猕猴桃细菌性溃疡病生防放线菌的筛选与鉴定

对分离自新疆巴里坤盐湖和达坂城盐湖的179株放线菌进行拮抗丁香假单胞杆菌猕猴桃致病变种筛选,从中筛选到8株具有拮抗作用的放线菌,其中ZBW8-1、E14-3和ZB5-6抗菌活性最显著,其抑菌圈直径分别为27.5 mm、23.6 mm和26.2 mm。针对这3株放线菌进行菌落形态观察,16s rRNA扩增、测序及系统进化分析,并对其进行抗生素合成相关基因的PCR鉴定。结果表明,放线菌ZBW8-1、E14-3和ZB5-6分别与糖霉菌属（Glycomyces）的Glycomyces fuscus TRM 49117、原小单孢菌属（Promicromonospora）的维也纳原小单孢菌（Promicromonospora vindobonensis）V45^T以及拟诺卡氏菌属（Nocardiopsis）的Nocardiopsis terrae YIM 90022^T 16s rRNA序列同源性最高,分别为99.86%、99.10%和99.86%。在系统进化关系上,这3株菌也分别与上述菌株聚类在同一个分支上。ZBW8-1、E14-3和ZB5-6菌株中均携带有NRPS基因,其中ZBW8-1和ZB5-6菌株中还含有PKS-II基因,且ZBW8-1菌株中还含有PKS-I基因。对这3株抗菌活性放线菌进行进化地位分析及抗生素合成相关功能基因的鉴定,将为研究这些菌株中的活性次生代谢产物奠定理论基础。     

番茄对细菌性斑点病的抗性遗传规律研究

利用细菌性斑点病的抗病番茄品种(红珍珠和美味樱桃)和感病品种(中蔬四号、美国大红樱桃和MR),通过常规田间抗感杂交、回交,接种病原菌鉴定子代的抗病性分离情况,统计分析的结果表明,杂交F2代全部抗病,F2代和BC1代的抗感分离比分别符合3：1和1：1的理论分离比,说明了红珍珠和美味樱桃对番茄细菌性斑点病的抗病性为单基因显性遗传。     

转鞭毛蛋白基因水稻细菌性条斑病抗性研究

该研究从生防菌枯草芽胞杆菌Bs-916中克隆了鞭毛蛋白基因,利用转基因载体pCAMBIA1300转入水稻,筛选得到98株阳性转基因植株。分子检测结果表明,有12个转基因株系可检测到目的基因的表达。随后抗病性鉴定表明,有3个转基因株系对水稻细菌性条斑病具有较高的抗性。该研究为目前水稻抗细菌性条斑病转基因研究拓宽了可应用基因资源的范围。     

丁香假单胞菌猕猴桃致病变种的遗传多样性及进化关系

猕猴桃细菌性溃疡病是一种严重威胁猕猴桃生产的毁灭性病害,在世界各大猕猴桃产区发病严重。引起该病的病原菌为丁香假单胞菌猕猴桃致病变种(Pseudomonas syringae pv.Actinidiae,Psa)。综合当前国内外关于该病原菌的相关研究,就其分类地位、遗传多样性及起源进化等方面进行了系统综述。     

病原菌Pst DC3000侵染拟南芥导致自噬和光合作用的抑制

在病原菌侵染下,植物体是如何通过超敏反应限制病原菌扩增的,到目前为止还不清楚。最近的研究显示自噬起了必要的作用,可见,研究自噬和超敏反应的机制非常重要。本研究通过观察在非寄主病原菌突变体丁香假单胞菌番茄致病变种(Pseudomonas syringae pv.tomatoDC3000,Pst DC3000)的侵染下,拟南芥光合功能的变化及自噬现象出现的情况,以为进一步研究植物抗病机理提供实验基础。以野生型拟南芥为材料,采用光谱分析和叶绿素荧光成像分析手段,研究不同浓度的Pst DC3000侵染对拟南芥离体叶片光合功能的影响;以转基因野生型拟南芥(绿色荧光蛋白标记的自噬基因8a)幼根为材料,应用共聚焦显微镜,研究不同浓度的Pst DC3000诱导拟南芥自噬的情况。实验发现,OD600=0.2的Pst DC3000侵染,可显著诱导拟南芥叶片活性氧的积累和迸发,并会引起拟南芥叶片光合作用效率的下降。同时发现,该病原菌处理2 h,可导致拟南芥根中自噬小体的产生。用2',7'-二氯二氢荧光素二乙酯(2',7'-dichlorodihydrofluorescein diacetate,H2DCFDA)标记活性氧,检测了P...     

芽胞杆菌BJ-6的鉴定及对甜瓜细菌性果斑病的防治

芽胞杆菌是目前植物病害生物防治研究最多的一类微生物,其在自然界中分布广泛,开发潜力大。【目的】为了探究从杏树根际土壤分离的芽胞杆菌BJ-6的分类地位及其防病促生作用。【方法】本研究测定了芽胞杆菌BJ-6的形态和生理生化特征,通过PCR扩增了该菌的16S rRNA、gyrA和gyrB基因并进行了序列测定,通过多基因聚类分析确定其分类地位,平板对峙法测定抗菌谱,盆栽幼苗实验验证其对甜瓜细菌性果斑病的防治效果和对甜瓜的促生作用。【结果】结合形态特征、生理生化特性及多基因序列分析建立的系统进化树,确定菌株BJ-6为解淀粉芽胞杆菌(B. amyloliquefaciens),抑菌实验发现该菌株对15种植物病原菌均有不同程度的抑菌活性,盆栽实验结果发现该菌株发酵液对甜瓜细菌性果斑病有很好的防治效果,并对甜瓜苗有很好的促生作用。【结论】BJ-6属于解淀粉芽胞杆菌,抑菌谱广,且具有防病促生作用,具有进一步开发为生防制剂的前景。     

一种新的蜜蜂细菌性幼虫病病原的分离鉴定

2005年早春,在浙江部分地区出现了一种严重的蜜蜂细菌性幼虫病,该病导致蜜蜂幼虫颜色发黄,失去光泽;严重时幼虫死亡腐烂。从10批发病死亡幼虫样品中,分离得到并保存了5类纯培养物。通过蜂群接种试验和实验室人工培养的幼虫接种,确定L2菌株能引起与自然发病相似的症状,且能从接种发病的幼虫上再次分离到相同菌株,证明L2菌株是该蜜蜂细菌性幼虫病的致病菌。进一步对分离到的该致病菌从发病特征、病原形态学、生理生化特性、16SrRNA序列等方面进行了分析鉴定,结果显示:该菌株属于肠球菌属的屎肠球菌(Enterococcusfaecium),不是目前报道的任何一种已知蜜蜂细菌性幼虫病的病原。     

陕西省猕猴桃溃疡病病原菌分离鉴定及分型研究

明确陕西省猕猴桃主产区丁香假单胞菌猕猴桃致病变种（Pseudomonas syringae pv. actinidiae,Psa）的变型,从而有针对性地制定猕猴桃溃疡病的综合防治策略,采集陕西省猕猴桃主产区周至、眉县、灞桥、渭南、长安和鄠邑不同年份发病样本进行病原菌的分离,对病原菌进行初步鉴定后再应用特异性PCR进行分子鉴定,并进行回接实验。从鉴定出的Psa菌株中选出16株不同年份不同地区的代表性菌株应用7个管家基因进行MLSA分型分析。共分离鉴定出86株不同地区不同年份的Psa菌株,并通过回接实验验证了其致病性。MLSA分型结果表明16株代表性Psa菌株均属于Psa3,且Psa3与Psa6的遗传距离最近。陕西省猕猴主产区的Psa变型为Psa3,近十年未发生变异。研究结果为今后Psa的检测鉴定及防治研究提供参考。     

湘西地区猕猴桃溃疡病病原菌分离及其全基因组分析

通过了解湘西地区猕猴桃溃疡病致病菌分类地位和基因类型,初步探讨其致病的分子机理。采用纯培养法分离猕猴桃溃疡病菌;基于16S～23S rRNA基因内转录间隔序列进行病原菌的系统发育分析;通过基因组测序和生物信息学分析解析其致病的分子机理。从“米良1号”和“红阳”猕猴桃感病枝条中分离获得5株溃疡病菌,编号为L211、L212、L321、L322、L323;通过形态特征和16S～23S rRNA基因内转录间隔序列分析,鉴定5株细菌均为丁香假单胞菌猕猴桃致病变种(Pseudomonas syringae pv.actinidae,Psa)。以菌株L211为代表进行体外猕猴桃枝条接种实验表明能引起典型溃疡病症状。通过菌株L211的全基因组测序和生物信息学分析,获得5 741条基因数目,长5 412 072 bp;基因功能注释发现菌株L211携带121种毒力因子、71个植物互作因子和77个耐药基因;同时,基因组单核苷酸多态性分析发现病原菌L211为基因Ⅲ型Psa。引起湘西地区猕猴桃溃疡病的病原菌是丁香假单胞菌猕猴桃致病变种基因Ⅲ型,与国内外报道的引起猕猴桃溃疡病大流行的致病菌一致。猕猴桃溃疡病发病...     

植物病原细菌Pseudomonas syringae pv. tomato基因组中的信号肽分析

应用SignalP 3.0 对植物病原细菌Pseudomonas syringae pv. tomato DC3000菌株基因组中的全部5 615个ORFs进行了分析,确定其中679个ORFs所编码蛋白质的N-端有信号肽序列,其中已经命名并有注释的有107个ORFs。信号肽的长度以19 ~31 个氨基酸居多,其中最多的是23 个氨基酸的信号肽。具有信号肽的ORFs编码蛋白的长度大多为101~400 个氨基酸之间。同时,对组成信号肽的氨基酸种类作了系统的分析,发现组成信号肽的氨基酸中非极性氨基酸占48.54%,极性氨基酸占18.67%,带负电荷氨基酸占24.54%,带正电荷氨基酸仅占8.00%,出现最多的3种氨基酸依次为亮氨酸、丙氨酸和丝氨酸,最少的氨基酸是异亮氨酸,在切割位点-1端的氨基酸中83.211%均为丙氨酸,在切割位点后3位的氨基酸中最多的氨基酸也是丙氨酸。通过分析确定628个分泌类信号肽,36个信号肽具有RR-motif的保守区段,15个脂蛋白类信号肽,未发现Prepilin-like 信号肽和Bacteriocin and Pheromone信号肽。     

The improvement of resistance to bacterial speck in transgenic tomato plants by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> mediated transformation

The pto gene, responsible for resistance to Pseudomonas syringae pv. tomato, was transferred to tomato genotype Urfa-2 by the LBA4404 strain of A. tumefaciens harboring the plasmid pPTC8. The presence of nptII and pto genes in transgenic plants was proved by PCR analysis. Insertion of the pto gene into the genome of transgenic plants and expression of the gene were confirmed by southern and northern hybridizations, respectively. The pathogen P. syringae pv. tomato was applied to all leaves of transgenic and control plants. While typical bacterial speck symptoms developed on the leaves of control plants, the transgenic plants did not display any typical symptoms of bacterial speck upon inoculation with strains 1 and 0. Some of these transgenic plants had thicker leaves than the control plants and produced abnormal flowers. The pollen of transgenic plants was used for crossing with control plants to produce F1 transgenic lines. Fruits from crossed transgenic and control plants were obtained, and F1 seeds germinated on Murashige and Skoog medium in the presence of kanamycin have developed F1 seedlings. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 1, pp. 102–110. The text was submitted by the authors in English.     

In vitro Antibiosis by Pseudomonas syringae Pss22d,Acting Against the Bacterial Blight Pathogen of Soybean Plants,Does Not Influence In planta Biocontrol

The epiphyte Pseudomonas syringae pv. syringae 22d / 93 (Pss22d), isolated from soybean leaves, had been characterized as a promising and species‐specific biocontrol strain in vitro and in planta against the plant pathogen P. syringae pv. glycinea (Psg), which causes bacterial blight of soybean. Three toxins are known to be produced by Pss22d: syringomycin, syringopeptin and 3‐methylarginine (MeArg). In contrast to syringopeptin and syringomycin, MeArg inhibited the growth of Psg in vitro. To examine if the toxins produced by Pss22d are responsible for antagonistic effects in planta, the pathogen Psg was co‐inoculated with either Pss22d wild‐type, a syringopeptin/syringomycin‐negative double mutant (Pss22d.ΔsypA/syrE), or a MeArg‐negative mutant (Pss22d.1) into wounds of pin‐pricked leaves of greenhouse‐grown soybean plants, respectively. In all three cases, the wild‐type Pss22d and its toxin‐deficient mutants prevented development of disease symptoms normally caused by Psg. These results indicated that neither syringopeptin, nor syringomycin, nor MeArg was required for Pss22d’s antagonistic activity in planta. Consequently, factors other than the three toxins may contribute to the intra‐species antagonism in planta.     

The phytotoxin coronatine induces light-dependent reactive oxygen species in tomato seedlings

The phytotoxin coronatine (COR), which is produced by Pseudomonas syringae pv. tomato DC3000 (DC3000), has multiple roles in virulence that lead to chlorosis and a reduction in chlorophyll content. However, the physiological significance of COR-induced chlorosis in disease development is still largely unknown. Global expression analysis demonstrated that DC3000 and COR, but not the COR-defective mutant DB29, caused reduced expression of photosynthesis-related genes and result in a 1.5- to 2-fold reduction in maximum quantum efficiency of photosystem II (F(V)/F(M)). Tomato (Solanum lycopersicum) seedlings inoculated with DC3000 and incubated in a long daily photoperiod showed more necrosis than inoculated seedlings incubated in either dark or a short daily photoperiod. The accumulation of reactive oxygen species (ROS) was detected in cotyledons inoculated with either purified COR or DC3000 but not in tissues inoculated with DB29. Interestingly, COR-induced ROS accumulated only in light and was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and diphenylene iodonium, which function to inhibit electron transport from PSII. Furthermore, COR and DC3000 suppressed expression of the gene encoding the thylakoid Cu/Zn superoxide dismutase but not the cytosolic form of the same enzyme. In conclusion, these results demonstrate a role for COR-induced effects on photosynthetic machinery and ROS in modulating necrotic cell death during bacterial speck disease of tomato.     

Age as a factor in induction of systemic acquired resistance in tomato against bacterial speck by aqueous fruit extracts of Azadirachta indica

In the present study, the effect of aqueous fruit extracts of Azadirachta indica on activity of Peroxidase (POX) at different ages of Tomato (Lycopersicon esculentum) leading to induction of systemic resistance against Pseudomonas syringae pv. tomato was evaluated. For this evaluation, four ages, that is, 6, 8, 10 and 12?weeks of plants were selected. A single leaf at the third node from base of each plant was treated either singly or with different combinations of Neem extract and pathogen. Samples were collected at an interval of 24?h for up to five days and after two weeks of the treatment from both treated and untreated nodes. The change in the activity of defence enzyme POX and expression of its isoforms was studied. The results demonstrate that systemic acquired resistance induced by the Neem fruit extract increases as the plant matures but it is not only the limiting factor.