不同种类的细菌对大白菜软腐欧氏杆菌根表吸附的竞争作用

用吸附竞争试验,测定了8种细菌共10个菌株对大白菜软腐欧氏杆菌(Erwinia carotovorasubsp.carotovora)菌株 RL4在大白菜根表吸附的竞争作用。竞争菌的吸附竞争作用与它们对大白菜根表的吸附能力相一致,在等量混合接种时,只有能对大白菜根表发生吸附的7个菌株表现出程度不同的吸附竞争能力,其中以菌株 BCE 4(RL 4是从它筛选出的抗利福平菌株)竞争最强,它也是对寄主根表吸附能力最强的菌株,其吸附竞争作用最高可达98%,不表现1∶1的数量关系。RL4和 BCE4的热杀死菌体不发生吸附竞争。发生吸附竞争的有效时间为竞争菌对根表预吸附15分钟以后,或病菌(RL4)吸附15分钟以前。     

大白菜根表结构与软腐欧氏杆菌吸附和侵入的关系

 用大白菜6个品种和软腐欧氏杆菌(Erwinia carotovora subsp.carotovora)致病力等性状不同的3个菌株测定表明,病菌对根毛吸附的菌量与对根系侵入的菌量间有正相关关系。吸附表现出可逆和不可逆两种状态,试验中用改进的方法对它们作了定量研究。可逆吸附发生和完成的最早时间分别是1~5和10~15分钟,此时用水洗法可使28~100%的菌体解吸附。不可逆吸附发生和完成的时间分别为15和30分钟,菌体以静止不动为特征,只能由解吸附剂处理而部分地解吸附。在接种后15分钟,可逆吸附逐渐向不可逆吸附转化,条件是病菌对根毛表面的种群压力必须保持在最初接种液含菌量的50%以上。病菌吸附还表现出根毛区段的差别,常以根毛顶端吸附菌量较高。     

苦瓜活性组分物质抑菌活性测定

以苦瓜(Momordica charantia L.)种仁中提取的凝集素备提物和苦瓜籽粗蛋白对8种植物病原真菌,5种植物病原细菌进行了抑菌活性测定。结果表明:两种提取物对小麦赤霉病菌、棉花黄萎病菌和苹果炭疽病菌表现出较好的抑菌活性。凝集素备提物(0.67 mg/mL)对小麦赤霉病菌和棉花黄萎病菌的抑菌率分别为69.12%和64.86%;苦瓜籽粗蛋白(6.67 mg/mL)对苹果炭疽病菌的抑菌率达68.61%。两种提取物对大白菜软腐病菌、花生青枯病菌、水稻白叶枯病菌和根癌农杆病菌等植物病原细菌均无抑菌活性,苦瓜籽粗蛋白(0.1 g/mL)对水稻细条病菌有一定抑制作用。     

三种环境因子对大白菜软腐欧氏杆菌根毛吸附的影响

 温度、pH和离子条件是影响病原细菌对寄主吸附的基本因子,文章测定了它们对大白菜软腐欧氏杆菌(Erwinia carotovora pv.carotoyora)在寄主根毛上吸附的影响。病菌在5个给定温度下对寄主根毛吸附量的变化,呈现为由吸附起始温度(18℃)、温度稳恒渐近范围(18-22℃)和温度饱和点(22℃)组成的吸附动力曲线。病菌在12个pH梯度下的运动状态、生活力及对寄主根毛吸附量的变化表明,病菌吸附以pH5.8-7.2为适宜范围,pH4.6和8.2为低限和高限;偏酸(pH5.0以下)和偏碱(pH7.2以上)可通过影响病菌的生活力和运动性而影响吸附。5×10^-6M的Ca++可使病菌吸附量提高2.5-4.2倍,同浓度的Mg++可使吸附量提高2.7倍。文章讨论了温度和pH条件影响吸附的动力学,以及吸附调节的可能机制。     

抑制植物病原细菌的植物筛选

以3种重要的植物病原细菌—辣椒青枯病菌(Ralstonia solanacearum)、柑橘溃疡病菌(Xanthomonas citri)和大白菜软腐病菌(Erwinia carotovorapv.carotovora)为供试菌,对43种植物甲醇提取物进行离体抑菌活性测定。研究结果表明,漆树对辣椒青枯病菌抑菌活性最强,其次是金秀清明茶;抑制柑橘溃疡病菌活性最高的植物有漆树和十大功劳;对大白菜软腐病菌没有筛选到抑菌活性较好的植物;垫状卷柏、七叶一枝花、少花龙葵、水半夏和豆瓣菜粗提物对3种植物病原细菌均无抑制作用。     

北京地区蔬菜软腐细菌病原的研究

 近年来京郊蔬菜软腐病害有所增加.经对夏季各类主要蔬菜和窖藏后期的大白菜研究鉴定,欧氏软腐细菌为害大白菜、蕃茄、青椒、马铃薯、黄瓜、冬瓜、南瓜和丝瓜等。经过形态、生理生化特性、致病性等一系列的测定,发现蔬菜上腐烂细菌的种类有所变化,窖藏后期大白菜腐烂主要病原是假单孢杆菌(Pseudomonas spp)夏季蔬菜上主要是胡萝卜软腐欧氏杆菌胡萝卜软腐致病型(Erwinia carotovora pv.carotovora),在蕃茄和丝瓜上还发现有胡萝卜软腐欧氏菌马铃薯黑胫病致病型(Erwinia carotovora pv.atroseptica).     

Alternaria alternata菌丝细胞壁凝集素的纯化与特性研究

 Alternaria alternata是接触性活体营养菌寄生真菌纤细齿梗孢Olpitrichum tenellum的寄主真菌之一,该寄生过程中菌寄生真菌与寄主真菌的识别机制未见报道。本文分离、纯化了A.alternata菌丝细胞壁上的一种参与识别作用的特异性蛋白——凝集素,并对其特性以及在菌寄生过程中的作用做了初步研究,粗提液经硫酸铵分级沉淀,DEAE-Sepharose阴离子交换柱层析和SephacrylS 100分子筛层析等步骤便可获得凝胶电泳均一的凝集素AAL,经凝胶过滤层析法测得分子量为37.2kDa,经糖染色鉴定为一种糖蛋白。孢子凝集试验表明,AAL对O.tenellum的孢子具有极强的凝集力,在含量为3 325μg/mL时,30min内90%的O.tenellum孢子发生凝集作用,对照(不含AAL的缓冲液)凝集力则显著下降。上述结果说明AAL在A.alternata细胞壁蛋白抽提液和O.tenellum的孢子吸附中扮演着重要角色,可能起识别因子的作用。     

大白菜软腐细菌从幼根侵染的组织病理学研究

 将大白菜种子置于培养皿内的滤纸上发芽。滤纸用不同浓度的洗涤与不洗涤的软腐欧氏杆菌(Erwinia carotovora pv.carotovoar Dye)悬浮液,离心上清液,培养基浸出液和灭菌生理盐水浸润。在25℃下培养26小时后,用细菌悬浮液处理的幼芽发病率为8-95%不等。病苗生长量为对照(用生理盐水处理)的17-80%。扫描电镜观察的结果指出,接种后病原细菌首先在根毛区发现,以后才在伸长区和成熟区发现大量病菌。成熟区表皮细胞与细胞交界处的细胞壁有被分解的迹象。在幼苗的导管及其附近的薄壁细胞内均发现了病原细菌,检出率分别为16%和3%。在病苗组织内和幼根表面的病菌可产生微纤丝。病苗幼茎经压气处理24小时后,对其断面进行扫描电镜观察,结果表明,病菌可将导管的外壁与纹孔膜分解而使导管解体仅剩螺蚊加厚部分或呈网孔状。     

棉铃虫唾液凝集素的初步研究

昆虫唾液在成功取食寄主中发挥关键作用。而有关植食性昆虫唾液(反吐液)的研究多集中在与寄主植物的互作关系上,鲜有涉及唾液免疫因子的研究。凝集素作为模式识别受体,在病原物的识别中发挥重要作用,是昆虫重要的免疫因子。棉铃虫幼虫血淋巴中已报道的凝集素种类至少有8种,但其唾液中是否存在凝集素却未见报道。本文通过与鸡血红细胞的血凝反应检测到棉铃虫幼虫唾液中含有凝集活性物质,凝集效价高达29。显微观察显示,唾液对鸡血红细胞具有明显的凝集作用。经35%和45%硫酸铵盐析沉淀获得的组分具有明显凝集活性,表明唾液中的凝集活性物质为蛋白质。通过糖抑制试验,发现棉铃虫唾液凝集素与其血淋巴凝集素的糖结合谱存在差异,表明他们是不同的凝集素种类。棉铃虫唾液凝集素的揭示为理解植食性昆虫唾液的免疫功能提供了新的证据。     

软腐欧氏杆菌产细菌素菌株的筛选和细菌素类型的研究

采用Echandi方法,测定了从国内不同植物上分离,经鉴定的3个种和亚种的软腐欧氏杆菌(Erwiniacarotovora var.carotovora Dye,E.carotovora var.otroseptica Dye,E.chrysanthemi Burkholder.etal.)的224个菌株产生细菌素的能力。指示菌为代表3个不同种的菌株。测定结果表明:224个试验菌株中能产细菌素的有97个,占总数的43.3％。经测定产生细菌素的专化性有所不同,约各有1/3菌株分别对3种、2种、1种指示菌有抑制作用。有些广谱的细菌素甚至对其它属的植物病原细菌也有抑制作用。少数种内专化的产细菌素菌株可望用于软腐欧氏杆菌种鉴定的辅助手段。电镜观察初步表明,这些细菌素可能是分子大小不同的两种类型的细菌素:小分子细菌素在电镜下不可见,大分子细菌素具有短杆状结构,颇似无头壳的噬菌体的尾部。     

大白菜软腐病菌游动性突变体在大白菜体内增殖扩展研究

 用培养皿滤纸吸附测定法和不伤根土壤拌菌处理及针刺接种法,测定了大白菜软腐病菌游动性突变体进入大白菜体内、并在其中侵染定殖和扩展的特性。结果表明,游动性丧失和增强的突变体都可以通过种子萌发和主动接触进入大白菜体内、并可以在体内有短期的繁殖,但菌量远低于野生菌。大白菜叶片接种实验说明,这两种突变体也都可以进行短距离扩展,但扩展距离和菌的繁殖量低于野生菌。     

Phenotypic and genetic characterization of Erwinia carotovora from mulberry (Morus spp.)

Phenotypic and genetic characteristics of nine bacterial strains isolated from mulberry ( Morus spp.), which were originally described as Erwinia carotovora ssp. carotovora (Ecc), were investigated. Based on the results of biochemical tests, these bacterial strains were divided into two different types, type 1 and type 2. Two strains of type 1 were similar to Ecc, whereas seven strains of type 2 were distinct from Ecc. A polyphasic study that included serological assay, specific PCR assay for E. carotovora ssp. atroseptica (Eca), PCR-RFLP of a pectate lyase ( pel ) gene and RAPD-PCR was performed on the type 2 strains, and the data were compared with those of related E. carotovora subspecies. The results of serological and specific PCR assays for Eca showed that the type 2 strains were distinct from Eca. In RFLP analysis of the pel gene using Sau 3AI, the type 2 strains showed a unique RFLP pattern. On the basis of RAPD analysis, similarity of RAPD patterns within the type 2 strains was very high. A unique RAPD fragment was isolated from the type 2 strains and used as a probe for Southern hybridization. This probe hybridized only with PCR products from the type 2 strains. Based on phenotypic, serological and genetic characteristics, the type 2 strains isolated from mulberry may belong to a distinct E. carotovora subspecies other than Eca or Ecc.     

Homolog of FlhDC, a master regulator for flagellum synthesis: required for pathogenicity in Erwinia carotovora subsp. carotovora

 Erwinia carotovora subsp. carotovora (Ecc) is a causal agent of soft-rot diseases in a wide variety of plants. Here, we have isolated nonmotile mutants in Ecc by in vivo insertional mutagenesis using a transposon Tn5. The sequence disrupted by the Tn5 insertion in YMU1 and YMU5 mutants was highly homologous to that of flhC and flhD genes, respectively. They are involved in the initiation of the expression of flagellum-related genes in many gram-negative bacteria such as Escherichia coli and Salmonella. With electron microscopy, the flhC and the flhD homolog mutants were shown to be aflagellate. Furthermore, the virulence of these mutants was greatly reduced in Chinese cabbage and potato compared to that of the parental strain. These results suggest that flagellar formation is required for the pathogenicity of Ecc. Received: November 5, 2002 / Accepted: December 2, 2002 Acknowledgments This research was supported in part by Grant-in-Aid (12052210) and by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (13073).     

软腐欧氏杆菌的蛋白酶与致病作用的关系

 胡罗卜软腐欧氏杆菌(Erwinia carotovora var.carotovora Dyc)在合成培养基MS中产生两种蛋白酶,但在无细胞滤液中测不到果胶裂解酶和果胶水解酶的活性。用等电聚焦电泳测得这两种蛋白酶都是碱性蛋白酶,等电点分别为8.3和8.9。变性温度为49-50℃,对酸或碱具有较高的稳定性。纯化的蛋白酶单独作用马铃薯块、能使其变软,切片观察表明细胞壁被明显降解。通过滤膜结合法,将供体菌1830/pJB4J1::Tn5的转座子转入到野生型StEcc-12中,从2000个接合子中获得了11个缺少产蛋白酶能力的突变体。这些突变体的分泌性蛋白质等电聚焦图谱与野生型菌株明显不同,其中一个变突体蛋白酶带完全缺失,另外一个则出现8条新的蛋白带,其他5个属部分缺失。这些突变体对马铃薯块组织离析、腐烂的能力都有不同程度的减弱。研究结果认为在由胡罗卜软腐欧氏杆菌引起的软腐病程中蛋白酶和果胶酶是协同作用的,果胶酶降解植物细胞间和胞壁中的果胶质,而蛋白酶则降解植物细胞壁和膜中的蛋白质。     

Detection of soft rot Erwinia spp. on seed potatoes: conductimetry in comparison with dilution plating, PCR and serological assays

Automated conductance measurements in polypectate medium were used for the detection of pathogenic soft rot Erwinia spp. in potato peel extracts. The detection threshold for Erwinia carotovora subsp. atroseptica (Eca) in inoculated peel extracts was ca. 10⁴ colony forming units (cfu) ml^-1 when samples were considered positive on the basis of a response within 48 h at 20 °C. Detection of E. chrysanthemi (Ech) was less sensitive, only 10⁵ cfu ml^-1 peel extract were detected within 36 h at 25 °C. The linear correlation between detection times in conductimetry and inoculum levels of Eca and Ech in peel extracts was used for a quantitative estimation of Eca and Ech in naturally contaminated peel extracts. Samples giving a positive conductimetric response had to be confirmed with an enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) for the presence of Eca and Ech, because E. carotovora subsp. carotovora (Ecc) also generated a conductance response. Conductimetry was sensitive and efficient for detection of contamination levels of Eca higher than 10⁴ cfu ml^-1 peel extract. For Ech, conductimetric detection was less sensitive and inefficient due to low contamination levels of Ech and the presence of high numbers of Ecc in many samples after enrichment, which interfered with the test. Immunofluorescence cell staining (IF) combined with enrichment and immunofluorescence colony staining (IFC) were suited to detect and quantify low numbers of Eca and Ech at less than 10⁴ cfu ml^-1 in peel extracts. However, since false positive and negative reactions in serology were observed, the use of PCR after enrichment, or in combination with IFC to confirm positive results, was required for accurate detection.     

Overwintering of Erwinia carotovora subsp. carotovora in Diseased Tissues in Soil and Its Role as Inoculum for Soft Rot of Chinese Cabbage (Brassica campestris, Pekinensis Group)

To examine whether the pathogenic bacterium, Erwinia carotovora subsp. carotovora, causal agent of soft rot of Chinese cabbage (Brassica campestris L., pekinensis group), can overwinter in plant debris and soil and serve as inoculum the following year, we monitored field populations of rifampicin-resistant, phage-sensitive strains of the bacterium. Chinese cabbage (cv. Matsushima Kohai W1116) were planted in field soil in pots that were sunk into the field on Aug. 2, 1996 and eventually reduced to one plant per pot. Outer petioles of the plants were inoculated with mixture of 13 bacterial strains of E. carotovora subsp. carotovora on Sept.5, 1996. After the soft rot spread throughout the plant, the diseased plant was buried in the potted soil. New seeds were sown in the pots on April 30, 1997, and the disease was observed in June and July. The bacterial strains were re-isolated from the potted soil, diseased tissue and rhizosphere soil by the dilution plating method on modified Drigalski's medium containing 100 ppm rifampicin and by the enrichment technique. In addition to rifampicin resistance, phage sensitivities of some of the re-isolated strains were identical to those of the strains buried in the soil with the diseased plant in the previous year. From these results, some of the 13 strains overwintered in the soil and infested plant tissue and acted as primary inoculum the following year. The frequency of re-isolation varied among the strains, perhaps because of competition among the strains, differences in epidemiological behavior and stabilizing selection among the strains, and the presence of different ecotypes of the organism. Received 21 July 2000/ Accepted in revised form 19 September 2000     

胡萝卜软腐欧氏杆菌胡萝卜亚种游动性突变体的筛选

 用转座子Tn5对胡萝卜软腐欧氏杆菌胡萝卜亚种(Erwinia carotovora subsp.carotovora,Ecc)进行诱变,获得9个游动性改变了的突变体。M432游动性变大;M143、M451和M574游动性完全丧失;M43、M49、M330、M725和M726游动性较野生型变小。这9个突变体在大白菜叶柄上的致病力均减弱。游动性变小或丧失的突变体鞭毛数目减少或未发现有鞭毛。