经系列亚克隆和对hrp^-突变体的功能互补验证,4.5-kb BamHI-KpnI为最小功能片段,该片段可使X. o. pv. oryzicola的hrp^-突变体恢复在烟草上激发产生HR和在水稻上具致病性。序列测定和分析显示,4.5-kb hrp片段中含hrpXooc和hrpG_Xooc基因。单独的hrpG_Xooc和hrpXooc不能功能互补hrp^-突变体。hrpXooc与其它黄单胞菌中已克隆的hrpX的同一性达83%以上,推测的蛋白质水平上的差别主要在32、141、164、175、213、247和357位点上。HrpX序列中α-螺旋-转-α-螺旋结构在黄单胞菌中高度保守。hrpG_Xooc与水稻白叶枯病菌的hrpGXoo同一性达96%,与X. campestris pv. vesicatoria的hrpG_Xcv同一性达87%,与Ralstonia solanacearum的hrpG_Rs同源性较低,4种HrpG蛋白质水平上的差别主要集中在22、29、115和252位点上。HrpGXooc和HrpGXcv同列比较显示,3~9和216~220区域的氨基酸序列有所不同,可能反映了HrpG蛋白在感知环境信号和调节hrp基因表达方面的差别。     

Rep-PCR技术对中国水稻条斑病菌的遗传多样性初析

 采用Rep-PCR技术,对30个水稻细菌性条斑病菌株(Xanthomonas oryzae pv.oryzicola)进行遗传多样性分析,同时对李氏禾条斑病菌等其它10个参试菌株也进行了比较。Rep-PCR是利用一些基于细菌的短的重复序列引物(ERIC和BOX)的DNA扩增特性,2种引物组合的电泳图谱结合并分析,以水稻细菌性条斑病菌各自的指纹谱型在相似率80%时可分为6簇,初步表明我国水稻细菌性条斑病菌群体的遗传分化明显;发现自然界存在的弱或无毒性菌株与毒性菌株的Rep-PCR指纹图谱差异很大;毒性菌株的遗传分簇与其致病性具有一定的相关性。用ERIC扩增水稻条斑病菌基因组DNA的指纹比BOX更为多样,两者对菌株的分辨率不同。因此,Rep-PCR技术可有效地用于监测水稻细菌性条斑病菌的遗传变异,还可应用于菌株的鉴定和分类学研究。