黄柏果实提取物对植物病原真菌的抑制作用

在室内测定了芸香科植物黄柏(Phellodendron chinese Schneid.)果实乙醇粗提物及其4个不同极性溶剂的萃取部分在浓度为1 mg/mL时对小麦纹枯病菌(Rhizoctonia cerealis)、稻纹枯病菌(Rhizoctonia solani)、番茄镰刀菌萎焉病菌(Fusarium oxysporum f. sp.lycopersici)、黄瓜枯萎病菌(Fusarium oxysporum f.sp.cucumerinum)、棉花枯萎病菌(Fusarium vasinfectum)、棉花黄萎病菌(Verticillium dahliae)、小麦赤霉病菌(Fusarium graminearum)、玉米小斑病菌(Bipolaris maydis)、西瓜枯萎病菌(Fusarium oxysporum f.sp.niverum)、梨黑星病菌(Venturia piri-na)、稻瘟病菌(Magnaporthe grisea)等11种植物病原真菌的抑制作用.乙酸乙酯部分和正丁醇部分对植物病原菌均表现出较强的抑制活性,其中乙酸乙酯部分对两种丝核菌小麦纹枯病菌和稻纹枯病菌的生长抑制作用最强,抑制率分别为100.00%和89.36%;正丁醇部分对两者的抑制率分别为97.32%和61.32%.实验结果表明,黄柏果实中的抗真菌活性成分主要存在于乙醇粗提物中的乙酸乙酯和正丁醇萃取部分中.     

苹果树内生菌筛选及其对苹果腐烂病防治效果

为了获得苹果腐烂病菌的内生拮抗菌株,并初步研究其拮抗特性以及防治效果,用平板对峙法从苹果树根部、茎部、叶片筛选到具有拮抗作用的内生菌,明确其无菌滤液的抑菌效果,并测定拮抗菌株对苹果离体果实腐烂病害的防治效果。从分离纯化的56株内生菌中筛选出12株苹果腐烂病菌的拮抗菌,其中G2、G9、J32和Y40的抑菌效果比较明显,分别为69.64%、58.93%、67.86%、67.88%,当G2和G9的无菌滤液浓度达到8%时,其抑制率最高,分别达到了78.26%、76.29%,J32和Y40的无菌滤液浓度达到8%时抑制率均达到72.33%;在苹果果实离体试验中,G2、Y40的抑制率分别达到32.56%和26.89%;所筛选的这4株菌株对小麦赤霉病菌(Fusa Hum graminearum)、番茄灰霉病菌(Botrytis cinerea)、烟草赤星病菌(Alternaria longipes)、棉花枯萎病菌(Fusarium oxysporun f.sp.vasinfectum)、稻瘟病菌(Pyricularia oryzae Cav)、黄瓜枯萎病菌(Fusarium oxysporum f.sp.cucumerium)、西瓜枯萎病菌(Fusarium oxysporum f.sp.niveum)均有一定的抑制作用,其中抑制率最高达79.65%;4株菌对苹果腐烂病病原菌的抑制大多数会导致病原菌菌丝畸形。试验结果表明拮抗真菌G2、G9、J32和Y40均对苹果腐烂病菌有较强拮抗作用,为进一步开展苹果主要病害的生物防治研究提供了潜在资源菌。     

抑制水稻细菌性条斑病菌的没食子酸分离及其对水稻细菌性条斑病的防治作用

通过液—液萃取、硅胶和凝胶柱层析法,从佛甲草(Sedum lineare)分离出一种可以抑制水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)生长的单体化合物,经质谱分析,确定该化合物为没食子酸(gallic acid,GA)。在30 mg·m L-1浓度下,GA能抑制一些植物病原细菌如桃细菌性穿孔病菌(X.campestris pv.pruni)、水稻细菌性条斑病菌(X.oryzae pv.oryzicola)、水稻白叶枯病菌(X.oryzae pv.oryzae)、柑橘溃疡病菌(X.axonopodis pv.citri)、大豆细菌性斑点病菌(Pseudomonas syringae pv.glycinea)、番茄细菌性斑点病菌(P.syringae pv.tomato)和胡萝卜软腐果胶杆菌(Pectobacterium carotovora subsp.carotovora)的生长;GA还对11种植物病原真菌如烟草疫霉(Phytophthora nicotianae)、指状青霉(Penicillium digitatum)、滇刺枣褐腐病菌(Streptobotrys streptothrix)、瓜果腐霉(Pythium aphanidermatum)、芒果拟盘多毛孢(Pestalotiopsis mangiferae)、新月弯孢霉(Curvularia lunata)、立枯丝核菌(Rhizoctonia solani)、(Fusarium oxysporum f.sp.niverum)、西瓜专化型尖孢镰刀菌(F.oxysporum f.sp.nicotianae)、番茄灰霉病菌(Botrytis cinerea)和齐整小核菌(Sclerotium rolfsii)的生长具有一定的抑制作用。在300 mg·m L-1浓度下,GA对水稻细菌性条斑病的田间防治效果达到64.62%。该研究结果表明没食子酸具有开发成为一种防治水稻细菌性条斑病的杀菌剂的潜力。     

抑制水稻细菌性条斑病菌的没食子酸分离及其对水稻细菌性条斑病的防治作用（英文）

通过液—液萃取、硅胶和凝胶柱层析法,从佛甲草(Sedum lineare)分离出一种可以抑制水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)生长的单体化合物,经质谱分析,确定该化合物为没食子酸(gallic acid,GA)。在30 mg·mL~(-1)浓度下,GA能抑制一些植物病原细菌如桃细菌性穿孔病菌(X.campestris pv.pruni)、水稻细菌性条斑病菌(X.oryzae pv.oryzicola)、水稻白叶枯病菌(X.oryzae pv.oryzae)、柑橘溃疡病菌(X.axonopodis pv.citri)、大豆细菌性斑点病菌(Pseudomonas syringae pv.glycinea)、番茄细菌性斑点病菌(P.syringae pv.tomato)和胡萝卜软腐果胶杆菌(Pectobacterium carotovora subsp.carotovora)的生长;GA还对11种植物病原真菌如烟草疫霉(Phytophthora nicotianae)、指状青霉(Penicillium digitatum)、滇刺枣褐腐病菌(Streptobotrys streptothrix)、瓜果腐霉(Pythium aphanidermatum)、芒果拟盘多毛孢(Pestalotiopsis mangiferae)、新月弯孢霉(Curvularia lunata)、立枯丝核菌(Rhizoctonia solani)、(Fusarium oxysporum f.sp.niverum)、西瓜专化型尖孢镰刀菌(F.oxysporum f.sp.nicotianae)、番茄灰霉病菌(Botrytis cinerea)和齐整小核菌(Sclerotium rolfsii)的生长具有一定的抑制作用。在300 mg·mL~(-1)浓度下,GA对水稻细菌性条斑病的田间防治效果达到64.62%。该研究结果表明没食子酸具有开发成为一种防治水稻细菌性条斑病的杀菌剂的潜力。     

几株农用拮抗链霉菌的初步研究

从山西蟒河自然保护区分离到5株链霉菌。琼脂移块法和发酵液扩散法试验表明,它们所产生的抗生素对棉花枯萎病菌(Fusarium oxysporum f.sp.vasinfectum)、棉花黄萎病菌(Verticillium dahliae)、棉花立枯病菌(Rhizoctonia solani)、水稻纹枯病菌(Pellicularia sasakii)、黄瓜枯萎病菌(Fusarium oxysporum f.sp.cucumerinum)等十多种植物病原真菌,对果蔬贮藏期间的病原真菌,如木霉(Trichodermasp.)、意大利青霉(Penicillium italicum)、根霉(Rhizopussp.)等都有明显的抑制作用。     

杀真菌素链霉菌AL-04的筛选与鉴定

对13株来源于青藏高原土壤的放线菌进行活化,再分别采用琼脂扩散法、生长速率法、孢子萌发抑制法和离体叶片法筛选出一株抗菌活性高且抗性稳定的菌株AL-04。抗菌活性结果表明,该菌株对4种常见的土传病害病原真菌:辣椒疫霉病菌(Phytophthora capsici)、黄瓜枯萎病菌(Fusarium oxysporum f.sp.cucumerinum)、西瓜枯萎病菌(Fusarium oxysporum f.sp.niveum)、番茄灰霉病菌(Botrytis cinerea)都有较强的抑制效果,抑菌率均在70.0%以上,其中对辣椒疫霉病菌(Phytophthora capsici)抑菌率高达93.0%。通过形态特征、生理生化特征分析和16S rDNA序列分析,将该菌株鉴定为杀真菌素链霉菌(Streptomyces fungicidicus)。     

红火蚁共生链霉菌Streptomyces sp. DF-5的分离培养及其发酵液对植物病原真菌的抑制活性研究

自入侵中国以来红火蚁Solenopsis invicta Buren受到广泛关注,明确与该蚁共生或者伴生的微生种类及其生物学功能具有重要意义。本研究从红火蚁工蚁体中筛选出一个共生菌株,观察、分析了其生长、生理生化特性和16S rRNA同源序列特征,应用菌丝生长速率法测定了该菌株发酵液滤液对3种植物病原真菌的抑菌作用。鉴定结果表明该菌株属于链霉菌属Streptomyces,命名为Streptomyces sp.DF-5,其发酵液滤液对常见植物病原真菌如荔枝霜疫霉病菌Peronophythora litchii、香蕉枯萎病菌Fusarium oxysporum f.sp.cubense和稻瘟病菌Magnaporthe oryzae具有较好的抑制效果,抑制率均在70%以上,其中对荔枝霜疫霉病菌的抑制效果高达94.5%。本研究可为利用红火蚁共生菌开发新型生物农药提供参考。     

花魔芋软腐病原真菌分离鉴定

魔芋软腐病是魔芋生产过程中的重要病害,也是限制魔芋产业发展的主要因素。目前,已有报道魔芋软腐病主要由细菌引起,鲜有真菌引起魔芋球茎软腐发病的报道。为明确云南曲靖市花魔芋(Amorphophallus konjac)软腐病的病原种类和侵染特征,该研究通过组织分离法,对采集自云南曲靖市的花魔芋病样进行了真菌的分离,通过形态学结合基于ITS与LSU序列分析的分子鉴定方法对分离真菌进行鉴定,并根据柯赫氏法则进行致病性测定,并对鉴定出的病原真菌同魔芋软腐病原细菌进行了双回接试验分析。结果表明:(1)从形态学和分子水平鉴定了轮纹镰刀菌(Fusarium concentricum)、尖孢镰刀菌(F. oxysporum)和F. ambrosium 3种镰刀菌,1种毛霉属真菌(Mucor sp.),1种根霉属真菌(Rhizopus sp.),1种青霉属真菌(Penicillium sp.)和1种粉红螺旋聚孢霉属真菌(Clonostachys sp.)。(2)统计分析发现,轮纹镰刀菌的相对丰度最高,为45.45%。(3)柯赫氏法则检测发现轮纹镰刀菌具有致病性。(4)轮纹镰刀菌和病原细菌胡萝卜果胶杆菌(Pectobacterium aroidearum)双接种魔芋球茎发现软腐病发病更快,病变组织重量显著高于单接种轮纹镰刀菌或果胶杆菌处理。综上表明,魔芋软腐病可能是由真菌和细菌复合侵染引发。该研究结果为魔芋软腐病的防治提供了理论依据。     

生姜共生真菌的分离及其体外抑菌效应初探

为筛选得到优良植物病害生防菌,对广西生姜Zingiber officinale种植区健康生姜根系和叶片中的共生真菌进行了组织分离,以生姜茎腐病菌群结腐霉Pythium myriotylum和香蕉枯萎病菌尖孢镰刀菌古巴专化型4号生理小种Fusarium oxysporum f. sp. cubense race 4为指示菌,通过平板对峙培养法和发酵液菌落直径法试验进行筛选评价,并结合形态学观察及ITS序列分析对筛选出的生防效果最好的共生真菌进行了鉴定。结果表明,从生姜植株共分离得到34株共生真菌,其中根系分离22株,叶片分离12株;对峙培养发现有6株共生真菌对生姜茎腐病菌和香蕉枯萎病菌均有抑制作用;其中菌株SBM-11拮抗作用最强,对生姜茎腐病菌抑制率达到93%,对香蕉枯萎病菌抑制率达到82%;SBM-11的发酵液对生姜茎腐病菌和香蕉枯萎病菌抑制率分别为82%、73%,与其他菌株发酵液抑制效果相比差异明显;结合形态和分子鉴定结果表明SBM-11菌株为绿色木霉Trichoderma viride,极具生防潜力。     

茶多酚对几种植物病原真菌的抑制作用及机理研究

用不同浓度的茶多酚液对玉米小斑病菌(Bipolaris maydis)、香蕉炭疽病菌(Colletotrichum musae)和莲腐败病菌(Fusarium oxysporum f. sp.)进行抑菌测定.结果表明:茶多酚对三种植物病原真菌生长和分生孢子萌发都具有极显著的抑制作用(P<0.01);不同浓度的茶多酚液对同种植物病原真菌的抑制作用不同,随着茶多酚浓度的增大,其抑制力增强,其中10和5 mg/mL抑制力最强;茶多酚对三种不同的植物病原真菌的抑制程度也不同,其中对玉米小斑病菌的抑制效果最好,10和5 mg/mL茶多酚稀释液的分生孢子萌发抑制率达100%,且原生质外溢,细胞畸变.其作用机理是破坏了菌体的细胞膜结构和抑制了CAT、POD酶活,使其丧失细胞膜的屏障和酶系的保护功能.     

A rapid method for identifying and quantifying soft rot erwinias directly from plant material based on their temperature tolerances and sensitivity to erythromycin

A method is described for identifying and quantifying three soft rot erwinias directly from plant tissue and from other sources that is particularly useful in epidemiological studies. Colonies of these bacteria form characteristic deep cavities on selective-diagnostic crystal violet pectate (CVP) medium. Bacteria from individual presumptive erwinia colonies on CVP plates spot inoculated on plates of CVP medium with or without erythromycin (35 μg/ml) added and incubated at 27, 33°5 and 37°C can be identified according to the pattern of cavity formation. Erwinia carotovora pv. atroseptica forms the characteristic cavities only at 27°C and E. carotovora pv. carotovora at 27 and 33.5°C but not at 37°C on CVP with or without erythromycin. Erwinia chrysanthemi forms cavities at all temperatures and can also be identified by failure to grow at 27°C on CVP with erythromycin. Similarly, erwinias in mixed populations can be quantified by dilution plating on CVP with or without erythromycin and incubating at the different temperatures. Using this method, ca 80% of 183 erwinia strains in a culture collection were correctly identified, the precision increasing to over 95% when recently isolated erwinia strains were examined.     

Application of amplified fragment length polymorphism fingerprinting for taxonomy and identification of the soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi

The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (clusters 1 to 4) resulted. Cluster 1 contained Erwinia carotovora subsp. carotovora (subclusters 1a and 1b) and Erwinia carotovora subsp. odorifera (subcluster 1c) strains, while cluster 2 contained Erwinia carotovora subsp. atroseptica (subcluster 2a) and Erwinia carotovora subsp. betavasculorum (subcluster 2b) strains. Clusters 3 and 4 contained Erwinia carotovora subsp. wasabiae and E. chrysanthemi strains, respectively. While E. carotovora subsp. carotovora and E. chrysanthemi showed a high level of molecular diversity (23 to 38% mean similarity), E. carotovora subsp. odorifera, E. carotovora subsp. betavasculorum, E. carotovora subsp. atroseptica, and E. carotovora subsp. wasabiae showed considerably less (56 to 76% mean similarity), which may reflect their limited geographical distributions and/or host ranges. The species- and subspecies-specific banding profiles generated from the AFLPs allowed rapid identification of unknown isolates and the potential for future development of diagnostics. AFLP fingerprinting was also found to be more differentiating than other techniques for typing the soft rot erwinias and was applicable to all strain types, including different serogroups.     

Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica

Erwinia spp. that cause soft-rot diseases in plants produce a variety of extracellular pectic enzymes. To assess the correlation between patterns of pectic enzyme production and taxonomic classification, we compared the enzymes from representative strains. Supernatants obtained from polygalacturonate-grown cultures of nine strains of Erwinia chrysanthemi, three strains of E. carotovora subsp. carotovora, and three strains of E. carotovora subsp. atroseptica were concentrated and subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. Pectate lyase, polygalacturonase, and exo-poly-alpha-D-galacturonosidase activities were visualized by staining diagnostically buffered pectate-agarose overlays with ruthenium red after incubation of the overlays with the isoelectric focusing gels. The isoelectric focusing profiles of pectate lyase and polygalacturonase were nearly identical for strains of E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica, showing three pectate lyase isozymes with isoelectric points higher than 8.7 and a polygalacturonase with pI of ca. 10.2. Isoelectric focusing profiles of the E. chrysanthemi pectic enzymes were substantially different. Although there was considerable intraspecific heterogeneity, all strains produced at least four isozymes of pectate lyase, which could be divided into three groups: basic (pI, ca. 9.0 to 10.0), slightly basic (pI, ca. 7.0 to 8.5), and acidic (pI, ca. 4.0 to 5.0). Several strains of E. chrysanthemi also produced a single form of exo-poly-alpha-D-galacturonosidase (pI, ca. 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid identification and differentiation of the soft rot erwinias by 16S-23S intergenic transcribed spacer-PCR and restriction fragment length polymorphism analyses

Current identification methods for the soft rot erwinias are both imprecise and time-consuming. We have used the 16S-23S rRNA intergenic transcribed spacer (ITS) to aid in their identification. Analysis by ITS-PCR and ITS-restriction fragment length polymorphism was found to be a simple, precise, and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from Erwinia and other genera using universal PCR primers. After PCR, the banding patterns generated allowed the soft rot erwinias to be differentiated from all other Erwinia and non-Erwinia species and placed into one of three groups (I to III). Group I comprised all Erwinia carotovora subsp. atroseptica and subsp. betavasculorum isolates. Group II comprised all E. carotovora subsp. carotovora, subsp. odorifera, and subsp. wasabiae and E. cacticida isolates, and group III comprised all E. chrysanthemi isolates. To increase the level of discrimination further, the ITS-PCR products were digested with one of two restriction enzymes. Digestion with CfoI identified E. carotovora subsp. atroseptica and subsp. betavasculorum (group I) and E. chrysanthemi (group III) isolates, while digestion with RsaI identified E. carotovora subsp. wasabiae, subsp. carotovora, and subsp. odorifera/carotovora and E. cacticida isolates (group II). In the latter case, it was necessary to distinguish E. carotovora subsp. odorifera and subsp. carotovora using the alpha-methyl glucoside test. Sixty suspected soft rot erwinia isolates from Australia were identified as E. carotovora subsp. atroseptica, E. chrysanthemi, E. carotovora subsp. carotovora, and non-soft rot species. Ten "atypical" E. carotovora subsp. atroseptica isolates were identified as E. carotovora subsp. atroseptica, subsp. carotovora, and subsp. betavasculorum and non-soft rot species, and two "atypical" E. carotovora subsp. carotovora isolates were identified as E. carotovora subsp. carotovora and subsp. atroseptica.     

Genetic regulation of pathogenicity and virulence factors in bacteria Erwinia carotovora subsp. atroseptica: identification of kduD gene

A mutant that cannot utilize pectin substances of plant cell walls was obtained via insertion of mini-mini-Tn5xylE transposon into the chromosome of phytopathogenic bacteria Erwinia carotovora subsp. atroseptica. The inability of mutant cells to utilize these substrates was caused by a failure to accomplish the catabolism of unsaturated digalacturonic acid (UDA). Study of enzymatic activities has established that mutant bacteria lost the ability to produce 2,5-diketo-3-deoxygluconate dehydrogenase, an enzyme of intracellular UDA utilization. Molecular cloning of the mutant gene was conducted, and its nucleotide sequence was determined. It was shown that the nucleotide sequence of this gene had an 82% homology with the sequence of Erwinia chrysanthemi EC3937 kduD gene encoding 2,5-diketo-3-deoxygluconate dehydrogenase. The intergene kdul-kduD region in bacteria Erwinia carotovora subsp. atroseptica is shorter in length by 98 nucleotides than the corresponding region of Erwinia chrysanthemi and does not contain promoter sequences. The kduD gene was located at 126.8 min of the Erwinia carotovora subsp. atroseptica genetic map.     

Comparative study of regulatory mechanisms for pectinase production by Erwinia carotovora subsp. carotovora and Erwinia chrysanthemi

The production of pectinase, the major virulence determinant of soft-rot Erwinia species, is controlled by many regulatory factors. We focused on the major regulatory proteins, KdgR, CRP, Pir, and PecS, characterized mainly in E. chrysanthemi, and tested for their presence and function in the control of pectate lyase (Pel) and polygalacturonase (Peh) production in E. carotovora subsp. carotovora. Homologues of kdgR and crp but not of pir and pecS were detected by Southern blot analyses in E. carotovora subsp. carotovora. In fact, KdgR and CRP homologues of E. carotovora subsp. carotovora had high amino acid identities to those of E. chrysanthemi, including a complete match of the hypothetical helix-turn-helix DNA-binding motif. However, in Western blot analyses using anti-Pir (E. chrysanthemi) antibodies, a cross-reacting protein was present in both Erwinia species, although Pel production in E. carotovora subsp. carotovora was not further stimulated by adding plant extract into the medium containing PGA (polygalacturonic acid) in which hyperinduction by Pir has been reported in E. chrysanthemi EC16. When plasmids that contained each of these regulatory genes from E. chrysanthemi were introduced into E. carotovora subsp. carotovora, Pel production was controlled as predicted from their roles in E. chrysanthemi, except for PecS. PecS exerted a positive control in E. carotovora subsp. carotovora, in contrast to a negative control in E. chrysanthemi. DNA-binding assays demonstrated that KdgR, CRP, Pir, and PecS of E. chrysanthemi and KdgR and CRP homologues of E. carotovora subsp. carotovora could bind to the promoter regions of pel-1, pel-3, and peh of E. carotovora subsp. carotovora. Taken together, KdgR and CRP homologues of E. carotovora subsp. carotovora may regulate Pel and Peh production as in E. chrysanthemi. However, the presence of Pir and PecS homologues in E. carotovora subsp. carotovora was not identified in this study, though these proteins of E. chrysanthemi were functional on the promoter regions of the pectinase genes of E. carotovora subsp. carotovora.     

Characterization of a monoclonal antibody against active pectate lyase from Erwinia carotovora

A monoclonal antibody (2E2) produced against pectate lyase from Erwinia carotovora ssp. carotovora reacted with a 41- and a 44-kilodaltion protein on Western blots of concentrated Erwinia culture supernatants resolved by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. It was unequivocally shown that monoclonal 2E2 reacted with an active form of pectate lyase by affinity purifying the antigen with the monoclonal. The affinity-purified antigen was enzymatically active and moved as a single protein band in a nonequilibrium isoelectric focusing gel. Monoclonal 2E2 reacted with the pectate lyases of a diverse range of E. carotovora ssp. carotovora, ssp. atroseptica, and ssp. betavasculorum strains, as well as with one of three strains of E. chrysanthemi. The electrophoretic mobility of the major protein (44 kilodaltons) that reacted with 2E2 was identical within a subspecies but differed among subspecies.     

Differentiation of Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica isolated from potato by Western blot and subsequent indirect ELISA

Electrotransfer of protein bands from a polyacrylamide gel to a hydrophobic poly-vinylidene difluoride (PVDF) membrane (Western blot) and their serological determination by indirect ELISA (immunoblotting) were used to differentiate Erwinia carotovora subsp. carotovora (Ecc) from Erwinia carotovora subsp. atroseptica (Eca). Ninety strains: 69 Ecc, 19 Eca and two Erwinia chrysanthemi (Echr) were examined. Eight polyclonal antisera against whole cells, glutaraldehyde fixed cells, glycopro-teins, and somatic antigens were prepared. Antisera produced with glutaraldehyde fixed cells did not recognize any band of the protein pattern. The remaining antisera recognized a limited number of bands. Two protein bands allowed differentiation of the two subspecies by the antisera against glycoproteins. One band with an estimated molecular weight of 36000 Da was present in the 19 Eca strains tested and another band with an estimated molecular weight of 35 000 Da was present in the 69 Ecc strains, except for three cases. The strains of Echr showed a band with an estimated weight of 33 000 Da.     

Verification of ELISA results by immunomagnetic isolation of antigens from extracts and analysis with SDS-PAGE and Western blotting, demonstrated for Erwinia spp. in potatoes

Isolation of antigens on immunomagnetic beads and subsequent analysis with SDS-PAGE and Western blotting (immunomagnetic isolation-Western blotting (IMI-WB)) was used to verify positive ELISA results for Erwinia chrysanthemi and Erw. carotovora subsp. atroseptica in potato peel extracts. Direct analysis of highly contaminated extracts by Western blotting without previous immuno-isolation resulted in background reactions, whereas immunomagnetic isolation resulted in distinct bands of specific antigens. Target cells as well as antigenic cell products were captured in IMI-WB. Band patterns on IMI-WB of cell-free culture filtrates and cell suspensions were highly similar, but the removal of cells lowered the detection level by 10- to 100-fold. Threshold levels of IMI-WB were generally comparable with those of ELISA.
No differences in threshold levels and band patterns were found between a direct format and an indirect format of immuno-isolation.
In IMI-WB, blotting patterns differed between Erw. chrysanthemi and Erw. carotovora subsp. atroseptica. The patterns were identical for 15 Erw. chrysanthemi strains, isolated from potato peel extracts in The Netherlands. However, one of 15 strains of Erw. carotovora subsp. atroseptica from potato peel extracts in The Netherlands gave an aberrant pattern. Target bacteria could be easily distinguished from those of cross-reacting strains on the basis of band patterns.
Potato peel extracts naturally contaminated with Erw. chrysanthemi gave IMI-WB patterns that were similar to pure cultures of the homologous strains.     

Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil

AIMS: To determine the characteristics of bacteria associated with the blackleg disease of potato in Brazil and compare them with species and subspecies of pectolytic Erwinia. METHODS AND RESULTS: Biochemical and physiological characteristics of 16 strains from blackleg-infected potatoes in State of Rio Grande do Sul, Brazil, were determined and differentiated them from all the E. carotovora subspecies and E. chrysanthemi. Pathogenicity and maceration ability of the Brazilian strains were greater than those of E. carotovora subsp. atroseptica, the causal agent of potato blackleg in temperate zones. Analyses of serological reaction and fatty acid composition confirmed that the Brazilian strains differed from E. carotovora subsp. atroseptica, but the sequence of 16S rDNA gene and the 16S-23S intergenic spacer (IGS) region confirmed the Brazilian strains as pectolytic Erwinia. Restriction analysis of the IGS region differentiated the Brazilian strains from the subspecies of E. carotovora and from E. chrysanthemi. A unique SexAI restriction site in the IGS region was used as the basis for a primer to specifically amplify DNA from the Brazilian potato blackleg bacterium in PCR. CONCLUSIONS: The bacterium that causes the blackleg disease of potato in Brazil differs from E. carotovora subsp. atroseptica, the blackleg pathogen in temperate zones. It also differs from other subspecies of E. carotovora and from E. chrysanthemi and warrants status as a new subspecies, which would be appropriately named E. carotovora subsp. brasiliensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The blackleg disease of potato is caused by a different strain of pectolytic Erwinia in Brazil than in temperate potato-growing regions. The Brazilian strain is more virulent than E. carotovora subsp. atroseptica, the usual causal agent of potato blackleg.