贵州半夏块茎腐烂病拮抗细菌的筛选与鉴定

【目的】从半夏根际土壤筛选对半夏块茎腐烂病致病菌有拮抗活性的细菌并进行鉴定。【方法】采用稀释分离法和平板对峙法分离拮抗菌,然后根据菌落形态、生理生化特性,结合分子生物学方法进行鉴定。【结果】从半夏根际土壤共分离到228株细菌,其中菌株GZDF2、GZDF3、GZDF4对病原细菌及病原真菌均有拮抗活性,抑菌圈大小达23 mm,并且抑菌谱广。经形态学观察、生理生化特性分析和分子生物学方法,将菌株GZDF2、GZDF3、GZDF4鉴定为短短芽胞杆菌(Brevibacillus brevis)。进一步采用gyr B和rpo B基因对3株短短芽胞杆菌进行聚类分析,结果显示GZDF3菌株与其他2株短短芽胞杆菌存在遗传差异。【结论】半夏根际土壤中分离到的3株短短芽胞杆菌抑菌活性强、抑菌谱广,具有开发成生防剂的潜力。     

一株马铃薯干腐病拮抗菌的筛选、鉴定及其生物防效

【背景】马铃薯干腐病是一种由镰刀菌引起的田间和储藏期都普遍发生的病害,主要引起块茎腐烂,致使马铃薯品质和产量降低,严重影响其食用价值和经济价值。【目的】发掘有效的生防菌株以控制马铃薯干腐病,并探究其抑菌作用。【方法】从甘肃定西地区马铃薯根际土壤中分离到109株细菌,以硫色镰刀菌(Fusarium sulphureum)为靶标菌,采用平板对峙法筛选拮抗菌,并通过形态学、生理生化特征及16S r RNA基因序列分析对拮抗菌株进行鉴定。检测拮抗菌无菌发酵液对F.sulphureum菌丝生长、孢子萌发、马铃薯块茎损伤接种病斑直径、干腐病发病率及对绿豆种子发芽的影响。【结果】筛选到一株对马铃薯干腐病有较强抑制作用的菌株YL11,经鉴定其为假单胞菌属(Pseudomonas sp.)菌株。YL11菌株无菌发酵液对F.sulphureum菌丝生长、孢子萌发、马铃薯块茎病斑扩展、干腐病发病率、毒素活性均有显著抑制作用。20%无菌发酵液对F.sulphureum菌落生长的抑制率达到87.3%;75%无菌发酵液可完全抑制孢子萌发;无菌发酵液浸泡能有效抑制马铃薯干腐病病斑的扩展,14 d时对病斑扩展的抑制率达到67.1%;90 d后干腐病的发生率降低了68.4%;同时降低了F.sulphureum毒素的活性。【结论】拮抗菌株YL11能显著抑制F.sulphureum的生长,对马铃薯干腐病有较强的生物防治效果,具有潜在的应用价值。     

宁海地区香鱼弧菌病病原菌鉴定

摘要：【目的】香鱼弧菌病对中国沿海地区的香鱼养殖业造成了巨大的危害,然而,病原不明导致了防治上的许多问题。本文鉴定了引起宁海地区香鱼爆发性弧菌病的病原。【方法】采用TCBS平板分离优势菌;采用回归感染试验确认病原菌,采用改进的寇氏法计算LD50;采用形态学观察、生理生化特征测定、细菌特异性引物PCR扩增检测及细菌16S rRNA和金属蛋白酶（MP）基因序列分析鉴定细菌;采用药敏实验测定它对部分抗生素的敏感性。【结果】分离并鉴定优势菌株ayu-H080701为宁海地区香鱼弧菌病的病原菌,它对香鱼的半致死量为1.2×104 CFU。形态学观察和生理生化特征测定表明,ayu-H080701与鳗利斯顿氏菌最为接近。PCR扩增检测表明,细菌16S rRNA 基因通用引物和鳗利斯顿氏菌MP基因特异引物均能扩增到预期大小的特异性条带。ayu-H080701与鳗利斯顿氏菌16S rRNA基因核苷酸序列同源性最高,为99.4%～99.5%,与同属的海弧菌和美人鱼发光杆菌分别为94.3%和91.9%;ayu-H080701与鳗利斯顿氏菌MP氨基酸序列同源性高达97.6%～98.8 %,与其它弧菌科成员则低于75.6 %,系统进化树分析也揭示ayu-H080701与鳗利斯顿氏菌进化相关性最高。【结论】引起宁海地区香鱼弧菌病的菌株ayu-H080701被鉴定为鳗利斯顿氏菌。     

甘肃大葱贮藏期镰孢菌腐烂病病原鉴定

【目的】大葱在贮藏期频繁发生镰孢菌腐烂病,损失严重。明确该病害病原种类对病害防治具有重要意义。【方法】利用组织分离法对采集自甘肃省兰州市(区)蔬菜市场的16份大葱贮藏期镰孢菌腐烂病病样进行病原物的分离、纯化培养,经单孢分离后根据形态学特征,再结合r DNA-ITS、EF-1a(tef)基因序列分析的方法进行鉴定。【结果】共分离得到80株镰孢菌,经鉴定分属3个种,即层出镰孢菌(Fusarium proliferatum)、尖孢镰孢菌(F.oxysporum)和燕麦镰孢菌(F.avenaceum),其中层出镰孢菌为大葱镰孢菌腐烂病的优势致病菌,分离频率为52.50%。对兰州白葱不同部位进行致病性测定,结果表明层出镰孢菌对大葱鳞茎的致病力最强,而燕麦镰孢菌对大葱鳞茎的致病力最弱。【结论】3种镰孢菌作为该病害的病原,属国内首次报道。     

百合枯萎病拮抗细菌的筛选、鉴定及其抑菌物质研究

【目的】筛选对百合枯萎病具有抑菌活性的拮抗细菌,对其抑菌活性物质进行初步分离纯化分析。【方法】以强致病力的百合尖孢镰刀菌(Fusarium oxysporum)为靶菌,采用系列稀释法和平板对峙法初筛拮抗细菌,并通过产铁载体能力、水解酶活性、土壤定殖力等多种生防特性指标进行复筛,结合形态学特征、生理生化指标和16S rRNA基因序列比对鉴定其分类地位;利用百合尖孢镰刀菌作为靶菌进行活性追踪,结合酸沉淀、快速柱色谱、HPLC等分离纯化手段,对菌发酵液中的抑菌活性物质进行纯化分析。【结果】在64株百合根际细菌和386株海洋细菌中进行初筛,得到9株对百合镰刀菌具有较强拮抗活性的菌株,最后筛选了1株拮抗活性较强且产铁载体能力和水解酶活性、土壤定殖能力较高的菌株11B91,鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其抑菌活性物质初步推测可能为iturin和fengycin脂肽类化合物。【结论】菌株11B91在百合枯萎病的生物防治中具有潜在的应用价值,证实海洋来源的微生物也具有防治陆地植物病原菌的潜力,为植物病害的防治拓宽了思路。     

禾谷镰刀菌拮抗菌21-6的鉴定及其抑菌活性测定

【背景】禾谷镰刀菌（Fusarium graminearum）是一种危害小麦生产的重要病原真菌。【目的】筛选对禾谷镰刀菌具有拮抗活性的链霉菌菌株,为该病原的生物防治提供理论基础。【方法】采用稀释涂布法分离链霉菌,利用平板对峙法筛选高活性拮抗菌株;通过形态学、生理生化特征和16S rRNA基因序列分析确定其分类地位;采用生长速率法分析其发酵条件及无菌发酵液的稳定性;并测定该菌株的防病效果和抑菌谱。【结果】筛选到一株对禾谷镰刀菌具有较强抑制作用的链霉菌菌株21-6,抑菌率为75.2%±2.1%。根据形态学、生理生化特征及16S rRNA基因序列分析,将其鉴定为Streptomyces stelliscabiei。菌株21-6在pH为中性条件下的PDB培养基中培养5 d能够产生更好的抑菌效果。无菌发酵液能够抑制禾谷镰刀菌的菌丝生长和孢子萌发过程。无菌发酵液不受高温、胃蛋白酶、胰蛋白酶及蛋白酶K的影响,耐酸但对碱性条件敏感。发酵液对禾谷镰刀菌侵染小麦胚芽鞘具有抑制效果。菌株21-6具有聚酮合酶pks-Ⅰ和pks-Ⅱ基因。此外,该菌株对5种植物病原真菌均具有抑制效果。【结论】链霉菌菌株21-6对禾谷镰刀菌具有较好的生防潜力。     

林下参内生生防细菌的分离鉴定及定殖能力

【背景】多年生林下参在自然环境下生长多年,其体内存在的内生菌具有更强的适应性和定殖性,可以提高植物自身抗性,抑制病原菌的生长,更好地发挥与植物的互作。【目的】筛选定殖能力强、繁殖能力快且对病原菌具有拮抗作用的优势菌株。【方法】采用常规组织分离方法,从健康林下参根部组织中分离内生菌,通过对峙试验筛选出对人参病原菌有拮抗作用的内生细菌并对其以传统的鉴定方法进行鉴定。【结果】在得到的6株内生细菌中,菌株LXS-N2对人参立枯病病原菌、人参猝倒病病原菌均有明显抑菌性,而且具有定殖性好、繁殖快的特点,通过破坏病原真菌细胞壁和细胞膜以及改变菌丝形态从而抑制病原真菌生长。【结论】经形态学观察、生理生化反应及16S rRNA基因序列分析鉴定内生菌LXS-N2为贝莱斯芽孢杆菌,具有良好的应用开发潜力。     

上海市进口火龙果软腐病病害分析

应用病原形态学和真菌rDNA-ITS分子标记及Fusarium oxysporum种特异性序列分析, 对引起上海市水果市场销售的进口火龙果软腐的市场病害进行了分离与鉴定, 明确了两种主要的致病真菌为尖孢镰刀菌(Fusarium oxysporum)和单隔镰刀菌(Fusarium dimerum)。其中, F. oxysporum为引起进口火龙果采后软腐的最主要病原真菌, 该菌最适生长温度和致病温度均为25 °C, 光照条件下的致病性强, 在15 °C和35 °C条件下致病性明显降低, 5 °C和45 °C条件下该菌无法正常生长和致病, 并且该菌还能够引起香蕉、番茄、葡萄等多种水果腐烂。系统研究引起进口火龙果采后软腐病病原真菌的生物学特性, 为进口火龙果采后病原真菌有效控制方法的制定提供了有益的参考。     

偏肿栓菌内生木霉菌株18-21抑菌效果研究

为了筛选获得对植物根腐病及食用菌腐败等有效的生防菌,采用抑菌圈法和对峙培养法筛选生防菌株,通过形态学和16S r DNA分子鉴定确定生防菌分类地位。从1株野生偏肿栓菌Trametes gibbosa子实体中分离获得1株具有抑菌效果的生防菌,代号为18-21。筛选获得的这株生防菌对植物根腐病及食用菌腐败病原菌腐皮镰刀菌(Fusarium solani)、三线镰刀菌(Fusarium tricinctum)的抑制率分别为56.7%、65.8%,并对铜绿假单胞菌(Pseudomonas aeruginosa)、枯草芽胞杆菌(Bacillus subtilis)、侧芽胞杆菌(Bacillus latersprorus)、表皮葡萄球菌(Staphylococcus epidermidis)等病原细菌均具有显著的抑菌效果,通过形态学和分子生物学分类鉴定,确定为渐绿木霉(Trichoderma viridescens)。研究结果拓宽了生防菌的分离材料来源,丰富了生防资源,具有很大的应用潜力。     

新疆野果林苹果腐烂病病原菌鉴定及药用植物内生细菌对其抑菌效果

【背景】药用植物内生细菌能产生与寄主植物相同或相似的化合物及一些新的次级代谢产物等,具有促进宿主植物生长、抵抗病虫害、降解有毒有害化合物等作用。【目的】进一步提高苹果腐烂病生物防治的效率,丰富新疆药用植物内生细菌拮抗功能菌株的资源库。【方法】从新疆伊犁新源县和塔城额敏县野果林中采集带腐烂病病斑的果树枝条,分离鉴定苹果腐烂病病原菌,并采用平板对峙法从药用植物内生细菌中筛选对苹果腐烂病具有抑制作用的拮抗菌株。【结果】从两地共分离获得234株分离株,筛选鉴定出25株Valsa malicola和2株Valsa mali;同时,筛选出92株具有抑菌效果的内生细菌菌株,其中70株来自甘草植物内生细菌。【结论】药用植物甘草中富含较为丰富的抗苹果腐烂病病原菌的微生物菌株资源。本研究在新疆野果林苹果腐烂病的生物防治及药用植物内生细菌的开发利用等方面具有重要意义。     

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。     

Characterization of Pectobacterium carotovorum subsp. carotovorum Causing Watermelon Soft Rot Disease in Iran

The aim of this study was characterized Pectobacterium carotovorum subsp. carotovorum (Pcc) the causal pathogen of watermelon soft rot disease in Iran. Of fifty bacterial isolates with white grey and convex colonies on nutrient agar obtained from symptomatic watermelon, ten isolates were selected for further tests. Pathogenicity tests results showed that all test isolates developed typical water‐soak symptoms after 2 days and signs of soft rot began 4 days after inoculation on watermelon fruits. Based on the phenotypic properties, the isolates were identified as Pectobacterium carotovorum subsp. carotovorum. The 16S rDNA sequences of isolates were 99% similar to the corresponding 16S rDNA sequence of the reference Pcc isolate. BOX and ERIC‐PCR analysis indicated that genetic diversity was present among the isolated Pcc isolates did not relate to the geographic location isolated from. To the best of our knowledge, this is the first study of biochemical and genotypic characterization of Pcc isolates the causal agents of soft rot disease on watermelon, in Iran.     

胡萝卜软腐果胶杆菌鞭毛钩基因flgK 的功能

【目的】为了研究鞭毛钩基因flgK在胡萝卜软腐果胶杆菌胡萝卜亚种(Pectobacterium carotovorum subsp.carotovorum,P.c.c)的功能。【方法】本研究采用两亲同源交换法构建了基因缺失突变体ΔflgKpcc并构建了互补菌株ΔflgKpcc-KH,测定突变体及其互补菌株的菌体形态、运动性、致病因子、致病性等表型。【结果】与野生菌株PccS1相比,ΔflgKpcc鞭毛缺失,菌体易沉降,在0.3%半固体培养基上运动能力明显降低,生长速率无明显变化,但是纤维素酶和蛋白酶的活性、生物膜形成能力明显下降,对感病寄主的致病力显著减弱。基因互补可以使上述突变表型恢复。【结论】实验表明,鞭毛基因flgK突变导致了菌体的运动性降低、病原菌毒性相关的酶活力下降,从而导致致病力下降。     

Combining flow cytometry and gfp reporter gene for quantitative evaluation of Pectpbacterium carotovorum ssp. carotovorum in Ornithogalum dubium plantlets

Aims: Ornithogalum dubium is a natural host of the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc). The present study was aimed to develop a quantification system for Pcc expressing a gfp reporter gene, using fluorescent activated cell sorter (FACS) in planta. Methods and Results: Several calibration steps were required to distinctly gate the GFP‐labelled bacteria at FL1 mode and count the bacteria. To validate the bacterial counts obtained by FACS analysis, an internal standard of polystyrene green fluorescent microsphere beads was employed, resulting in high correlation with serial dilutions and plate counting. This allowed quantification of the bacteria, with no further need to culture, dilute or plate the cells. Micropropagation tools were developed to produce uniform plantlets of O. dubium, which were either inoculated with increasing concentrations of Pcc or elicited for resistance towards Pcc using methyl jasmonate. The rapid counting procedure allowed recovering, gating and counting the bacterial population in planta, separately from the plant cells background and from the microsphere beads. Conclusions: The FACS based quantification approach of Pcc was found accurate, reproducible and time saving, thus useful for counting bacteria in planta. Significance and Impact of the Study: The combination of time‐ and cost‐saving approach for Pcc quantification with efficient screening tools during early stages of micropropagation may facilitate the preliminary process of selection for resistant cultivars.     

Efficacy of potent antagonistic yeast Wickerhamiella versatilis against soft rot disease of potato caused by Pectobacterium carotovorum subsp. carotovorum

Abstract

In this study, an antagonistic yeast isolate, Wickerhamiella versatilis was considered as a promising biocontrol agent against Pectobacterium carotovorum subsp. Carotovorum (Pcc) the causal agent of soft rot disease of potato. Antagonistic yeast inhibited the growth of Pcc in vitro, and reducing the soft rot severity of infected potato tubers (cv. Diamant) under greenhouse conditions. Consequently, cellulase and pectinase hydrolytic activities in infected potato tubers with yeast?+?Pcc were decreased compared with infected tubers with Pcc. The histological characterization of treated potato tubers with antagonistic yeast W. versatilis using scanning electron microscope showed the accumulation of extracellular substances that may induce plant resistant and protects potato tubers from hydrolysis and damages. This study may introduce the possibility of using the antagonistic yeast isolate, as a biocontrol agent against soft rot of potato tubers.     

Bacterial Stem Rot in Greenhouse Pepper (Capsicum annuum L.) in Sardinia (Italy): Occurrence of Erwinia carotovora subsp. carotovora

An unusual bacterial disease was observed in pepper plants during research carried out in greenhouses in central‐north Sardinia. The characteristics were: the presence of lesions and exudates on stems, soft rot of the pith, and a brownish‐black colour in the petioles and leaf‐veins. Only two isolates of 21 were pathogens. One was obtained from exudate present on the stem and the other from pith. Experimental infections revealed that the bacterial isolates were particularly aggressive in the stems and fruit of pepper and tomato. Biochemical, physiological and serological tests in conjunction with fatty acid profile analysis confirmed that they were Erwinia carotovora subsp. carotovora (Jones) Bergey et al. The product of 434 bp polymerase chain reaction (PCR) enabled a preliminary identification of isolates to be made. Restriction fragment length polymorphism (RFLP) analysis of amplification products showed that the isolates DPP 23_ef and DPP 24_m, strain type CFBP 2046 and DPP 28₁, isolated from pepper fruit, belonged to the RFLP group 12, whereas DPP 29, also isolated from pepper fruit, was included in RFLP group 1. Measures to prevent and control this recently introduced disease are suggested in the conclusion of this paper.     

Identification of a DNA restriction-modification system in Pectobacterium carotovorum strains isolated from Poland

AIMS: Polish isolates of pectinolytic bacteria from the species Pectobacterium carotovorum were screened for the presence of a DNA restriction-modification (R-M) system. METHODS AND RESULTS: Eighty-nine strains of P. carotovorum were isolated from infected potato plants. Sixty-six strains belonged to P. carotovorum ssp. atrosepticum and 23 to P. carotovorum ssp. carotovorum. The presence of restriction enzyme Pca17AI, which is an isoschizomer of EcoRII endonuclease, was observed in all isolates of P. c. atrosepticum but not in P. c. carotovorum. The biochemical properties, PCR amplification, and sequences of the Pca17AI restriction endonuclease and methyltransferase genes were compared with the prototype EcoRII R-M system genes. Only when DNA isolated from cells of P. c. atrosepticum was used as a template, amplification of a 680 bp homologous to the gene coding EcoRII endonuclease. CONCLUSIONS: Endonuclease Pca17AI, having a relatively low temperature optimum, was identified. PCR amplification revealed that the nucleotide sequence of genes for EcoRII and Pca17AI R-M are different. Dcm methylation was observed in all strains of Pectobacterium and other Erwinia species tested. The sequence of a DNA fragment coding Dcm methylase in P. carotovorum was different from that of Escherichia coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Pca17AI is the first psychrophilic isoschizomer of EcoRII endonuclease. The presence of specific Dcm methylation in chromosomal DNA isolated from P. carotovorum is described for the first time. A 680 bp PCR product, unique for P. c. atrosepticum strains, could serve as a molecular marker for detection of these bacteria in environmental samples.     

Bacteriophages Isolated in China for the Control of Pectobacterium carotovorum Causing Potato Soft Rot in Kenya

Soft rot is an economically significant disease in potato and one of the major threats to sustainable potato production. This study aimed at isolating lytic bacteriophages and evaluating methods for and the efficacy of applying phages to control potato soft rot caused by Pectobacterium carotovorum. Eleven bacteriophages isolated from soil and water samples collected in Wuhan, China, were used to infect P. carotovorum host strains isolated from potato tubers showing soft rot symptoms in Nakuru county, Kenya. The efficacy of the phages in controlling soft rot disease was evaluated by applying individual phage strains or a phage cocktail on potato slices and tubers at different time points before or after inoculation with a P. carotovorum strain. The phages could lyse 20 strains of P. carotovorum, but not Pseudomonas fluorescens control strains. Among the 11 phages, Pectobacterium phage Wc5 r, interestingly showed cross-activity against Pectobacterium atrosepticum and two phage-resistant P. carotovorum strains. Potato slice assays showed that the phage concentration and timing of application are crucial factors for effective soft rot control. Phage cocktail applied at a concentration of 1 9 109 plaque-forming units per milliliter before or within an hour after bacterial inoculation on potato slices, resulted in C 90%reduction of soft rot symptoms. This study provides a basis for the development and application of phages to reduce the impact of potato soft rot disease.     

Pectobacterium carotovorum subsp. brasiliense Causing Soft Rot on Paprika in Korea

During summer 2011 in South Korea, severe fruit rot of paprika was observed, causing severe economic losses in paprika production. Symptoms of fruit and pedicel decay were consistent with symptoms caused by Pectobacterium carotovorum subsp. brasiliense (Pcb) as recently described in Brazil, the United States, Israel and South Africa. Physiological analysis and pathogenicity test of strains isolated from paprika fruit revealed that the pathogen was the bacterium Pcb. Sequencing and phylogenetic analysis of the 16S rDNA and partial 16S–23S rDNA intergenic spacer region confirmed that the isolates were Pcb. This is the first report of Pcb in Korea, which has a significant economic impact on Korean paprika production.     

Natural Fusarium Grain Infection Level in Wheat, Barley and Oat after Early Application of Fungicides and Herbicides

In Norway, early application of fungicides against cereal leaf diseases (before Zadoks 60) is common practice amongst farmers. Whether this procedure has any effect on Fusarium infection of the mature grain has been little investigated. To evaluate effects on Fusarium grain infection, cereal grains were sampled during 1996, 1997 and 1998 from 12 field trials where early spraying against fungal diseases in spring wheat, spring barley and oats was carried out. Percentage infected grains and frequency of different Fusarium species was analysed in every grain sample. The effect of fungicides, glyphosate and postemergence herbicides on Fusarium grain infection was studied. Significant increase in Fusarium infection was detected in fungicide‐treated plots compared with untreated plots. Fusarium avenaceum and F. tricinctum were the most frequent species detected. The internal ranking of Fusarium species remained the same after spraying. No significant effects were found on the level of Fusarium infection after glyphosate treatment in autumn or herbicide treatment during the growing season.