水稻细菌性叶鞘褐腐病研究

从浙江嘉兴市和余杭市的晚稻褐腐叶鞘及谷粒的156份样本中分离出363个荧光细菌，经菌落形态和致病性测定后获得19个致病菌株，从中选出10个进行细菌学和Biolog鉴定，并与6个标准菌株比较，结果证实，这种水稻叶鞘褐腐病是由Pseudomonas fusouaginae引起，种子能带菌传病。     

小麦细菌性叶鞘褐腐病及其病原鉴定

为搞清杭州小麦上一新发生病害的病原，从病区获得6份小麦病株样本的症状，经初步的植物细菌学特性分析，鉴定为细菌病害。6个分离细菌经32项生理生化反应、茵落形态、致病性、Biolog、脂肪酸分析及与6个标准菌株比较，证实了这种小麦叶鞘褐腐病是由Pseudomonasfuscovaginae．,Miyajima，Tanii＆Akita引起，与水稻细菌性叶鞘褐腐病为同一病原。     

美澳型核果褐腐病菌的检疫鉴定方法

本文根据美澳型核果褐腐病菌的形态学、生物学培养性状和分子生物学等方面的特征,确立了检疫鉴定美澳型核果褐腐病菌的各项技术要求.     

进口智利李子上核果褐腐病菌鉴定

从进口智利李子上分离到一种引起李子果实腐烂的病原真菌,接种李子能引起果实变褐和腐烂。该真菌菌丝在PDA培养基上的生长最适温度为25℃,产孢少,菌落初始为浅灰色,边缘浅裂状,多次传代后逐渐变为浅白色,边缘整齐。分生孢子无色,单孢,柠檬形或卵圆形,大小11．5（9．5～14．7）μm×7．4（5．3～9．2）μm。ITS序列分析结果表明该分离物ITS区序列与GenBank中登录的16株舱laxa序列相似性分别为99．8％～100％,碱基的差异为0～1bp;与21株旭frueticola序列相似性为99．0％～99．2％,碱基的差异为4～5bp;与10株M.fructigena的序列相似性为97．5％～97．7％,碱基的差异为13～14bp。基于ITS序列的系统发育关系分析表明分离物sh675和GenBank中登录的16个M.laxa菌株属于同一个聚类群,相关的Mfructieola和M.fructigena属于另一个聚类群。M.laxa特异性引物ITS1Mix／ITS4Mlx能扩增分离物sh675的菌丝DNA,得到预期的356bp扩增产物。根据该真菌的菌落形态特征、分生孢子形态特征、ITS序列和PCR检测结果,将其鉴定为核果褐腐病菌（Monilinia laxa）。     

褐腐病菌三种分子鉴定方法的比较

为了筛选出适用于口岸检疫的、可快速鉴定三种褐腐菌,即美澳型核果褐腐菌(Monilinia fructicola)、核果褐腐菌(M. laxa)和仁果褐腐菌(M. fructigena)的方法,采用Lane等基于形态学特征的方法对采自北京、山东、河北等省市的58株褐腐菌进行了种类鉴定,并以这些菌及三个种的标准菌株为材料比较了已报道的三种分子检测方法的可靠性。研究发现,58株菌中有53株是美澳型核果褐腐菌,2株为核果褐腐菌,3株为仁果褐腐菌。采用Ioos等的PCR方法,从3个标准菌株、53株美澳型核果褐腐菌和2株核果褐腐菌中都只扩增出相应种的特征条带,而从3株仁果褐腐菌中的2株中扩增出了两个种的特征条带。采用Ma等的方法,从美澳型核果褐腐菌标准菌株、采自国内的53株美澳型核果褐腐菌和核果标准菌株中,只扩增到相应种的特征条带,而从采自国内的2株核果褐腐菌株中不仅扩增出了核果褐腐菌的条带还扩增出了美澳型核果褐腐菌的特征条带,3株仁果褐腐菌株产生了核果褐腐菌的特征条带,仁果褐腐菌标准菌株中没有得到产物。采用Cote等的方法,只从16株美澳型核果褐腐菌中扩增出该种的特征条带,从其余菌株(包括标准菌株)中没有获得产物。这些研究结果表明:Ioos等的检测方法可用于检测美澳型核果褐腐菌和核果褐腐菌;Ma等的检测方法可用于检测美澳型核果褐腐菌,而不适用于检测其它两个种;而仁果褐腐菌的分子检测方法需要进一步研究和完善。     

MALDI-TOF质谱技术鉴定植物病原体研究进展

基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术具有快速和高通量特点,适合分析微量蛋白质、多肽、多糖和核酸,该技术已在生命科学领域广泛应用。本文综述了 MALDI-TOF MS 技术原理以及近年来在植物病原真菌和细菌鉴定方面的研究进展。MALDI-TOF MS 作为一种简便和快捷的检测技术,必将在植物病原菌鉴定领域发挥更大的作用。     

中国猕猴桃细菌性花腐病菌的鉴定

 从福建、湖南和湖北猕猴桃病花上分离到能引起花腐病的32个细菌菌株,经细菌学和BiologGN测试板测定,可以看出中国的猕猴桃细菌性花腐病菌与新西兰的猕猴桃花腐病菌、丁香假单胞菌丁香致病变种Pseudomonas syringae pv.syringae和绿黄假单胞菌P.viridiflava相似,与萨氏假单胞菌P.savastanoi和猕猴桃溃疡病菌P.syringae pv.actinidiae有更多的不同,但是DNA/DNA同源性测定结果却显示出中国的菌株可分为2个类型:第1个类型与新西兰猕猴桃花腐病菌和萨氏假单胞菌有很高的同源性,第2类型与绿黄假单胞菌有很高的同源性,说明中国菌株分别属于这2个种。第1类型来自于福建和湖北,第2类型来自于湖南。     

云南省水稻细菌性条斑病菌的致病型划分和水稻抗性资源的鉴定

 为明确云南省水稻细菌性条斑病菌(Xanthomonas oryzae pv. oryzicola, Xoc)的致病力分化以及不同类型水稻品种对Xoc的抗感特性,通过针刺接种法将云南省8个稻区采集的86株Xoc菌株,接种于6个携带不同抗性基因的水稻鉴别品种(IRBB4、IRBB5、IRBB14、IRBB18、IRBB21和IR24)。根据这些菌株在鉴别品种上的毒力差异进行了UPGMA聚类分析,将其划分为9个致病型(Ⅰ型 ~ Ⅸ 型)。其中,Ⅰ型为优势菌群,分布频率为50.5%。对不同稻区的优势菌群进行分析,发现云南省各稻区Xoc的致病型呈多样性分布,以强毒力的Ⅰ型为高频率致病型。选用 Ⅰ 型、Ⅱ 型和 Ⅵ 型代表菌株对云南省的80个主栽和区试水稻品种进行抗性评价,对3个致病型表现抗性的材料比例分别为30.0%、35.0%和57.5%。筛选出9个对3种致病型都表现为抗性的品种,其中“Deyou16”和“Changgui2”表现为高抗。研究结果可为云南省防治水稻细菌性条斑病的水稻区域性布局和抗性品种的利用提供理论依据。     

大白菜对褐腐病抗性的快速鉴定方法研究

为了建立一套大白菜对褐腐病抗性的快速鉴定方法,本研究对菌丝、菌核作为接种物进行了比较,并分析了菌丝浓度、接种苗龄、接种温度以及接种后空气湿度对褐腐病发病的影响。结果表明：菌饼回接土壤表面为最佳接种方法,菌丝浓度对致病力无影响;最佳接种苗龄为1~2叶期,最佳接种温度25?℃,最佳接种湿度90%~100%;病情指数调查均在接种后第7天。     

水稻细菌性谷枯病菌和水稻白叶枯病菌荧光RPA快速检测方法的建立

本研究分别针对我国进境植物检疫性有害生物水稻细菌性谷枯病菌及水稻白叶枯病菌建立了光RPA检测方法,并对其灵敏度、特异性以及对实际种子样本的检测能力进行了测试评价。结果表明,本研究建立的两种方法均能够在20 min内特异性检测到目标菌株,对水稻细菌性谷枯病菌(Burkholderia glumae)菌液的检测下限达到了11.4 CFU/反应,DNA检测下限达到了4.83×10^-5ng/反应;对水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)菌液的检测下限达到了4.42 CFU/反应,DNA检测下限达到了3.83×10^-4ng/反应。两种方法均能够成功应用于带菌种子样品的快速检测。     

烟草黑胫病和根黑腐病生防假单胞杆菌的筛选与鉴定

为获得对烟草黑胫病和根黑腐病具有双重防病效果且能够促进烟草生长的假单胞杆菌,采用稀释涂布法从40份土壤样品中分离出201株细菌,通过平板对峙和含毒介质法,筛选出对烟草黑胫病和烟草根黑腐病病原菌均具有良好拮抗作用的菌株PA2101和PG3402。盆栽促生试验表明,菌株PA2101和PG3402能协调地改善烟草地上部分的生长和烟草根系发育,均在一定程度上增加了烟草的株高、叶面积、株鲜重、根鲜重、叶片数和根长,提高了烟草的根冠比和根活力。盆栽试验结果表明,菌株PA2101对烟草黑胫病和根黑腐病的防效分别为70.11%和62.67%,均高于其对照药剂;菌株PG3402对两种病害的防效分别为60.92%和60.00%,与对照药剂相当。抗性标记菌株的定殖试验结果表明,菌株PA2101和PG3402在接种后第29 d能定殖于烟草根际土壤和根内,在烟草茎和叶内也能长时间存在,表明两菌株能够良好定殖。16Sr DNA序列、菌落形态和生理生化性状分析表明,菌株PA2101为铜绿假单胞杆菌Pseudomonas aeruginosa,菌株PG3402为格拉纳达假单胞杆菌Pseudomonas granadensis。综上所述,菌株PA2101和PG3402对烟草具有良好的促生作用,并对烟草黑胫病和根黑腐病有较好的防病效果,是具有生防潜力的菌株。     

Implications of bacterial contaminated seed lots and endophytic colonization by Pseudomonas fuscovaginae on rice establishment

The economic impact of seedborne bacterial diseases on rice production provides a major motivation for research on seed health. This paper reports on the endophytic growth of a rifampicin‐marked strain of the seedborne rice pathogen Pseudomonas fuscovaginae. The bacterium was found in most tested seeds indicating that, even without visible discolouration, seed transmission is possible. Crushed discoloured seeds contained more bacterial cells than did non‐crushed discoloured seeds. These bacteria were released during seed soaking, contaminating clean seed and lowering seed germination. Cells of a rifampicin‐resistant strain of P. fuscovaginae, which had been inoculated onto rice seeds, were subsequently recovered from different growth stages and from different rice tissues, thereby indicating endophytic colonization. These results have implications for seedling establishment, as symptomless seeds do not assure disease‐free seeds, and the presence of seedborne bacteria results in poor germination and poor seedling establishment. Elimination of seedborne bacteria by soaking in sodium hypochlorite can increase seed germination. This could be used in developing control strategies, and, if practised regularly, reduce entry of seedborne disease‐causing organisms into crops, resulting in lower disease pressure.     

梨灰霉病菌拮抗细菌Pseudomonas extremorientalis的鉴定、发酵条件优化及防效评价

灰霉病是梨贮藏期的主要病害,发病率高,为害严重。为了有效控制该病害发生,利用平皿稀释法从梨根际土壤中分离内生细菌,并采用琼脂平板扩散法进行抑菌活性初筛,筛选出1株拮抗细菌L21,基于形态特征、生理生化特性及16S rRNA和gyrB序列分析,鉴定为Pseudomonas extremorientalis。采用生长速率法测定菌株L21的无菌发酵液对12种病原真菌均有抑制作用,采用Salkowski比色法测定菌株L21能够分泌吲哚-3-乙酸（IAA）,采用酪蛋白培养基平板检测菌株L21可以产生胞外蛋白酶。在单因素试验的基础上,通过Plackett-Burman试验设计筛选出pH、硫酸锰和转速3个影响发酵的重要因素,利用最陡爬坡试验和Box-Behnken试验设计,获得菌株L21的最佳发酵条件为蔗糖20 g/L、牛肉膏20 g/L、硫酸锰3.00 g/L、甘油1 g/L、K₂HPO₄ 1.50 g/L、接种量8%、温度34℃、pH 7.31、转速200.66 r/min和培养时间15 h。处理96 h后,菌株L21对梨灰霉病的防效为74.23%,与多菌灵的防效差异不显著。本研究表明菌株L21具有广谱抑菌活性,为植物真菌病害的生物防治提供新的生防资源。     

假单胞菌产生的抗生素

假单胞菌属Pseudomonas群体庞大,广泛分布于土壤、水体和动植物中,并产生大量的次生代谢产物以适应环境的变化。抗生素为假单胞菌在自然生态条件下的生存提供了重要的竞争手段,同时也是生防假单胞菌防治植物病害的主要机制之一。本文介绍假单胞菌产生的抗生素种类、作用机制、不同浓度下抗生素的生物学功能,以及假单胞菌抗生素在医学和农业病害防治中的应用。     

Identification of cold-tolerant Pseudomonas viridiflava and P. marginalis causing severe carrot postharvest bacterial soft rot during refrigerated export from New Zealand

In 1999, New Zealand carrots ( Daucus carota ) exported to the Middle East incurred substantial damage due to bacterial soft rot that resulted in major financial loss to farmers. A modified carrot tissue bioassay was developed under standard conditions to provide an economical and rapid means of monitoring export carrots for bacteria able to cause severe bacterial soft rot. Using this bioassay, two bacterial isolates causing severe degradation were detected and subsequently identified as Pseudomonas viridiflava (designated NZCX09) and P. marginalis (NZCX27), using the Biolog system and 16S rRNA phylogenetic analysis. Investigation of disease epidemiology of NZCX09 and NZCX27 at low temperatures showed that tissue degradation occurred at temperatures approaching 0°C. These findings emphasize the importance of postharvest sanitization, and the efficacy of refrigeration methods used in controlling soft rots in carrots stored over time.     

黄瓜细菌性茎枯萎病病原鉴定及温度对其致病力的影响

自2003年以来, 在我国北京?辽宁和山东等地的黄瓜上发现了一种重要细菌病害—黄瓜细菌性茎枯萎病, 对黄瓜生产造成了严重危害?从发病的黄瓜叶片?茎部和果实上分离的菌株接种到黄瓜后, 症状与自然发病症状完全一致?经过多位点序列分型和BOX-PCR技术, 以及LOPAT?GATTa和Ayers碳源利用试验等, 确定该病害的病原菌为丁香假单胞流泪致病变种 Pseudomonas syringae pv. lachrymans?以黄瓜细菌性茎枯萎病菌A2为供试菌株, 黄瓜细菌性角斑病菌丁香假单胞菌流泪致病变种 P.syringae pv. lachrymans pslb8为对照菌株, 对其进行不同温度下致病力?体内和体外生长能力测定, 结果表明:20℃条件下, A2菌株的致病力?体内和体外生长能力均强于pslb8菌株, 相对低温的条件更有利于黄瓜细菌性茎枯萎病病原菌的侵染和病害的发生?     

A novel rhizobacterium Bk7 for biological control of brown sheath rot of rice caused by Pseudomonas fuscovaginae and its mode of action

The study presents the first report on biocontrol of brown sheath rot disease of rice caused by Pseudomonas fuscovaginae using rhizo-bacterial isolate Bacillus amyloliquefaciens Bk7. Four potential bioactive antagonists were selected from 120 Bacillus isolates. Results obtained from in vitro laboratory assay showed that rhizosphere bacterial strain Bk7 and its metabolites significantly suppressed the growth of Pseudomonas fuscovaginae with 93 % efficacy. In glasshouse experiments, strain Bk7 exhibited biocontrol efficacy of 76.6 % by reducing the disease incidence to 16.9 %, compared to 72.8 % observed in control treatment. In addition, the isolate Bk7 showed the growth promotion efficacy of plant height (GPE, 46.4 %) and fresh weight (GPE, 84.3 %). Characterization of isolate Bk7 revealed its strong capability for biofilm formation, inorganic phosphate solubilization and production of high amounts of Indole-3 acetic acid, siderophores and ammonia in vitro. Results obtained from multiplex PCR assay confirmed the presence of five lipopeptide biosynthetic gene markers (srfAA, fenD, bmyB, bacA and ituC) in the genome of strain Bk7. Moreover, Real-time qPCR of these genes demonstrated that surfactin, iturin and bacylisin coding genes were highly expressed in response to P. fuscovaginae exposure in vitro. Rhizosphere bacterial strain Bk7 was identified as B. amyloliquefaciens strain Bk7 based on the analysis of 16S rDNA internal transcribed spacer sequences and a fatty acid methyl ester analysis. The results obtained from this study showed the potential usefulness of Bk7 as a biocontrol agent in disease control of rice brown sheath rot.     

Comparison of ethylene-producing Pseudomonas syringae strains isolated from kudzu (Pueraria lobata) with Pseudomonas syringae pv. phaseolicola and Pseudomonas syringae pv. glycinea

The relationships among strains of Pseudomonas syringae pv. glycinea (Psg) and Pseudomonas syringae pv. phaseolicola (Psp) isolated from kudzu ( Pueraria lobata) and bean ( Phaseolus vulgaris) were investigated. All strains tested showed a close phenotypic similarity, with the exception of the utilization of inositol and mannitol as well as the production of toxins. On this basis the strains could be divided into three groups. Group 1 consists of all strains of pathovar glycinea, group 2 includes all Psp strains isolated from kudzu, and all Psp strains isolated from bean belong to group 3. This grouping was also reflected in the genetic fingerprints using the polymerase chain reaction (PCR) with primers that anneal to dispersed repetitive bacterial sequences (rep-PCR). The rep-PCR generated fingerprints were unique for each of the three groups. The strains of group 2, Psp strains isolated from kudzu, possess certain characteristics of group 1 (ethylene production) and group 2 (phaseolotoxin production). The Psp strains from kudzu can be clearly differentiated from Psp strains isolated from bean. They utilize mannitol, produce ethylene, and are strongly pathogenic to kudzu, bean, and soybean. The results obtained show that the Psp strains from kudzu should be separated from the pathovar phaseolicola and should represent their own pathovar.     

Identifying resistance in wild and ornamental cherry towards bacterial canker caused by Pseudomonas syringae

Bacterial canker is a major disease of stone fruits and is a critical limiting factor to sweet cherry (Prunus avium) production worldwide. One important strategy for disease control is the development of resistant varieties. Partial varietal resistance in sweet cherry is discernible using shoot or whole tree inoculations; however, these quantitative differences in resistance are not evident in detached leaf assays. To identify novel sources of resistance to canker, we used a rapid leaf pathogenicity test to screen a range of wild cherry, ornamental Prunus species and sweet cherry × ornamental cherry hybrids with the canker pathogens, Pseudomonas syringae pvs syringae, morsprunorum races 1 and 2, and avii. Several Prunus accessions exhibited limited symptom development following inoculation with each of the pathogens, and this resistance extended to 16 P. syringae strains pathogenic on sweet cherry and plum. Resistance was associated with reduced bacterial multiplication after inoculation, a phenotype similar to that of commercial sweet cherry towards nonhost strains of P. syringae. Progeny resulting from a cross of a resistant ornamental species Prunus incisa with susceptible sweet cherry (P. avium) exhibited resistance indicating it is an inherited trait. Identification of accessions with resistance to the major bacterial canker pathogens is the first step towards characterizing the underlying genetic mechanisms of resistance and introducing these traits into commercial germplasm.