土壤稀有放线菌的选择性分离及其抗菌活性研究

在优化土壤稀有放线菌选择性分离方法的基础上,对从安徽、浙江、山东、陕西等地采集的31份土样中的稀有放线菌进行了选择性分离。结果表明:以改进的HV培养基为分离培养基,将土壤样品稀释100倍,添加2×10-5 g/mL重铬酸钾＋2×10-5 g/mL萘啶酮酸＋1×10-5 g/mL卡那霉素作为抑制剂分离效果最好。以常见植物病原真菌和细菌为指示菌,从分离的417株放线菌中筛选出H32、H75、H223、H227、H238等5株可代谢抗菌活性物质的菌株。离体条件下,5株放线菌菌株发酵液对小麦根腐病菌Bipolaris sorokiniana、玉米大斑病菌Exserohilum turcicum、烟草赤星病菌Alternaria alternate的菌丝生长和孢子萌发均有强烈的抑制作用,其中H223和H238菌株发酵液对病原真菌菌丝生长的抑制中浓度(EC50)值均小于10 mL/L;H32菌株发酵液对小麦根腐病菌、玉米大斑病菌、烟草赤星病菌孢子萌发的EC50值分别为25.5、28.9和29.9 mL/L。5株放线菌发酵液对枯草芽孢杆菌、大肠杆菌、金黄色葡萄球菌抑制作用显著,其中H223菌株发酵液抑制作用最强,抑菌圈达30 mm以上。盆栽实验结果表明,5株放线菌发酵液对小麦白粉病的保护效果为73.45％~82.35％,治疗效果为67.74％~70.80％。     

土壤放线菌的选择性分离及其代谢产物抗菌活性评价*

从土壤中选择性分离放线菌,并进行抗菌活性评价,以期筛选到具有潜在应用开发价值的菌株。以改良的HV培养基为分离培养基,添加萘啶酮酸、制霉菌素作为抑制剂,对从安徽、重庆、河北、湖北、江苏、吉林、山东、四川、天津、云南及德国莱比锡等地采集的45份土壤样品中的放线菌进行了选择性分离。以植物病原菌黑曲霉、棉花枯萎病菌、稻瘟病菌、辣椒疫霉、茄青枯病菌为指示菌,通过平板琼脂块初筛,并结合摇瓶复筛和盆栽防效进行评价。共选择性分离到570株放线菌;初筛显示,212个菌株代谢产物显示了抗真菌或抗细菌活性,占总菌株数量的37.2%,33个菌株代谢产物兼具抗真菌活性和抗细菌活性,占菌株数量的5.8%;摇瓶复筛显示,其中2株放线菌TJ231、TJ430发酵液对黑曲霉、棉花枯萎病菌、稻瘟病菌、辣椒疫霉、茄青枯病菌显示了强烈的抑制效果;1株放线菌TJ561发酵液对黑曲霉、棉花枯萎菌、稻瘟菌、辣椒疫霉显示了强烈的抑制效果;盆栽试验表明,放线菌TJ231、TJ430、TJ561发酵液对辣椒疫霉病的保护效果分别为56.2 %、87.2%、61.4%。     

土壤拮抗放线菌的分离及其抗菌活性筛选

以常见植物病原真菌和细菌为指示菌,从分离的422株放线菌中筛选出28株可代谢抗细菌活性物质的菌株,其中N331、N393菌株发酵液对供试12种真菌有显著的抑制作用。离体条件下,N331、N393菌株发酵液对供试真菌菌丝生长的抑制中浓度(EC50)均小于15 mL/L;对玉米弯孢病菌、棉花枯萎病菌、小麦根腐病菌、黄瓜霜霉病菌孢子萌发的EC₅₀亦小于15 mL/L。盆栽试验结果表明,N331、N393菌株发酵原液对黄瓜霜霉病的保护效果分别为97.4%和85.6%,治疗效果分别为83.3%和59.3%。     

阿特拉津高效降解菌的分离与筛选

土样采自河北、山东一些农药厂排污口,通过室内阿特拉津无机盐培养基的驯化培养,分离得到4株在无机盐培养基上对阿特拉津有明显降解圈的降解细菌。底物阿特拉津浓度为1000mg/L,反应体系50ml,体系菌浓度为8.9×107cfu/ml,恒温30℃,180r/min,培养7d,其室内降解效率分别为40.6%、75.7%、82.3%、96.9%。其中菌株BZB-11的降解效率最高。对菌株BZB-11进行降解动态考察,结果显示,BZB-11菌株在无机盐液体培养基中(阿特拉津底物浓度为1000mg/L,反应体系50ml,体系菌浓度6.59×109cfu/ml,恒温30℃,180r/min),对阿特拉津1~3d的降解速度较快,3d可达89.5%,7d的降解率达100%。综合来看,该菌株是一株很有应用前景的高效菌株。     

Isolation and characterization of a novel simazine-degrading beta-proteobacterium and detection of genes encoding s-triazine-degrading enzymes

A moderately persistent herbicide, simazine, has been used globally and detected as a contaminant in soil and water. The authors have isolated a simazine-degrading bacterium from a simazine-degrading bacterial consortium that was enriched using charcoal as a microhabitat. The isolate, strain CDB21, was gram-negative, rod-shaped (0.5-0.6 microm x 1.0-1.2 microm) and motile by means of a single polar flagellum. Based on 16S rRNA sequence analysis, strain CDB21 was identified as a novel beta-proteobacterium exhibiting 100% sequence identity with the uncultured bacterium HOClCi25 (GenBank accession number AY328574). PCR using primers that were specific for the genes of the atrazine-degrading enzymes (atzABCDEF) of Pseudomonas sp. strain ADP showed that strain CDB21 also possessed the entire set of genes of these enzymes. Nucleotide sequences of the atzCDEF genes of strain CDB21 were 100% identical to those of Pseudomonas sp. strain ADP. Sequence identity of the atzA genes between these bacteria was 99.7%. The 398-nucleotide upstream fragment of the atzB gene of strain CDB21 was 100% identical to ORF30 of Pseudomonas sp. strain ADP, and the 1526-nucleotide downstream fragment showed 99.8% sequence similarity to the atzB gene of the pseudomonad.     

拮抗农业致病菌放线菌的分离、筛选与鉴定

采用梯度稀释法从土壤中分离出放线菌G5。该菌株对苹果轮纹病菌、油菜菌核病菌等多种农业致病真菌有很强的抑制作用。用16SrDNA序列分析法对G5进行序列分析,该菌株16SrDNA序列与链霉菌属的13个放线菌的同源性为99％。用传统细菌学的方法对G5菌株进行鉴定,其形态特征和生理生化特性与链霉菌的特征和特性相一致,表明该菌株属于链霉菌。     

Polyphasic characterization of xanthomonas strains from onion

ABSTRACT Xanthomonas leaf blight has become an increasingly important disease of onion, but the diversity among Xanthomonas strains isolated from onion is unknown, as is their relationship to other species and pathovars of Xanthomonas. Forty-nine Xanthomonas strains isolated from onion over 27 years from 10 diverse geographic regions were characterized by pathogenicity to onion and dry bean, fatty acid profiles, substrate utilization patterns (Biolog), bactericide resistance, repetitive sequence-based polymerase chain reaction fingerprinting, rDNA internally transcribed spacer (ITS) region, and hrp b6 gene sequencing. Multiplication of onion Xanthomonas strain R-O177 was not different from X. axonopodis pv. phaseoli in dry bean, but typical common bacterial blight disease symptoms were absent in dry bean. Populations from each geographical region were uniformly sensitive to 100 mug of CuSO(4), 100 mug of ZnSO(4), and 100 mug of streptomycin sulfate per ml. Biolog substrate utilization and fatty acid profiles revealed close phenoltypic relatedness between onion strains of Xanthomonas and X. axonopodis pv. dieffenbachiae (57% of strains) and X. arboricola pv. poinsettiicola (37% of strains), respectively. A logistic regression model based on fatty acid composition and substrate utilization classified 69% of strains into their geographical region of origin. Sequencing of a portion of the hrp B6 gene from 24 strains and ITS region from 25 strains revealed greater than 97% sequence similarity among strains. DNA fingerprinting revealed five genotype groups within onion strains of Xanthomonas and a high degree of genetic diversity among geographical regions of origin. Based on pathogenicity to onion, carbon substrate utilization, fatty acid profiles, rDNA genetic diversity, and genomic fingerprints, we conclude that the strains examined in this study are pathovar X. axonopodis pv. allii. Implications of genetic and phenotypic diversity within X. axonopodis pv. allii are discussed in relation to an integrated pest management program.     

Isolation and characterisation of a bacterial strain degrading the herbicide sulcotrione from an agricultural soil

BACKGROUND: The dissipation kinetics of the herbicide sulcotrione sprayed 4 times on a French soil was studied using a laboratory microcosm approach. An advanced cultivation‐based method was then used to isolate the bacteria responsible for biotransformation of sulcotrione. Chromatographic methods were employed as complementary tools to define its metabolic pathway. RESULTS: Soil microflora was able quickly to biotransform the herbicide (DT₅₀ ≈ 8 days). 2‐Chloro‐4‐mesylbenzoic acid, one of its main metabolites, was clearly detected. However, no accelerated biodegradation process was observed. Eight pure sulcotrione‐resistant strains were isolated, but only one (1OP) was capable of degrading this herbicide with a relatively high efficiency and to use it as a sole source of carbon and energy. In parallel, another sulcotrione‐resistant strain (1TRANS) was shown to be incapable of degrading the herbicide. Amplified ribosomal restriction analysis (ARDRA) and repetitive extragenic palendromic PCR genomic (REP‐PCR) fingerprinting of strains 1OP and 1TRANS gave indistinguishable profiles. CONCLUSION: Sequencing and aligning analysis of 16S rDNA genes of each pure strain revealed identical sequences and a close phylogenetic relationship (99% sequence identity) to Pseudomonas putida. Such physiological and genetic properties of 1OP to metabolise sulcotrione were probably governed by mobile genetic elements in the genome of the bacteria. Copyright © 2011 Society of Chemical Industry     

Adsorption and degradation of four acidic herbicides in soils from southern Spain

BACKGROUND: Pesticide degradation and adsorption in soils are key processes determining whether pesticide use will have any impact on environmental quality. Pesticide degradation in soil generally results in a reduction in toxicity, but some pesticides have breakdown products that are more toxic than the parent compound. Adsorption to soil particles ensures that herbicide is retained in the place where its biological activity is expressed and also determines potential for transportation away from the site of action. Degradation and adsorption are complex processes, and shortcomings in understanding them still restrict the ability to predict the fate and behaviour of ionisable pesticides. This paper reports the sorption and degradation behaviour of four acidic pesticides in five soils from southern Spain. Results are used to investigate the influence of soil and pesticide properties on adsorption and degradation as well as the potential link between the two processes. RESULTS: Adsorption and degradation of four acidic pesticides were measured in four soils from Spain characterised by small organic matter (OM) contents (0.3-1.0%) and varying clay contents (3-66%). In general, sorption increased in the order dicamba < metsulfuron-methyl < 2,4-D < flupyrsulfuron-methyl-sodium. Both OM and clay content were found to be important in determining adsorption, but relative differences in clay content between soils were much larger than those in OM content, and therefore clay content was the main property determining the extent of herbicide adsorption for these soils. pH was negatively correlated with adsorption for all compounds apart from metsulfuron-methyl. A clear positive correlation was observed for degradation rate with clay and OM content (P < 0.01), and a negative correlation was observed with pH (P < 0.01). The exception was metsulfuron-methyl, for which degradation was found to be significantly correlated only with soil bioactivity (P < 0.05). CONCLUSIONS: Both OM and clay content were found to be important in determining adsorption, but relative differences in clay content between soils were much larger than those in OM content, and therefore clay content was the main property determining the extent of herbicide adsorption for soils of this type. pH was negatively correlated with adsorption for all compounds apart from metsulfuron-methyl. The contrasting behaviour shown for these four acidic pesticides indicates that chemical degradation in soil is more difficult to predict than adsorption. Most of the variables measured were interrelated, and different behaviours were observed even for compounds from the same chemical class and with similar structures.     

番茄内生细菌的分离及拮抗菌株的筛选

生物防治以其安全、高效及无污染等特点已经成为植物病害防治的重要途径。植物内生细菌在植物体内具有稳定的生存空间、不易受外界环境的影响等特点,近年来受到广泛关注[1]。番茄灰霉病和叶霉病是番茄重要病害,多年来一直依赖化学药剂防治。据报道,这两种病害的生防菌株主要是从土壤中分离的,与植物内生细菌相比,这些“外来”微生物在与植物根际土壤中大量习居微生物的竞争中难以占居优势,田间防治效果大多不明显甚至无效。作者从健康番茄植株体内分离内生细菌,筛选出对灰霉病菌和叶霉病菌有拮抗作用的菌株,以期探索新的生物防治途径。1材料…     

黄河中上游半干旱区典型盐渍土中拮抗性放线菌筛选

对采自陕西、宁夏和内蒙等地典型盐渍土中放线菌组成及拮抗性进行了研究。结果表明:(1)该地区盐渍土中放线菌以链霉菌属为主,非链霉菌仅占9.27%,链霉菌属中以球孢类群,粉红孢类群,灰褐类群,黄色类群为主,占到放线菌总数的55.62%。(2)供试的706株放线菌中有拮抗活性的占到76.44%,但广谱性拮抗菌株较少;对细菌拮抗能力较真菌强;拮抗性放线菌以粉红孢类群、灰褐类群和球孢类群为优势类群;(3)发酵滤液拮抗性试验筛选出4株能产生较强抑菌活性物质的菌株,其中3株都是粉红孢类群。     

An Endophytic Actinomycete, Streptomyces sp. AOK-30, Isolated from Mountain Laurel and Its Antifungal Activity

To survey endophytic actinomycetes as potential biocontrol agents against fungal diseases of mountain laurel, young plants of mountain laurel (Kalmia latifolia L.) were used as an isolation source. From a total of 73 actinomycetes isolates obtained from leaves, stems and roots of test plants, Streptomyces sp. AOK-30 was selected, because i) it had a broad and intense antimicrobial spectrum against various yeasts and fungal pathogens of Ericaceae, ii) it grew on the multiplication and rooting media for tissue culture for this plant, and iii) the tissue-cultured seedlings that had been treated with this isolate became resistant to Pestalotia disease without any adverse growth effects such as malformation, dwarfing, discolorization and defoliation. This isolate was identified as Streptomyces sp., based on cultural, physiological and morphological characteristics. The present results indicate that isolate AOK-30 is a potential biocontrol agent against diseases of mountain laurel. Received 10 September 2002/ Accepted in revised form 22 November 2002     

一株辣椒尖孢炭疽病菌拮抗菌株的分离鉴定与发酵条件优化

 为了探寻辣椒尖孢炭疽病的生防药剂,从而控制该病在辣椒生产上的扩散。以辣椒尖孢炭疽病菌（Colletotrichum acutata）为指示菌,从浏阳河风光带土样中分离筛选出6株拮抗放线菌株,其中菌株ND045对指示菌的拮抗效果最好,菌丝生长抑制率高达71.6%。经形态观察、培养性状、生理生化鉴定,并结合16S rDNA序列分析,将ND045菌株鉴定为吸水链霉菌（Streptomyces hygroscopicus）。抗菌谱研究表明,该菌株对稻瘟病菌(Magnaporthe oryzae)、辣椒疫霉病菌(Phytophthora capsici)、黄瓜枯萎病菌（Fusarium omysporum）和辣椒白绢病菌（Sclerotium rolfsii）都具有较强的拮抗作用,抑菌率达40.58%～72.00%。通过单因子变量法和均匀设计法对菌株ND045进行发酵条件研究,结果表明,其最佳的发酵条件为：15 g大米粉、5 g大豆粉、0.5 g MgSO₄、0.01 g FeSO₄·7H₂O、1.5 g NaCl、水1 000 mL、30℃、pH 8。经优化后,菌丝生长抑制率最高达到82.61%,比优化前提高了11.01%。该研究结果为辣椒尖孢炭疽病的生物防治提供科学依据。     

野生药用植物内生真菌的分离及拮抗菌株筛选

为挖掘生防新资源,采用组织分离法从21种野生药用植物不同组织部位分离纯化内生真菌,以6种植物病原菌为靶标菌筛选拮抗菌株,并根据形态学及分子生物学对菌株进行鉴定,在此基础上研究了高效拮抗菌株对靶标菌菌丝生长及孢子萌发的影响。结果表明:从分离纯化的478株内生真菌中筛选出11株高活性拮抗菌株,分属于青霉属Penicillium、平脐蠕孢属Bipolaris、棘壳孢属Pyrenochaeta、镰孢属Fusarium、粒毛盘菌属Lachnum、垫壳孢属Coniella和Neonectria 7个属,其中青霉属占比最高,达36.36%。平皿对峙试验表明, 棘壳孢菌12-R-5对灰葡萄孢表现出高效性和专一性,其对菌丝抑制率达66.67%。含药平皿试验表明,菌株12-R-5发酵滤液10倍稀释液对灰葡萄孢菌丝生长抑制率达100%,与其他处理差异显著。菌株12-R-5 10倍发酵滤液处理灰葡萄孢10 h,孢子不萌发;100倍稀释液处理对孢子萌发抑制率为91.01%。显微镜观察发现,棘壳孢菌使灰葡萄孢菌丝扭曲变形,膨大肿胀及断裂,部分菌丝发生抑缩、消融现象。说明棘壳孢菌12-R-5菌株具有很好的生防潜力及应用前景。     

假单胞菌WX14群体感应淬灭酶基因的克隆及其功能研究

本研究采用报告菌平板法及β-半乳糖苷酶活性测定并筛选到10株具有较强降解酰基高丝氨酸内酯（AHLs）能力的细菌,其中菌株WX14降解AHLs能力较强,且菌体和菌体外泌物均具有降解活性。利用PCR法从菌株WX14中克隆到hacA和hacB两个群体感应淬灭酶基因,基因全长分别为2286和2370 bp,氨基酸序列比对分析结果表明,HacA属于阿库来菌素A酰化酶蛋白家族,HacB属于青霉素G酰化酶蛋白家族。这两个基因编码产物均为N末端亲核（N-terminal nucleophile,Ntn）水解酶家族成员。将hacA和hacB导入到软腐果胶杆菌Pectobacterium carotovorum后,可减弱该细菌在马铃薯块和白菜上的致病性,因此这2种AHLs降解酶对依赖于群体感应的软腐病有一定防病作用。经16S rDNA序列同源性分析鉴定,3株为假节杆菌Pseudarthrobacter spp.、1株节杆菌Paenarthrobacter sp.、4株假单胞菌Pseudomonas和2株芽胞杆菌Bacillus spp.。     

Studies on Endophytic Actinomycetes ( I ) <Emphasis Type="Italic">Streptomyces </Emphasis>sp. Isolated from Rhododendron and Its Antifungal Activity

To survey endophytic actinomycetes as potential biocontrol agents against fungal diseases of rhododendron, young plants of rhododendron were surface-sterilized for use as an isolation source. Nine, six and two isolates, with distinguishing characteristics based on the macroscopic appearance of colonies, were obtained from roots, stems and leaves, respectively, suggesting that various species of actinomycetes grow in the respective organs of this plant as symbionts or parasites. On an agar medium, only isolate R-5 commonly formed a clear growth-inhibition zone against two major fungal pathogens of rhododendron, Phytophthora cinnamomi and Pestalotiopsis sydowiana, indicating that this isolate can produce antifungal material(s). Acetone extracts of a liquid culture of R-5 had a broad antimicrobial spectrum against Gram-positive bacteria, yeast and filamentous fungi. Isolate R-5 was identified as a Streptomyces sp. based on morphological, physiological and chemotaxonomical characteristics. The present results indicate that isolate R-5 is a suitable candidate for the biocontrol of diseases of rhododendron. Received 25 March 2000/ Accepted in revised form 18 May 2000     

啶虫脒和阿维菌素在4种不同类型土壤中的吸附及迁移

采用薄层层析和平衡振荡法,分别测定了啶虫脒和阿维菌素在紫色土、砖红土、黄壤土和黑土4种不同类型土壤中的迁移率(Rf )和吸附常数(Kd),比较了2种农药在单用及混用下迁移和吸附行为的差异。结果表明:2种农药在单用及混用下的迁移行为几乎不存在差异,在4种土壤中的Rf 值大小顺序均为:黄壤土>紫色土>黑土>砖红土;2种农药的吸附过程均符合经典的Freundlich模型,混用时阿维菌素对啶虫脒的吸附无影响,但啶虫脒的存在会减少土壤对阿维菌素的吸附,表现为竞争吸附作用。