d -Alanine-d -alanine ligase gene ( ddl ) of Erwinia chrysanthemi strain EC16 II: analysis of pectate lyase regulation using ddl ? mutant

 Erwinia chrysanthemi (Ech) triggers soft rot disease mainly by secreting pectate lyase (Pel), which is regulated in a complex manner by many regulatory genes. In a previous study, we used a gene dosage method to show that the ddl gene, which encodes d-alanine-D-alanine ligase, reduced Pel production and tissue maceration by Ech strain EC16n. In this study, the ddl ⁻ marker-exchanged mutant was shown to overcome the long growth lag caused by various salts in the growth medium and to increase Pel production over that by EC16n, especially in a medium containing magnesium salts. Thus, ddl seems to regulate Pel production in a negative manner. Because the profiles of a gel shift assay using the pelE promoter region as the target DNA with crude extracts of EC16n and ddl ⁻ mutant were distinguishable, Ddl is thought to affect the binding of other regulatory proteins. Expression of the ddl gene was induced in the medium containing a low-molecular-weight fraction of potato extract, but it was reduced in that containing both polygalacturonic acid (PGA) and the fraction. The repression of ddl expression by PGA should contribute in part to the in planta hyperinduction of Pel. Received: May 15, 2002 / Accepted: June 20, 2002     

Simultaneous and selective detection of two major soft rot pathogens of potato: Pectobacterium atrosepticum (Erwinia carotovora subsp. atrosepticum) and Dickeya spp. (Erwinia chrysanthemi)

Dickeya spp. and Pectobacterium atrosepticum are major pathogens of potato. Current methods to detect these soft-rotting bacteria require separate identification steps. Here we describe a simple method allowing simultaneous detection of both pathogens based on multiplex PCR. The sensitivity of the primer sets was first examined on purified genomic DNA of the type strains Dickeya chrysanthemi 2048^T and P. atrosepticum 1526^T. The specificity and detection limits of the primer sets were successfully tested on 61 strains belonging to various Dickeya and Pectobacterium species, on artificially inoculated and on naturally contaminated potato plants. This new method provides a gain in time and materials, the main advantages for large-scale processes such as pathogen-free seed certification.     

Virulence, accumulation of acetyl-coenzyme A, and pectate lyase synthesis are controlled by PhoP-PhoQ two-component regulatory system responding to organic acids of Erwinia chrysanthemi 3937

The PhoP-PhoQ two-components regulatory system is involved in the pathogenesis of animal, plant, and insect pathogenic bacteria in response to various environmental factors. To elucidate how this system contributes to the plant pathogenesis of Erwinia chrysanthemi 3937 (Ech 3937), marker-exchanged mutants of phoP and phoQ were constructed. Their role in the regulation of a major virulent factor, pectate lyase (Pel), in response to various organic acids was then tested. These mutants synthesized more Pel than did the wild type in the medium containing acetate or citrate as the sole source of carbon, but they synthesized less Pel than did the wild type in pyruvate or malate as the sole source of carbon. Synthesis of Pel did not differ in succinate, fumarate, or glycerol from the wild type. The phoP and phoQ mutants grown and resuspended in acetate or citrate also caused more maceration, and the wild type pretreated in pyruvate or malate caused more maceration than did the mutants. The level of intracellular acetyl-coenzyme A (acetyl-CoA) almost paralleled the synthesis of Pel in the wild type and in the mutants of the phoP and phoQ. These results suggested that acetyl-CoA may be involved in regulation of Pel synthesis through two-independent regulatory cascades via the PhoP-PhoQ system (in an opposite manner) in response to acetate/citrate and pyruvate/malate. However, ackA and pta genes, involved in the synthesis of acetyl-CoA in Escherichia coli, were not expressed as predicted on the basis of the level of acetyl-CoA. Thus there may be an additional regulation or pathway for the synthesis of acetyl-CoA in Ech 3937.     

Application of RFLP analysis of recA, gyrA and rpoS gene fragments for rapid differentiation of Erwinia amylovora from Erwinia strains isolated in Korea and Japan

Restriction fragment length polymorphism (RFLP) analysis of the PCR amplified fragments of recA, gyrA and rpoS genes was applied for the characterization of Erwinia amylovora and Erwinia strains, which cause fire blight and Asian pear blight in orchards. Primers, constructed on the basis of the published recA, gyrA and rpoS gene sequences of Erwinia carotovora, allowed us to amplify DNA fragments for RFLP differentiation of E. amylovora and E. pyrifoliae and finally to distinguish strains within these species and relate them to pear pathogens from Japan. Three to seven restriction endonucleases were applied for RFLP analysis of each gene fragment. The electrophoretic patterns generated after PCR–RFLP for each of the tested genes, were characteristic and specific for each species and allowed their differentiation. The data show that PCR–RFLP analysis of the recA, gyrA and rpoS gene fragments may be considered as a useful tool for the identification and differentiation of E. amylovora and E. pyrifoliae. Almost identical restriction patterns of the analyzed gene fragments indicated a high relationship of E. pyrifoliae strains from Korea and pear pathogens from Japan and a divergence to E. amylovora. For quick and effective differentiation of E. amylovora strains from Erwinia strains from Asia without nucleotide sequencing we recommend the amplification of recA and rpoS gene fragments and digestion of each of them with restriction endonuclease Hin6I.     

Characterization and epidemiology of Colletotrichum acutatum sensu lato (C. chrysanthemi) causing Carthamus tinctorius anthracnose

In 2012, Colletotrichum isolates were collected from field‐grown safflower (Carthamus tinctorius) crops in central Italy from plants exhibiting typical anthracnose symptoms. Colletotrichum isolates were also collected from seed surfaces and from within seeds. The genetic variability of these isolates was assessed by a multilocus sequencing approach and compared with those from Colletotrichum chrysanthemi and Colletotrichum carthami isolates from different geographic areas and other Colletotrichum acutatum sensu lato‐related isolates. Phylogenetic analysis revealed that all of the strains isolated from C. tinctorius belonged to the species described as C. chrysanthemi, whereas all of the strains belonging to C. carthami had been isolated from Calendula officinalis. Phenotypic characterization of isolates was performed by assessing growth rates at different temperatures, morphology of colonies on potato dextrose agar (PDA) and the size of conidia. All C. chrysanthemi isolates from safflower had similar growth rates at different temperatures, comparable colony morphologies when grown on PDA and conidial sizes consistent with previously described C. chrysanthemi isolates. Pathogenicity tests were performed by artificially inoculating both seeds and plants and confirmed the seedborne nature of this pathogen. When inoculated on plants, C. chrysanthemi caused the typical symptoms of anthracnose on leaves. This is the first record of this pathogen on C. tinctorius in Italy, and it presents an updated characterization of Colletotrichum isolates pathogenic to safflowers in Europe.     

Biological control of red rot in sugarcane by native pyoluteorin‐producing Pseudomonas putida strain NH‐50 under field conditions and its potential modes of action

BACKGROUND: Rhizobacteria have a good potential to suppress soilborne diseases, but their efficacy against sugarcane pests is rarely reported. Bacterial strains isolated from the rhizosphere of sugarcane were evaluated for their potential to suppress red rot disease on two susceptible varieties, Co‐1148 and SPF‐234, under field conditions. The strains were also characterised for the production of secondary metabolites associated with their antagonistic activity. RESULTS: One out of four strains, the Pseudomonas putida strain NH‐50 (EU627168), reduced disease severity by 44–60% in different field trials. This potent antagonistic strain produced pyoluteorin antibiotic, as confirmed by high‐performance liquid chromatography (HPLC). The PltB gene involved in pyoluteorin synthesis was amplified from the P. putida strain NH‐50 and sequenced. The extracellular metabolites and volatile and diffusible antibiotics secreted by the tested strains inhibited mycelial growth of Glomerella tucumensis (Speg.) Arx & E Mull in vitro by 7–55%. CONCLUSION: The pyoluteorin‐producing bacteria P. putida strain NH‐50 significantly reduced disease severity on both sugarcane varieties, irrespective of fungal inoculation, i.e. either inoculated through stem or through soil. This strain also possesses other plant growth characteristics and can be used as a biopesticide for sugarcane crop. Copyright © 2011 Society of Chemical Industry     

三株球孢白僵菌对草地贪夜蛾的毒力比较

为筛选有效防治草地贪夜蛾Spodoptera frugiperda的微生物制剂,采用室内生物测定法测定从北京市、浙江省和海南省土壤样品中分离获得的3株球孢白僵菌Beauveria bassiana菌株bbbj、bbzj和bbhn对草地贪夜蛾3龄幼虫的毒力,并比较这3株菌株的产孢情况和合成白僵菌素的能力。结果显示,分离自北京市的菌株bbbj对草地贪夜蛾3龄幼虫的毒力最强,LC50为3.37×105个孢子/mL;其次为浙江省的菌株bbzj,毒力略逊于菌株bbbj;海南省的菌株bbhn毒力最弱,其在试验最高浓度108个孢子/mL处理7 d后对草地贪夜蛾3龄幼虫的致死率低于50%。菌株bbbj的产孢量远高于另外2株菌株,并且其菌丝结构上着生大量芽生孢子簇,而且菌株bbbj菌体中的白僵菌素含量最高,培养5 d后,分别为菌株bbzj和bbhn的40.08倍和65.85倍。虽然补充白僵菌素可以提高菌株bbhn的毒力,但是草地贪夜蛾幼虫对白僵菌素敏感度不高。表明球孢白僵菌菌株bbbj对草地贪夜蛾幼虫有较高的毒杀活性,具有作为草地贪夜蛾生防菌株的潜力。     

两株茶尺蠖核型多角体病毒毒株对灰茶尺蠖的毒力及基因组差异

为明确茶尺蠖核型多角体病毒（Ectropis obliqua nucleopolyhedrovirus,EcobNPV）毒株对灰茶尺蠖E.grisescens的毒力差异及其机制,以前期筛选获得的高效毒株EcobNPV-QF4和原始毒株EcobNPV-QV为研究对象,采用PCR扩增技术对其进行分子鉴定,通过叶盘法毒力生测试验测定毒力差异,并用定量PCR技术比较病毒拷贝数及基因组测序方法分析基因组差异。结果表明,EcobNPV-QV经分子鉴定存在3个基因型,而EcobNPV-QF4则存在2个基因型,2株毒株的基因型存在差异;EcobNPV-QV和EcobNPV-QF4对灰茶尺蠖的幼虫毒力分别为4.26×10⁶ PIB/头和9.72×10⁷ PIB/头,EcobNPV-QF4对灰茶尺蠖的毒力是EcobNPV-QV的22.8倍;饲毒0~48 h之间EcobNPV-QF4在灰茶尺蠖体内的增殖拷贝均显著高于EcobNPV-QV,EcobNPV-QF4比EcobNPV-QV具有更快的繁殖能力;比较2株毒株的基因组,EcobNPV-QF4比EcobNPV-QV长315 bp,与EcobNPV-QV相比EcobNPV-QF4基因组的hr1~hr3区域出现倒位,其中核苷酸还原酶小亚基基因在EcobNPV-QF4中重复1次,3个同源重复区的回文序列存在差异但2株毒株仍为α杆状病毒亚家族II亲缘种分离株。表明2株毒株的基因组在同源重复区的序列及回文个数间存在差异,推测这可能与导致其对灰茶尺蠖毒力差异有关。     

和田鞘氨醇杆菌AMCC 100218菌株的筛选、鉴定及杀线虫特性

为筛选对南方根结线虫具有致死效果的生防细菌,从山东省10个县市蔬菜主产区番茄根际土壤中分离可培养细菌,采用离体杀线虫试验测定分离菌株对南方根结线虫Meloidogyne incognita的致死活性,结合生理生化特征及分子生物学方法对高效杀线虫菌株进行分类鉴定,同时对其杀线虫特性进行表征,并通过盆栽试验进一步验证其生防潜力。结果显示,从山东省蔬菜主产区番茄根际土中分离到1株高效杀线虫菌株AMCC 100218,结合生理生化试验与16S rRNA序列分析,鉴定此菌株为和田鞘氨醇杆菌Sphingobacterium hotanense;该菌株对南方根结线虫2龄幼虫的致死效果可达88.87%,其杀线虫活性物质具有较好的热稳定性和储存稳定性,且耐碱不耐酸;盆栽试验结果表明,该菌能够显著减少土壤中的虫口密度,降低番茄发病率。表明和田鞘氨醇杆菌AMCC 100218菌株是1株具有防治根结线虫病潜力的生防细菌。     

双氧木脂素E对东方粘虫幼虫体壁肌超微结构及Na+-K+-ATPase、Ca2+-ATPase的影响

双氧木脂素E（haedoxane E）是从杀虫植物透骨草Phryma leptostachya L.中分离出的一种对多种农林害虫和卫生害虫具有杀虫活性的化合物。本研究以东方粘虫Mythimna separata为供试昆虫,对双氧木脂素E对其幼虫杀虫作用部位进行了初步研究。电镜观察表明,双氧木脂素E对东方粘虫幼虫体壁肌具有致毒作用,能使肌细胞发生明显病变,细胞核皱缩变形,染色质浓缩,线粒体肿胀、大面积空泡化,肌质网扩张,肌原纤维排列紊乱,胞质内出现大量多泡体。酶活力测定结果也显示,双氧木脂素E能不同程度抑制Na+-K+-ATPase和Ca2+-ATPase活力。因此推测双氧木脂素E是一种作用于东方粘虫肌肉组织的肌肉毒剂。     

Identification of potential virulence genes in Erwinia chrysanthemi 3937: transposon insertion into plant-upregulated genes

Erwinia chrysanthemi 3937 is a soft-rotting plant pathogen in Enterobacteriaceae. It attacks a wide range of plant host species. Previously, we identified dozens of E. chrysanthemi 3937 genes induced during plant infection by microarray differential display. Here, we have mutated plant-upregulated and putatively plant-upregulated genes in E. chrysanthemi 3937 using a transposon insertion method. Of 57 mutants produced, 8 were significantly reduced in maceration in African violet leaves. These 8 E. chrysanthemi genes are similar to Escherichia coli purU (formyltetrahydrofolate deformylase; ASAP20623) and wcaJ (undecaprenylphosphate glucosephosphotransferase; ASAP18556), Bacillus subtilis dltA (d-alanine-d-alanyl carrier protein ligase; ASAP19406), Pseudomonas syringae PSPTO2912 (ABC transporter, periplasmic glutamine-binding protein; ASAP15639), Pseudomonas aeruginosa pheC (cyclohexadienyl dehydratase; ASAP19773), P. syringae syrE (peptide synthase; ASAP19989), Vibrio vulnificus VV12303 (unknown protein; ASAP18555), and Yersinia pestis speD (S-adenosylmethionine decarboxylase; ASAP20536). In some of the genes, possible roles in virulence could be postulated based on the functions of their homologues. This work demonstrated that a low proportion of pathogenicity-related genes were among the plant-upregulated genes of E. chrysanthemi 3937. This study and further dissection of these putative virulence genes should lead to new insights into infection mechanisms in pathogens.     

紫花苜蓿根际芽胞杆菌YB-2的鉴定及其生防和促生能力分析

为获得对紫花苜蓿Medicago sativa生长有益的生防菌,以尖孢镰刀菌Fusarium oxysporum为指示菌从紫花苜蓿根际土壤中分离筛选拮抗菌株,基于形态学特征、生理生化特性和16S rDNA与gyrB基因序列分析对其进行鉴定,并评价其对紫花苜蓿根腐病的生防效果以及提高寄主植物耐盐碱胁迫的能力。结果表明,从紫花苜蓿根际土壤中筛选到1株对尖孢镰刀菌有较强拮抗作用的菌株YB-2,该菌株对尖孢镰刀菌菌丝生长的抑制率为71.37%;结合形态学特征、生理生化特性和16S rDNA与gyrB基因序列分析结果将菌株YB-2鉴定为枯草芽胞杆菌Bacillus subtilis。菌株YB-2具有溶解无机磷的能力,且具有产氰化氢、羧甲基纤维素酶、1-氨基环丙烷-1-羧酸脱氨酶、吲哚乙酸和嗜铁素的能力,在NaCl含量为1%～9%、pH 7～9条件下均能生长。菌株YB-2对紫花苜蓿根腐病的相对防效达56.83%;在盐碱条件下,与对照相比,接种YB-2的紫花苜蓿株高极显著增加了17.82%,根长显著增加了28.22%,地上部鲜重和地上部干重分别显著增加了51.12%和48.57%。表明菌株YB-2兼...     

棉花黄萎病拮抗细菌H14的筛选鉴定及其拮抗机理分析

为筛选对大丽轮枝菌Verticillium dahliae Kleb.具有拮抗活性的根际细菌,以大丽轮枝菌为靶菌,分离获得棉花根际细菌,利用平板对峙法和琼脂扩散法筛选具有较高拮抗活性的菌株,采用室内盆栽法测定筛选所得菌株对棉花黄萎病的防效,并通过形态特征、生理生化特性和16S r DNA基因序列分析确立其分类地位,采用底物降解法和抗菌肽基因克隆法检测其产生水解酶和抗菌肽的能力。结果显示,试验共分离获得182株棉花根际细菌,筛选得到3株对大丽轮枝菌抑菌率大于50.00%且抑菌圈直径大于15 mm的菌株,其中菌株H14的抑菌率和抑菌圈直径最大,分别为54.25%和18.10 mm。该菌能在0～9%NaCl和pH 4.5～9.0的NB培养基上生长;经形态特征观察、生理生化试验及16S rDNA基因序列分析,最终将其鉴定为莫哈韦芽胞杆菌Bacillus mojavensis;菌株H14对大丽轮枝菌孢子萌发的抑制率和对棉花黄萎病的盆栽防效分别为89.55%和74.57%;菌株H14能合成蛋白酶,含有srfAA、fenD、bacA脂肽类抗生素合成基因。表明莫哈韦芽胞杆菌菌株H14能够合成蛋白酶和脂肽类拮抗物质,具有良好的抑菌和防病能力。     

疏水表面诱导橡胶树炭疽菌侵染结构的发育分化过程

为了解橡胶树2种炭疽病菌的侵染结构发育分化过程,采用平板菌落生长速率法测定了3株胶孢炭疽菌Colletotrichum gloeosporioides和3株尖孢炭疽菌C.acutatum的菌丝生长速率,测量其分生孢子大小,显微观察2种炭疽菌在疏水表面诱导下侵染结构的发育分化过程。结果表明,胶孢炭疽菌菌丝生长速率为0.96~1.36 cm/d,显著高于尖孢炭疽菌的菌丝生长速率0.72~0.89 cm/d,但二者分生孢子大小无显著差异。在疏水表面诱导下,2种炭疽菌分生孢子在接种2~6 h后开始萌发,12 h孢子萌发率为71.70%~88.05%,13~16 h开始分化附着胞,24 h附着胞形成率为48.99%~70.74%,36 h菌丝诱发形成大量附着枝,48 h后分生孢子产生的次生菌丝也可诱发形成附着枝,附着枝呈圆形、姜瓣形、梨形或不规则形。分生孢子极易产生,可在菌丝顶端成簇或菌丝侧面排列产生,也可由分生孢子形成的芽管产生,或在芽管分化附着胞过程分枝形成分生孢子;附着胞多着生于芽管顶端,少数附着胞顶端可继续萌发类似短芽管结构,再次分化形成可黑色化的次级附着胞。表明橡胶树2种炭疽菌不同菌株间分生孢子萌发时间、孢子萌发率、附着胞形成时间和形成率有一定差异,但种间无明显差异;橡胶树炭疽菌分生孢子极易形成,在疏水表面容易分化形成附着胞和附着枝,说明具有极强的适生性。     

我国长江流域和南方地区花生青枯菌遗传多样性分析

为明确不同青枯菌的遗传多样性和其在花生植株上的致病力差异,采用国际上新的青枯菌演化型分类模式,对从我国长江流域和南方地区9个花生种植区分离的95株花生青枯菌Ralstonia solanacearum菌株进行遗传多样性分析,基于内源葡聚糖酶基因egl对青枯菌进行系统发育研究,并对供试青枯菌的致病力进行测定。结果表明,所有95株菌株均属于青枯菌演化型I型,即亚洲分支类型。在序列变种分类上,所检测的9个花生种植区中有8个种植区的花生青枯菌菌株属于序列变种14,仅有1个种植区(广西壮族自治区贺州市)的花生青枯菌菌株属于序列变种48,表明我国长江流域和南方地区花生青枯菌群体遗传多样性水平较低。青枯菌致病力测定结果表明,来自赣州市的菌株GZ-1、贺州市的菌株HZ-2和宜昌市的菌株YC接种到花生植株14 d后,花生的病情指数分别为43.8、75.0和87.5,而来自其它6个花生种植区的菌株接种花生后,其病情指数均为100.0,表明菌株GZ-1和HZ-2的致病力较弱,而其它7个花生种植区代表性菌株的致病力均较强。     

Virulence, resistance to magainin II, and expression of pectate lyase are controlled by the PhoP-PhoQ two-component regulatory system responding to pH and magnesium in Erwinia chrysanthemi 3937

Marker-exchanged mutants of phoP and phoQ of Erwinia chrysanthemi (Ech) strain 3937 became more sensitive to the cationic antimicrobial peptide (CAMP) magainin II than did the wild type at a low Mg²⁺ concentration and at either acidic or neutral pH. At high Mg²⁺ and acidic pH, only the phoQ mutant, but not the phoP mutant, became more sensitive to magainin II than did the wild type; both mutants were more sensitive at neutral pH. The hyperinduction of Pel synthesis in medium containing plant extracts and polygalacturonic acid (PGA) was confirmed in the wild type but not in the mutants at low Mg²⁺ and neutral pH. However, Pel was hyperinduced at high Mg²⁺ and neutral pH in these mutants but not in the wild type. Maceration was also greatly reduced by these mutants compared to the wild type when the inoculum was precultured and then resuspended in the medium with low Mg²⁺ at neutral pH. However, when bacteria were precultured and resuspended in the medium with high Mg²⁺ at neutral pH, severe maceration was observed in these mutants but not in the wild type. Thus, at low Mg²⁺, PhoP-PhoQ TCS seems to be stimulated for maceration and the hyperinduction of Pel synthesis. At high Mg²⁺, however, PhoP-PhoQ TCS may be repressed for these phenotypes, and PhoP may be controlled by a mechanism(s) other than PhoQ regulation.     

高毒力金龟子绿僵菌的筛选及其胞外蛋白酶产量测定

以大蜡螟Galleria mellonella L.幼虫为供试昆虫,对从果园土壤中筛选出的金龟子绿僵菌Metarhizium anisopliae的生物活性进行了测定,共获得4株高毒力菌株,其LT50值在1.85～2.56 d之间,LD50值在6.11×102～1.55×104 spores之间。利用明胶-琼脂平板分析法对筛选出的高毒力绿僵菌菌株产胞外蛋白酶的水平进行了测定,结果显示,各菌株间产酶水平差异较大,且菌株产酶水平的高低与其对大蜡螟幼虫的毒力之间存在明显的线性相关性。     

内蒙古自治区向日葵小核盘菌的菌丝亲和组类型及其菌株的生物学特性、致病力和交配型

为明确内蒙古自治区阴山北麓地区向日葵小核盘菌Sclerotinia minor的遗传变异,对自内蒙古自治区乌兰察布市、包头市和呼和浩特市向日葵上分离纯化的110株向日葵小核盘菌菌株进行菌丝亲和组(mycelium compatibility group,MCG)划分,并对5个主要MCG组间和组内各菌株的生物学特性、致病力和交配型进行测定。结果表明,供试的110株菌株被划分为14个亲合组,其中MCG1为主要类型,包含32株菌株,占总菌株的29.1%;MCG2包含来自7个地点的25株菌株;5个MCG仅包含1株菌株;在这14个MCG中,MCG1～MCG5包含92株菌株,占总菌株数的83.6%。MCG1～MCG5组间各菌株在菌落直径和草酸分泌量上存在差异,但在菌核形成量、多聚半乳糖醛酸酶活性和致病力上无显著差异;而MCG1～MCG5组内各菌株在菌落直径、草酸分泌量、菌核形成量、多聚半乳糖醛酸酶活性和致病力上均有一定差异。在MCG1～MCG5各菌株的交配型中,除MCG2中菌株的负反转型与正反转型比例接近1∶1外,其它4个MCG中菌株的负反转型与正反转型比例均偏离1∶1,表明内蒙古自治区向日葵小核盘菌具有较高的遗传变异程度。     

Characterization of an antibacterial substance produced by <Emphasis Type="Italic">Erwinia carotovora</Emphasis> subsp. <Emphasis Type="Italic">carotovora</Emphasis> Ecc 32

A total of 88 strains of Erwinia carotovora subsp. carotovora (Ecc) isolated from various host plants in several geographic regions were screened for production of antibacterial substances using the same strains as indicators. Of the 88 strains, 72 produced antibacterial substances. One of these 72 strains, a Brazilian strain Ecc 32, produced an antibacterial substance active against all tested Ecc strains on TSA medium. The antibacterial spectrum of the compound from Ecc 32 strain was limited to closely related strains of soft-rot Erwinia species. Such a narrow spectrum of activity is typical of bacteriocins. The compound produced by Ecc 32 strain, however, was resistant to some enzymes and detergents. Moreover, the compound was heat-stable and active over a wide pH range. The physical characteristics of the compound were not in agreement with those of bacteriocin or carotovoricin.     

Molecular cloning of two chitinase genes from Serratia marcescens and their expression in Pseudomonas species

Two chitinase encoding EcoRI fragments from the enteric soil bacterium Serratia marcescens were cloned. From a genomic library of 5686 transductants, 21 expressed chitinase activity as indicated by clearing of a chitin-containing medium. The chitinase encoding clones could be divided into two groups. Four had an 18kb EcoRI fragment and 17 had a 9·4 kb EcoRI fragment. In Southern hybridization experiments the 18kb fragment showed no homology to the 9·4 kb fragment and restriction enzyme maps indicated no similarity. Triparental mating with fluorescent Pseudomonas spp. yielded transconjugants that expressed chitinase activity, inhibited growth of Fusarium oxysporum f. sp. redolens germ tubes and reduced disease of radish caused by the same fungus.