香蕉枯萎病内生生防菌H4-3抑菌蛋白提取及抑菌活性研究

铜绿假单胞菌Pseudomonas aeruginosa H4-3菌株是福建省农科院农业生物资源研究所微生物研究中心从香蕉植株中分离到的一株内生菌,该菌株对香蕉枯萎病病原菌具有很强的抑菌活性,初步判断其活性物质为大分子蛋白.采用硫酸铵沉淀方法提取H4-3菌株的抑菌蛋白,研究不同硫酸铵浓度对H4-3菌株抑菌活性的影响,结果表明：随着硫酸铵浓度的提高,H4-3菌株抑菌蛋白粗提液的抑菌活性逐步提高,其盐析后的上清液抑菌活性则逐步降低,70％饱和度的硫酸铵处理即可完全沉淀.     

长薄鳅恶臭假单胞菌的分离鉴定及其感染的病理损伤

恶臭假单胞菌(Pseudomonas putida)是广泛分布于土壤、水体中的一种条件致病菌,可感染两栖类、鱼类和甲壳类等多种水生动物。2017年8月,四川省西昌市某养殖场的长薄鳅出现一种以体表溃烂、鳍条出血为主要特征的传染病。为探究其病因,本研究进行了病原菌分离、人工感染、分离菌表型特征测定、16S rRNA与gyrB基因序列分析。从病鱼肝、脾、肾中分离到优势菌株(BMH170820),人工感染试验证实了其病原性。根据其菌落形态、生理生化特性,结合16S rRNA与gyrB基因序列分析鉴定其为恶臭假单胞菌。组织病理学观察发现,其感染长薄鳅对鳃、肾、肝、心、脾与消化道等多处组织器官造成损伤,表现出明显的变性、坏死和炎症反应,致全身多器官功能障碍而死亡。药物敏感试验显示,该菌对新霉素、氧氟沙星以及强力霉素等敏感,而对阿莫西林、头孢氨苄与氟苯尼考等耐药。     

荧光假单胞菌与红绒盖牛肝菌共接种对杨树根际土壤酶活性及微生物多样性的影响

【目的】探究荧光假单胞菌与红绒盖牛肝菌共接种条件下NL-895杨根际土壤酶活性和土壤微生物功能多样性的变化,从微生态水平揭示溶磷细菌与外生菌根真菌互作对NL-895杨的促生机制。【方法】采用盆栽试验考察荧光假单胞菌JW-JS1与红绒盖牛肝菌(Xc)共接种后NL-895杨根际土壤酶(酸性磷酸酶、脱氢酶和转化酶)的活性变化,并通过BIOLOG微孔板法分析共接种对NL-895杨根际土壤微生物群落和功能多样性的影响。【结果】从整体趋势来看,不同接种处理后NL-895杨根际土壤酶活性均有所增强,土壤酸性磷酸酶、脱氢酶和转化酶活性均大于对照,其中,共接种JW-JS1+Xc处理对土壤酸性磷酸酶和转化酶活性的促进最为明显;接种150天后,不同接种处理土壤微生物的AWCD值均高于CK(培养24～48 h除外),且随着培养时间的延长呈先上升后稳定的趋势;培养96 h后,共接种JW-JS1+Xc处理土壤微生物McIntosh优势度指数和碳源利用丰富度指数显著高于单接种Xc或JW-JS1和CK(P<0.05),而Shannon丰富度指数和Simpson优势度指数显著低于单接种Xc或JW-JS1和CK(P<0.05);不同接种处理NL-895杨根际土壤微生物对BIOLOG微孔板中6大类碳源的利用能力差异显著(P<0.05)。【结论】荧光假单胞菌JW-JS1与外生菌根真菌Xc共接种对NL-895杨根际土壤酶活性和土壤微生物功能多样性的提高具有一定的促进作用,有利于其土壤肥力的改善,进而促进NL-895杨的生长。与单接种相比,溶磷细菌与外生菌根真菌共接种可更有效地提高杨树根际土壤酶活性和土壤微生物多样性。     

绿脓杆菌ATCC27853菌株外毒素A全长编码基因的克隆及其在 HEK 293T细胞中的表达

采用PCR方法从绿脓杆菌ATCC27853菌株基因组扩增其外毒素A（PEA）全长编码基因,并将PEA基因插入到pcDNA3.1A真核表达载体中,构建成pcDNA3.1/ PEA质粒表达载体。经磷酸钙介导将重组质粒转染 HEK293T 细胞进行表达。结果表明本文克隆的PEA基因与标准菌株PA103碱基序列基本相似,同源性达99%。表达产物经蛋白免疫印迹法（Western-blot）检测,表明本文构建的含有原基因信号肽的PEA基因能够在真核细胞进行分泌性表达,这为基于PEA的免疫毒素、疫苗佐剂和疫苗载体的研究奠定了基础。     

辣椒细菌性果实条斑病菌生物学特性及抑菌药剂筛选

研究辣椒细菌性果实条斑病菌的生物学特性,对病原菌进行室内药剂筛选,旨在为田间病害规律研究和病害防治提供依据。采用紫外分光光度计测定病原菌在不同温度、pH、NaCl浓度下的生长速率。采用滤纸片抑菌圈法对病原菌进行室内药剂筛选试验。结果表明,辣椒细菌性果实条斑病菌最适生长温度为28℃,最适pH 7.0,病原菌的耐盐性为12%。18种供试药剂在药剂推荐使用剂量范围内,四霉素、乙蒜素、77%志信、氯溴异氰尿酸4种药剂对病原菌有抑菌效果,对这4种药剂进行毒力测定,结果表明四霉素的毒力较强,EC₅₀值为11.75 mg/L,毒力强于其他3种试剂,四霉素可作为田间防治药剂的首选。     

产2,4-二乙酰基间苯三酚的微生物研究进展

在土壤环境中,大多数2,4-二乙酰基间苯三酚(2,4-DAPG)是由荧光假单胞菌(Pseudomonas fluorescens)产生的。它在生物防治中具有重要作用。对近年来2,4-DAPG的生物合成及调控机理,2,4-DAPG在诱导系统抗性(ISR)的机制,2,4-DAPG的生物反应模式、生态效应,荧光假单胞菌农田生物防治应用实例等相关研究进行了综述。     

适用于猪场沼液处理的好氧反硝化菌筛选及其脱氮特性评价

试验旨在筛选适合于猪场沼液处理的好氧反硝化菌。从活性污泥中分离获得 16 株好氧反硝化菌,其中菌株 ZH-14 的总氮(TN)和硝酸盐氮的去除效果最好,分别达到 50.73%和 99.99%。该菌株经过平板形态观察、功能基因和 16S rDNA 基因分析,鉴定为施氏假单胞菌(Pseudomonas stutzeri)。结果表明:菌株 ZH-14 在硝酸钾为唯一氮源培养基中生长时,48 h 后硝酸盐氮与 TN 的降解率分别为 100%和41.76%;以亚硝酸钠为唯一氮源培养时,亚硝酸盐氮降解率为 99.24%;以氨氮为唯一氮源培养时,氨氮的降解率达 94.1%;使用菌株 ZH-14 处理畜禽沼液,当其接种终浓度为 107 CFU/mL 时,经过 48 h 处理,氨氮、硝酸盐氮和化学需氧量的去除率分别为 54.4%、97.7%和 77.9%。综上表明,菌株 ZH-14 具有较强的反硝化和处理猪场沼液的能力,具有良好的应用开发前景。     

Biofilm production by pathogens associated with canine otitis externa,and the antibiofilm activity of ionophores and antimicrobial adjuvants

Otitis externa (OE) is a frequently reported disorder in dogs associated with secondary infections by Staphylococcus, Pseudomonas and yeast pathogens. The presence of biofilms may play an important role in the resistance of otic pathogens to antimicrobial agents. Biofilm production of twenty Staphylococcus pseudintermedius and twenty Pseudomonas aeruginosa canine otic isolates was determined quantitatively using a microtiter plate assay, and each isolate was classified as a strong, moderate, weak or nonbiofilm producer. Minimum biofilm eradication concentration (MBEC) of two ionophores (narasin and monensin) and three adjuvants (N‐acetylcysteine (NAC), Tris‐EDTA and disodium EDTA) were investigated spectrophotometrically (OD_570nm) and quantitatively (CFU/ml) against selected Staphylococcus and Pseudomonas biofilm cultures. Concurrently, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of planktonic cultures were assessed. 16/20 of the S. pseudintermedius clinical isolates were weak biofilm producers. 19/20 P. aeruginosa clinical isolates produced biofilms and were distributed almost equally as weak, moderate and strong biofilm producers. While significant antibiofilm activity was observed, no MBEC was achieved with narasin or monensin. The MBEC for NAC ranged from 5,000–10,000 µg/ml and from 20,000–80,000 µg/ml against S. pseudintermedius and P. aeruginosa, respectively. Tris‐EDTA eradicated P. aeruginosa biofilms at concentrations ranging from 6,000/1,900 to 12,000/3,800 µg/ml. The MBEC was up to 16‐fold and eightfold higher than the MIC/MBC of NAC and Tris‐EDTA, respectively. Disodium EDTA reduced biofilm growth of both strains at concentrations of 470 µg/ml and higher. It can be concluded that biofilm production is common in pathogens associated with canine OE. NAC and Tris‐EDTA are effective antibiofilm agents in vitro that could be considered for the treatment of biofilm‐associated OE in dogs.     

Solubilization of zinc phosphate by a strain of Pseudomonas fluorescens isolated from a forest soil

 A strain of Pseudomonas fluorescens, able to solubilize zinc phosphate, was isolated from a forest soil. Colonies of the microorganism produced clear haloes on solid medium incorporating zinc phosphate, but only when glucose was provided as the carbon source. Solubilization of zinc phosphate occurred by both an increase in the H⁺ concentration of the medium, probably a consequence of ammonia assimilation, and the production of gluconic acid. High concentrations of gluconic acid were produced when P. fluorescens 3a was cultured in the presence of zinc phosphate. Although under some conditions gluconic acid is purportedly able to solubilize metals by the formation of chelates, no evidence of zinc chelation was obtained in our experiments. Furthermore, the increased Zn²⁺ concentration caused by the solubilization process resulted in the manifestation of toxic effects on the culture. A sample of the culture, sonicated to disrupt cells, still possessed the ability to produce gluconic acid from glucose, in the presence and absence of zinc phosphate. The lack of gluconic acid overproduction in cultures of P. fluorescens 3a which were not amended with zinc phosphate suggests that at least some of the glucose oxidation required for the zinc solubilization occurred as a result of the toxic stress caused by the high Zn²⁺ concentration. Received: 16 December 1997     

A bacterium with close genetic identity to Pseudomonas mandelii associated with spring fish kills in wild bluegill Lepomis macrochirus Rafinesque and pumpkinseed sunfish Lepomis gibbosus (Linnaeus)

Pseudomonas fluorescens are known bacterial pathogens in fish. The P. fluorescens group contains at least nine different bacterial species, although species from fish have rarely been differentiated. Two isolated fish kills affecting wild bluegills, Lepomis macrochirus Rafinesque, and pumpkinseed sunfish, Lepomis gibbosus (Linnaeus), occurred in the spring of 2015 during cool water temperatures (12.5°C–15.5°C). Disease signs included severe bacteraemia with rare gross external signs. Pure bacterial cultures isolated from kidneys of all affected fish were identified as P. fluorescens using the API 20NE system, while no bacteria were isolated from asymptomatic fish. To further identify the species of bacterium within the P. fluorescens complex, genetic analysis of the 16S rRNA, rpoD and gyrB genes was conducted. DNA sequences of bacterial isolates from both mortality events were identical and had close identity (≥99.7%) to Pseudomonas mandelii. Although likely widespread in the aquatic environment, this is the first report of a bacterium closely resembling P. mandelii infecting and causing disease in fish. The bacterium grew at temperatures between 5°C and 30°C, but not at 37°C. It is possible that infections in fish were a result of immunosuppression associated with spring conditions combined with the psychrotrophic nature of the bacterium.