Isolation of antifungal bacteria from Japanese fermented soybeans, natto

An inhibitory effect of a traditional Japanese fermented food, natto, was found against plant pathogens such as Rhizoctonia solani and Fusarium oxysporum, and the bacteria which showed inhibition were isolated from the natto. Among isolated bacteria, BC-1 and GAc exhibited a strong antagonistic effect in vitro against plant pathogens on an agar medium. The supernatant of bacterial culture also showed strong activity against R. solani, which meant the antimicrobial substances were produced and secreted into the medium. Both of the bacteria were estimated as Bacillus amyloliquefaciens from a partial sequence of the 16s rRNA gene. High performance liquid chromatography analysis clearly showed the production of the lipopeptide antibiotic iturin A by BC-1 and GAc.     

Screening of endophytic bacteria against fungal plant pathogens

Bacterial endophytes were found from 6 plant leaves among 35 plant leaves screened. Two of the isolated bacteria showed antagonistic activity against fungal plant pathogens. An isolate named KL1 showed the clear inihibition against plant pathogens, Fusarium oxysporum and Rhizoctonia solani, on PDA as well as TSA plate. Supernatant of the bacterial culture also showed the clear inhibition against the fungal growth on the plate and the antibiotic substance was identified as iturin A by HPLC analysis. KL1 was identified as Bacillus sp. from the 16S rRNA gene analysis. Very thin hyphae of R. solani was miccroscopically observed when the fungus was co-cultivated with KL1.     

Screening of endophytic bacteria against fungal plant pathogens

Bacterial endophytes were found from 6 plant leaves among 35 plant leaves screened. Two of the isolated bacteria showed antagonistic activity against fungal plant pathogens. An isolate named KL1 showed the clear inihibition against plant pathogens, Fusarium oxysporum and Rhizoctonia solani, on PDA as well as TSA plate. Supernatant of the bacterial culture also showed the clear inhibition against the fungal growth on the plate and the antibiotic substance was identified as iturin A by HPLC analysis. KL1 was identified as Bacillus sp. from the 16S rRNA gene analysis. Very thin hyphae of R. solani was miccroscopically observed when the fungus was co-cultivated with KL1.     

应用FCM-qPCR方法定量检测水中常见病原体

以往对水体病原体的研究主要是通过监测粪大肠杆菌作为指示,然而研究表明粪大肠杆菌与水中病毒和细菌病原体呈现出较差的相关性.因此,选取水中典型病原体并对其进行定量检测是当前需要解决的技术问题.为此本研究建立了流式细胞术和定量PCR联合使用方法,用于快速获取水环境中总病毒、细菌以及几种典型病原体(大肠杆菌、军团菌、腺病毒、贾第虫和隐孢子虫等)的浓度水平,并将该方法应用到污水处理厂进出水及受纳河流上下游的病原体检测中.结果表明,该污水处理厂对总细菌和总病毒以及几种典型病原体都具有较高的去除率(93%);污水处理厂排水对受纳水体病原体浓度水平基本没有负面影响.研究为评估污水处理厂处理效果及排水对受纳水体的生态影响提供了技术支持.     

海洋细菌N3对几种赤潮藻的溶藻效应

从深圳大鹏湾南澳赤潮暴发海域的表层沉积物中分离得到1株海洋溶藻细菌(编号N3),以三角褐指藻(Phaeodactylumtricornutum)和常见赤潮生物锥状斯氏藻(Scrippsiella trochoidea)、海洋原甲藻(Prorocentrum micans)和中肋骨条藻(Skeletonemacostatum)为实验藻种,利用液体感染法研究了该株溶藻细菌的溶藻效应及溶藻方式.结果表明,菌株N3对中肋骨条藻和三角褐指藻均无溶藻活性,而菌悬液的体积分数为2%和10%时分别对锥状斯氏藻和海洋原甲藻有较好的溶藻效果,但溶藻现象不同.菌株N3可使锥状斯氏藻的细胞变形,胞内物质分布不均匀并最终死亡;使海洋原甲藻的藻细胞膨胀变形,细胞膜内物质聚集于一端,最终藻细胞破裂死亡.菌株N3对锥状斯氏藻和海洋原甲藻的溶藻方式相同,均以直接溶藻为主.菌株N3的菌悬液以2%、1%和0.1%的体积分数接种到锥状斯氏藻藻液中时,细菌的密度整体上呈下降趋势,2%处理组中藻细胞在120 h内全部死亡,而1%和0.1%处理组藻细胞的增长率略低于对照组.菌株N3的菌悬液以10%、5%和1%的体积分数接种到海洋原甲藻藻液中时,细菌的密度呈下降趋势,10%和5%处理组中藻细胞至120 h时死亡率分别为78%和70%,而1%处理组的藻细胞与对照组无显著差异.电镜观察、生理生化鉴定及16S rRNA鉴定结果表明,菌株N3为芽胞杆菌属(Bacillus sp.).     

Aqueous and organic extracts of Trigonella foenum-graecum L. inhibit the mycelia growth of fungi

Aqueous extracts from various plant parts of fenugreek (3%) (aerial parts: leaves and stems (LS), roots (R), ground seeds (GS) and not ground seeds (NGS)) and petroleum ether, ethyl acetate and methanolic fractions of the aerial parts were assayed to determine their antifungal potential against Botrytis cinerea, Fusarium graminearum, Alternaria sp., Pythium aphanidermatum, and Rhizoctinia solani. All fenugreek plant parts showed antifungal potential and the magnitude of their inhibitory e ects was species and plant parts dependent. R extract was shown less toxic (30.38%), whereas NGS extract expressed the strongest inhibition, with an average of 71.44%, followed by GS (58.56%) and LS (57.1%). Screening indicated that P. aphanidermatum was the most resistant species, with an average inhibition of 34.5%. F. graminearum, Alternaria sp. and R. solani were the most sensitive species, and were similarly inhibited (63.5%). The stability test indicated that the aqueous extracts of all plant parts lost approximately 50% of their relative activity after one month of storage at 4°C, whilst they lost 60%–90% of their activity when stored at ambient temperature for one month. The antifungal activity resided mainly in the methanol fraction and the minimum inhibitory concentration (MIC) of methanol fraction witch caused total inhibition of R. solani and Alternaria sp. was 60 g/ml. Results of current study suggested that the constituents of Trigonella foenum-graecum have potential against harmful pathogenic fungi. Therefore, fenugreek could be an important source of biologically active compounds useful for developing better new antifungal drugs.     

Indigenous bacteria may interfere with the biocontrol of plant diseases

Prodigiosin is a reddish antibiotic pigment that plays an important role in the biocontrol of plant diseases by the bacterium Serratia marcescens. However, its activity is unstable under agricultural conditions; further, it can be degraded by various environmental factors. To examine the effect of epiphytic microbes on the stability of prodigiosin used for biological control processes, we collected a total of 1,280 bacterial isolates from the phylloplane of cyclamen and tomato plants. Approximately 72% of the bacterial strains isolated from the cyclamen plants and 66% of those isolated from the tomato plants grew on minimal agar medium containing 100 μg ml⁻¹ prodigiosin. Certain isolates obtained from both plant species exhibited prodigiosin-degrading activity. We compared the 16S rRNA gene sequences derived from the isolates with sequences in a database. The comparison revealed that the sequences determined for the prodigiosin-degrading isolates were homologous to those of the genera Pseudomonas, Caulobacter, Rhizobium, Sphingomonas, Janthinobacterium, Novosphingobium, and Rathayibacter. These results indicate that indigenous epiphytic microorganisms may interfere with the interaction between plant pathogens and biocontrol agents by degrading the antibiotics produced by the agents.     

污水处理厂二级出水中四环素抗性菌的生长特性与耐药性

抗生素抗性菌的扩散已成为全世界面临的重要公共卫生安全隐患.本文从某城市污水处理厂二级出水中分离出21株四环素抗性菌,对其种属、生理生化特性、抗生素耐受性进行了研究.结果表明,21株四环素抗性菌均属于肠杆菌科,分别属于气单胞菌属(9株)、埃希氏菌属(5株)、肠杆菌属(3株)、克雷伯氏菌属(2株)、柠檬酸杆菌属(1株)和哈夫尼菌属(1株).这些菌大多属于条件致病菌,且有18株携带质粒.21株四环素抗性菌中,气单胞菌、埃希氏菌的平均最大生长量显著高于肠杆菌,大部分菌株的迟滞时间在2 h以内.抗生素耐受性试验结果表明,四环素抗性菌对氯霉素耐受能力最强,对β-内酰胺酶类抗生素(青霉素、氨苄青霉素、头孢噻吩)的耐受性次之,对利福平的耐受性最弱.75%以上的四环素抗性菌同时表现出对其它5种抗生素具有抗性.以上结果为我国污水处理厂抗生素抗性菌的风险分析与控制提供了参考数据.     

A2O工艺活性污泥中可培养丝状细菌的多样性

活性污泥法是应用最广泛的污水处理方法之一,但是由于丝状菌过度繁殖而引发的污泥膨胀是制约其发展的重大难题.本研究从A2O工艺城市污水处理系统的膨胀期活性污泥中分离培养出17株丝状细菌.对各菌株进行了16S rDNA测序和系统发育树分析,结果表明,这些分离的可培养丝状细菌均属于链霉菌属;利用rep-PCR指纹图谱技术进一步分析了所得菌种属内多样性,显示出活性污泥中链霉菌存在丰富的多样性.由于这些可培养丝状细菌与引起污泥膨胀的优势丝状菌(微丝菌)生理特征差别较大,不会在污水处理系统中过度繁殖,本研究向活性污泥中投加一定量的链霉菌分离菌株,发现部分菌株对污泥的沉降性能有明显改善作用,为污泥膨胀的防控提供了新的思路.     

Antibacterial potency of housefly larvae extract from sewage sludge through bioconversion

Use of the fly to convert sewage sludge into nutrient-rich soil conditioner and amendment is an attractive approach for sludge bioconversion. During this process, fecal coliforms, an indicating pathogen, in sludge were reduced to 5.3 × 102 most probable number /g dry solid from initial 3.32 × 106 MPN/g dry solid. It was also found that the extract of larvae grown in sludge during bioconversion have an observable inhibitory effect against bacteria compared to larvae grown in wheat bran as measured by minimum bacterial concentration tests. In vitro antimicrobial assay tests over time also showed that the extract had strong inhibitory efficiencies of ca. 99% against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens, while the efficiency was 69% and 57% against Bacillus subtilis and Klebsiella pneumoniae, respectively. The observed pathogenic bacterial cell membrane damage was found to be responsible for the phenomenon mentioned above, with nuclear acids leaching out quickly and alkaline phosphatase increasing in the outer membrane, followed by an increase of β-galactosidase in the inner membrane. Clearly, housefly larvae extract from sewage sludge through bioconversion possesses antibacterial potency against pathogenic bacteria.     

Colourimetric analysis and antimicrobial study of natural dyes and dyed silk

Colourimetric and antimicrobial activities of natural colourants and dyed silk have been studied. The colour strength (K/S) values were increased with increase in dye absorbance. The absorption of dyes on silk yarns was obtained from 10.56 to 39.48% at 5% concentration of dye from different plant extracts. The colourimetric parameters L¹, a¹, b¹, C, and H were measured for depth of the colour. The dyed silk with natural colourants displayed excellent antimicrobial activity (reduction rate: 25–65%) against the bacteria Escherichia coli and (reduction rate: 3–68%) against the fungal strain Aspergillus niger. The dyed silk exhibited good and durable fastness properties.     

一株架桥细菌的共凝集能力及强化生物膜反应器效能的研究

测定了架桥细菌Bacillus cereus G5与活性污泥中分离得到的13株土著细菌及3株复杂有机物降解菌配对组合后的共凝集能力和成膜能力.结果表明:凝集2 h和20 h时的共凝集率分别达到40%~70%和55%~80%;架桥细菌与其中15株细菌混合培养时的生物膜形成量高于单菌培养时的生物膜量.这表明在废水处理系统外G5能与土著菌和降解菌发生较强程度的共凝集,并可促进多数菌株生物膜量的增加.进一步考察了G5与3,5-二硝基苯甲酸降解菌Comamonas testosterone A3投加到序批式生物膜反应器(SBBR)中的生物强化效果.32 d的运行结果表明,同时投加A3与G5菌株的反应器,24 h时出水中3,5-二硝基苯甲酸由100.0 mg·L-1降解至10.1 mg·L-1,降解最快;3,5-硝基苯甲酸负荷由100 mg·L-1增加到1000 mg·L-1的运行过程中,平均降解率稳定在65.0%~88.1%的范围内,表现出最强的抗冲击能力;生物膜量在1.4~2.0 mg·cm-2之间,比其他两组反应器同期时的生物膜量略高.表明在废水处理系统内Bacillus cereus G5亦可能通过其广泛的共凝集能力,促进反应器中生物膜的形成,并辅助降解菌以自固定化方式定殖于生物膜,从而表现出快速的生物强化作用和较强的抗冲击能力.     

Innate humoural immunity is related to eggshell bacterial load of European birds: a comparative analysis

Fitness benefits associated with the development of a costly immune system would include not only self-protection against pathogenic microorganisms but also protection of host offspring if it reduces the probability and the rate of vertical transmission of microorganisms. This possibility predicts a negative relationship between probabilities of vertical transmission of symbionts and level of immune response that we here explore inter-specifically. We estimated eggshell bacterial loads by culturing heterotrophic bacteria, Enterococcus, Staphylococcus and Enterobacteriaceae on the eggshells of 29 species of birds as a proxy of vertical transmission of bacteria from mother to offspring. For this pool of species, we also estimated innate immune response (natural antibody and complement (lysis)) of adults, which constitute the main defence against bacterial infection. Multivariate general linear models revealed the predicted negative association between natural antibodies and density of bacteria on the eggshell of 19 species of birds for which we sampled the eggs in more than one nest. Univariate analyses revealed significant associations for heterotrophic bacteria and for Enterobacteriaceae, a group of bacteria that includes important pathogens of avian embryos. Therefore, these results suggest a possible trans-generational benefit of developing a strong immune system by reducing vertical transmission of pathogens.     

给水管网中耐氯性细菌的灭活特性研究

使用4种常见消毒剂对从实际管网中分离出来的7株耐氯性细菌进行消毒实验.结果表明,这7株细菌均具有较高的耐氯性,其中1株耐氯性最高的类龟分支杆菌自由氯99.9%灭活的CT值为120 mg.(L.min)-1,另外2株血红鞘氨醇单胞菌和甲基杆菌99.9%灭活的CT值分别为7 mg.(L.min)-1和4 mg.(L.min)-1.比较4种消毒剂的消毒效果发现,二氧化氯和单过硫酸氢钾的消毒效果较好,能够在30 min内使分支杆菌的灭活率达到5个数量级.自由氯由于衰减较快,消毒效果不佳.一氯胺能够维持一定的消毒剂浓度,但由于其氧化性较弱,因此需要提高浓度,才能满足消毒要求.能在1 h内灭活3个数量级以上分支杆菌的消毒剂投加量为:3.0 mg/L一氯胺、1.0 mg/L二氧化氯(以Cl2计)和1.0 mg/L单过硫酸氢钾(以Cl2计).考虑到我国水厂消毒的实际情况,建议采用间歇性提高一氯胺浓度或改换二氧化氯消毒的方法,提高对耐氯性细菌的灭活效果.     

攀枝花矿区糙野青茅根际耐铬放线菌筛选及促生能力评价

从攀枝花矿区糙野青茅根际土中分离放线菌,筛选获得对重金属铬具有强耐受性、抗菌促生和产胞外酶能力的放线菌,为微生物-植物联合修复重金属污染提供菌种资源.采用稀释涂布法进行放线菌的分离,通过放线菌在不同铬浓度高氏一号培养基上的生长状况判断其对重金属铬的耐受性,进一步采用原子吸收分光光度法分析菌株对铬耐受性强的菌株耐铬及去铬能力;通过对菌株产铁载体、溶磷、产吲哚乙酸(IAA)、产纤维素酶、产淀粉酶、产木糖酶、产酪蛋白酶能力及抗菌活性的筛选,综合评价菌株抗菌促生能力.结果显示,分离获得的27株根际菌株,光学显微形态及扫描电镜结果均显示为放线菌,其中有14株能在含铬的培养基上生长,具有耐铬能力.16S rRNA基因测序结果显示,14株放线菌分属于链霉菌属(streptomyces)、小单孢菌属(Micromonospora)和冢村氏菌属(Tsukamurella).菌株SCAU9002和SCAU9008在铬浓度为300mg·L~(-1)的处理下培养6 d,去铬率分别达到81.17%和78.41%;菌株SCAU9002、SCAU9006、SCAU9008、SCAU9009、SCAU9013同时具有3种促生功能,盆栽实验结果进一步表明,在重金属胁迫下,经菌株处理后的玉米植株更高,生物量更大,根系更发达,促生效果较好.此外,菌株SCAU9006对6种病原菌具有拮抗作用,5株菌同时具有3种及以上产胞外水解酶的能力,4株菌能在7%的NaCl浓度下生长,13株菌在pH5.0~8.0的范围内均可生长.结果表明,根际放线菌在促进矿区植物生长和铬尾矿污染土壤的生物修复研究中具有很大潜力.     

接种内生细菌对龙葵吸收积累镉的影响

通过在LB培养基中加入不同浓度的镉,从镉超富集植物龙葵植株内分离到3株很强的镉抗性内生菌株N1、N2、N4,最高耐受镉浓度达300 mg·L^-1.通过生理生化鉴定和16S rDNA序列分析,初步确定N1为芽孢杆菌属（Bacillus sp.）细菌,N2为肠杆菌属（Enterobacter sp.）细菌,N4为巨大芽孢杆菌（Bacillus megaterium）.将龙葵种植在含有一定镉（50 mg·kg^-1）的土壤中进行盆栽试验,以灌根的方式分别接种分离到的3株细菌及其混菌组合,并对其株高、生物量及镉吸收能力进行分析.结果显示,N4菌株处理的龙葵叶、茎、根部镉含量比不接菌对照分别增加32.94%、14.50%、23.44%,差异显著（p<0.05）;接种N1+N2+N4混菌的处理龙葵叶、茎和根干重分别比不接菌对照高出118.22%、109.83%和113.01%,植株地上和地下镉吸收总量分别增加109.53%和83.01%,差异显著.试验表明供试菌株能显著促进植株生长,强化龙葵吸收土壤中镉的能力.     

营养物质对铜绿微囊藻生长和藻际细菌的影响

菌-藻"体系在水生态平衡中发挥重要作用,为探明蓝藻水华时期的菌藻关系,研究了原位营养刺激后藻际微生物对铜绿微囊藻生长的影响.结果表明,LB培养基可抑制铜绿微囊藻生长,抑藻率为86.49%;而乙酸钠、葡萄糖和柠檬酸钠可促进藻细胞生长,增长率均在50%以上.对LB培养基和蛋白胨刺激后的藻际细菌进行溶藻菌的筛选,共分离出6株具有强效抑藻作用的菌株,其中Bacillus sp.A1抑藻率高达97.55%.16S rRNA高通量测序表明,向铜绿微囊藻中投加营养物质均可改变藻际细菌群落结构并增加物种丰富度,添加LB培养基和蛋白胨可促使藻际细菌群落数量剧增.生理生化响应表明,藻细胞受LB培养基和蛋白胨胁迫后,活性氧（ROS）水平和丙二醛（MDA）含量激增,细胞氧化损伤严重;超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性先急剧上升后下降,抗氧化酶系统受损.扫描电镜显示,LB培养基可使藻细胞逐渐萎缩,最终破碎;而藻际细菌显著增多.因此,在蓝藻水华暴发的水体进行适当的外部营养刺激可在一定程度上实现溶藻菌原位抑藻.     

多环芳烃降解真菌Fusarium solani对芘的吸收存储和降解特性研究

以芘为唯一碳源,从公路旁污染土壤中分离得到一株芘降解真菌,经鉴定为Fusarium solani(命名为Py F-1),荧光显微镜观察发现其菌丝能吸收储存芘.对该菌株在芘浓度为50 mg·L-1的液体培养基中对芘的吸收储存和降解特性,具体包括芘的生物降解、菌体吸收、菌体吸附和消解总量进行了39 d的动态研究,结果表明:生物降解芘在培养0~14 d内逐渐增加,培养14 d时占芘添加量的15.9%,此后基本达到稳定,培养结束时生物降解芘为芘添加量的17.6%;菌体吸收芘在0~21 d内含量较少且变化不显著,低于芘添加量的3%;而在21~39 d内,菌体吸收芘变化显著,芘的消解总量随着菌体吸收量的变化产生显著波动;培养28 d时,芘的消解总量达到最大值,其中菌体吸收芘占芘消解总量的一半,达到芘添加量的16.0%,菌体吸附芘不足芘添加量的0.5%,在研究中可以忽略不计.因此,培养前期培养基中芘的消解以生物降解为主,培养后期菌体吸收芘的变化是芘消解总量变化的主要原因.     

油气田土壤样品中可培养丁烷氧化菌多样性研究

分别采用丁烷或丁醇选择培养法,对采自四川省达州市普光气田和湖北省江汉油田的土壤样品中的丁烷氧化菌进行了分离、纯化、16S rRNA基因序列测定及系统发育研究,探讨不同油田不同环境中烃氧化菌数量、系统发育地位以及种群多样性的差异.结果表明,初步分离获得共25株丁烷氧化菌,分属于厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和变形菌门(Proteobacteria).2个油田样品分离出的丁烷氧化菌群落结构简单,均包括芽孢杆菌(Bacillus)、假单胞菌(Pseudomonas)、红球菌(Rhodococcus)和节杆菌(Arthrobacter)4个属的细菌,苍白杆菌属(Ochrobactrum)和分支杆菌属(Mycobacterium)的细菌分离自普光气田.普光气田分离出的丁烷氧化细菌数量和多样性略高于江汉油田.     

Antifungal activity of the pygidial gland secretion of <Emphasis Type="Italic">Laemostenus punctatus</Emphasis> (Coleoptera: Carabidae) against cave-dwelling micromycetes

The antifungal potential of the pygidial gland secretion of the troglophilic ground beetle Laemostenus punctatus from a cave in Southeastern Serbia against cave-dwelling micromycetes, isolated from the same habitat, has been investigated. Eleven collected samples were analyzed and 32 isolates of cave-dwelling fungi were documented. A total of 14 fungal species were identified as members of the genera Aspergillus, Penicillium, Alternaria, Cladosporium, Rhizopus, Trichoderma, Arthrinium, Aureobasidium, Epicoccum, Talaromyces, and Fusarium. Five isolates were selected for testing the antifungal activity of the pygidial gland secretion: Talaromyces duclauxi, Aspergillus brunneouniseriatus, Penicillium sp., Rhizopus stolonifer, and Trichoderma viride. The microdilution method has been applied to detect minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs). The most sensitive isolate was Penicillium sp., while the other isolates demonstrated a high level of resistance to the tested agent. L. punctatus has developed a special mechanism of producing specific compounds that act synergistically within the secretion mixture, which are responsible for the antifungal action against pathogens from the cave. The results open opportunities for further research in the field of ground beetle defense against pathogens, which could have an important application in human medicine, in addition to the environmental impact, primarily.