Multiplex detection and identification of bacterial pathogens causing potato blackleg and soft rot in Europe,using padlock probes

The objective of this study was to develop a multiplex detection and identification protocol for bacterial soft rot coliforms, namely Pectobacterium wasabiae (Pw), Pectobacterium atrosepticum (Pba) and Dickeya spp., responsible for potato blackleg and tuber soft rot. The procedures were derived from the phylogenetic relationships of these and other Enterobacteriaceae based on recA sequences. The group of Pw strains was highly homogeneous and could be distinguished from the other species. A ligation‐based method for detection of Pw was developed. Five padlock probes (PLPs) were designed, targeting recA sequences to identify the Pw, Pba or Dickeya spp., whereas a sixth probe recognised recA sequences of all soft rot coliforms including Pectobacterium carotovorum subsp. carotovorum (Pcc). Two PLP‐based applications were developed: one using real‐time PCR and one using universal microarrays. Assay sensitivity and specificity were demonstrated using 71 strains of Pw, Pcc, Pba and Dickeya spp. Both multiplex methods can be potentially used for seed testing and in ecological studies, but further validation is required.     

Estimation of Jordanian isolated Pectobacterium cellulase,pectinase concentrations,RAPD polymorphism and their maceration activity

Forty-two Pectobacterium isolates were recovered from contaminated soil and rotted vegetables in Jordan. Twenty of them were belonged to; Pectobacterium carotovorum subsp. Carotovorum (Pbc) (= Erwinia carotovora subsp. carotovora), 11 isolates were belonged to Pectobacterium atrospeticum (= Erwinia carotovora subsp. atroseptica) (Pba) and 11 isolates were not classifiable (Pbs). Maceration activity of the 42 proved their ability to macerate potato, carrot and radish slices. Maceration activity of the isolates either of the same subspecies or in between the isolates of different subspecies isolated from the same host or from different hosts was varied. The measured concentration in μM?ml^?1 of both cellulase and pectinase enzymes was variable too. The Rapid amplified polymorphic DNA-PCR finger printing of total genomic DNA using a pair of 10-mer oligonucleotide primers amplification showed similar DNA bands with some polymorphic variations amongst the isolates.     

A quorum sensing‐defective mutant of Pectobacterium carotovorum ssp. brasiliense 1692 is attenuated in virulence and unable to occlude xylem tissue of susceptible potato plant stems

Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post‐harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall‐degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild‐type expI gene in trans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and in vitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild‐type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild‐type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI‐inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild‐type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down‐regulated in the mutant compared with the wild‐type strain.     

胡萝卜软腐果胶杆菌CpxP蛋白纯化及抑菌功能鉴定

胡萝卜软腐果胶杆菌是世界十大植物病原菌之一,主要侵染十字花科的经济作物和观赏花卉。文中从胡萝卜软腐果胶杆菌的基因组中克隆1个抗菌基因cpxP(GeneID:29704421),将其构建在原核表达质粒pET-15b上,并转化至大肠杆菌Escherichia coli BL21 (DE3)进行表达,经纯化后进行稳定性和抑菌实验。结果显示,IPTG的诱导终浓度为1mmol/L,实现了蛋白的高效外源表达,纯化后电泳无杂蛋白残留,且该蛋白具有良好的热稳定性和pH稳定性。CpxP蛋白抑菌试验结果显示其对胡萝卜切片的抑菌率可达到44.89%,对马铃薯切片的抑菌率可达到59.41%。为进一步解释其抑菌机理,研究该蛋白的空间结构可为软腐病的防治和新型蛋白农药靶点研究提供新思路。     

Interactions of Ralstonia solanacearum and Pectobacterium carotovorum with Meloidogyne incognita on potato

Effect of interactions of Meloidogyne incognita with Ralstonia solanacearum and interaction of M. incognita with Pectobacterium carotovorum were studied in sequential and simultaneous inoculations on potato (Solanum tuberosum). Inoculation of M. incognita caused a lesser reduction in plant growth than caused by R. solanacearum. Inoculation of M. incognita plus R. solanacearum caused a greater reduction in plant growth than the damage caused by either pathogen. Inoculation of M. incognita prior to R. solanacearum resulted in a greater reduction in plant growth than R. solanacearum was inoculated prior to M. incognita. However, inoculation of M. incognita or P. carotovorum caused similar reduction in plant growth. Inoculation of P. carotovorum prior to M. incognita caused lesser reduction in plant growth than simultaneous inoculation of both pathogens. Inoculation of M. incognita caused galling in potato roots but the size of galls was small. Inoculation of P. carotovorum or R. solanacearum with M. incognita had adverse effect on galling and nematode multiplication. Wilting or soft rot index was 3 when R. solanacearum or P. carotovorum was inoculated alone. In other treatments, where R. solanacearum or P. carotovorum was inoculated with M. incognita, wilting or soft rot indices were 5.     

Bacteriophages Isolated in China for the Control of Pectobacterium carotovorum Causing Potato Soft Rot in Kenya

Soft rot is an economically significant disease in potato and one of the major threats to sustainable potato production. This study aimed at isolating lytic bacteriophages and evaluating methods for and the efficacy of applying phages to control potato soft rot caused by Pectobacterium carotovorum. Eleven bacteriophages isolated from soil and water samples collected in Wuhan, China, were used to infect P. carotovorum host strains isolated from potato tubers showing soft rot symptoms in Nakuru county, Kenya. The efficacy of the phages in controlling soft rot disease was evaluated by applying individual phage strains or a phage cocktail on potato slices and tubers at different time points before or after inoculation with a P. carotovorum strain. The phages could lyse 20 strains of P. carotovorum, but not Pseudomonas fluorescens control strains. Among the 11 phages, Pectobacterium phage Wc5 r, interestingly showed cross-activity against Pectobacterium atrosepticum and two phage-resistant P. carotovorum strains. Potato slice assays showed that the phage concentration and timing of application are crucial factors for effective soft rot control. Phage cocktail applied at a concentration of 1 9 109 plaque-forming units per milliliter before or within an hour after bacterial inoculation on potato slices, resulted in C 90%reduction of soft rot symptoms. This study provides a basis for the development and application of phages to reduce the impact of potato soft rot disease.     

Characterization of Iranian Pectobacterium carotovorum Strains from Sugar Beet by Phenotypic Tests and Whole-cell Proteins Profile

Thirty isolates of Pectobacterium carotovorum from soft rot‐affected sugar beet plants in the Fars province of Iran were characterized phenotypically and by analysis of whole‐cell protein electrophoresis patterns. The isolates were found to be heterogeneous based on the results of physiological and biochemical tests and protein profiles. The results of numerical analysis of phenotypic characteristics and protein patterns showed that only 27% of the collected isolates (phenon 4) could be identified as P. betavasculorum when compared with reference strains. Strains of the first, second, third and fifth phenon shared similar characters with those of P. carotovorum subsp. carotovorum, P. betavasculorum and P. carotovorum subsp. odoriferum, but were distinct from these subspecies. Inoculation of phenon 4 isolates into wounded sugar beet petioles led to black streaking, root rot and vascular necrosis. Other isolates were incapable of causing systemic symptoms in inoculated plants.     

Pectobacterium carotovorum subsp. brasiliense Causing Soft Rot on Paprika in Korea

During summer 2011 in South Korea, severe fruit rot of paprika was observed, causing severe economic losses in paprika production. Symptoms of fruit and pedicel decay were consistent with symptoms caused by Pectobacterium carotovorum subsp. brasiliense (Pcb) as recently described in Brazil, the United States, Israel and South Africa. Physiological analysis and pathogenicity test of strains isolated from paprika fruit revealed that the pathogen was the bacterium Pcb. Sequencing and phylogenetic analysis of the 16S rDNA and partial 16S–23S rDNA intergenic spacer region confirmed that the isolates were Pcb. This is the first report of Pcb in Korea, which has a significant economic impact on Korean paprika production.     

杓兰果胶杆菌海藻糖酶的克隆表达及应用

海藻糖酶在工业发酵和食品医药等领域应用广泛,我国缺乏性能优良工业品种的海藻糖酶,同时在应用研究领域还不够深入。本研究从自然界筛选获得一株产酸性海藻糖酶的杓兰果胶杆菌(Pectobacterium cypripedii),克隆获得其海藻糖酶基因PCTre,在大肠杆菌(Escherichia coli)BL21(DE3)体系中实现了重组表达,在5L发酵罐上28h产酶达到4130U/mL。酶学性质研究显示,PCTre能特异性水解海藻糖,最适反应温度和pH分别是35℃和5.5,在pH4.0、4.5、5.0处理8h后残余酶活仍超过80%,表现出良好的耐酸性。同时该酶还显示出优良的有机溶剂耐受性,在20%(V/V)乙醇溶液中处理24h仍能保留60%的酶活。进一步在含有20%(V/V)乙醇和7.5%海藻糖的模拟发酵体系中,当添加500U/L PCTre,可在16h内完全水解海藻糖,具有良好的应用于工业乙醇发酵的潜力。     

Identification of a DNA restriction-modification system in Pectobacterium carotovorum strains isolated from Poland

AIMS: Polish isolates of pectinolytic bacteria from the species Pectobacterium carotovorum were screened for the presence of a DNA restriction-modification (R-M) system. METHODS AND RESULTS: Eighty-nine strains of P. carotovorum were isolated from infected potato plants. Sixty-six strains belonged to P. carotovorum ssp. atrosepticum and 23 to P. carotovorum ssp. carotovorum. The presence of restriction enzyme Pca17AI, which is an isoschizomer of EcoRII endonuclease, was observed in all isolates of P. c. atrosepticum but not in P. c. carotovorum. The biochemical properties, PCR amplification, and sequences of the Pca17AI restriction endonuclease and methyltransferase genes were compared with the prototype EcoRII R-M system genes. Only when DNA isolated from cells of P. c. atrosepticum was used as a template, amplification of a 680 bp homologous to the gene coding EcoRII endonuclease. CONCLUSIONS: Endonuclease Pca17AI, having a relatively low temperature optimum, was identified. PCR amplification revealed that the nucleotide sequence of genes for EcoRII and Pca17AI R-M are different. Dcm methylation was observed in all strains of Pectobacterium and other Erwinia species tested. The sequence of a DNA fragment coding Dcm methylase in P. carotovorum was different from that of Escherichia coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Pca17AI is the first psychrophilic isoschizomer of EcoRII endonuclease. The presence of specific Dcm methylation in chromosomal DNA isolated from P. carotovorum is described for the first time. A 680 bp PCR product, unique for P. c. atrosepticum strains, could serve as a molecular marker for detection of these bacteria in environmental samples.     

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.     

Induced suppression of soft rot disease in tobacco by combined application of Bacillus subtilis strain B4 and chemical elicitor BTH

In this study, the utility of the combined application of Benzo-(1,2,3)-thidiazole-7-carbothioic acid S-methyl ester (BTH), a systemic acquired resistance (SAR) inducer, and the plant growth-promoting rhizobacteria (PGPR), Bacillus subtilis strain B4 (B4), was investigated for the suppression of soft rot disease caused by Pectobacterium carotovorum SCC1 (SCC1) in tobacco seedlings. Soft rot disease was completely suppressed in tobacco with combined application of B4 strain and 0.1 mM BTH when compared to individual application upon pathogen challenge. In addition, the population of bacterial cells of B4 strain was found to be greater when cultured in growth media in the presence of BTH, compared to the growth of B4 strain in the absence of BTH. Spectrophotometer analysis revealed that there was an increased broad range of compounds in the culture filtrate of B4 strain when grown with BTH, compared to the culture filtrate of B4 strain alone. The combined application of B4 strain and 0.1 mM BTH induced the increased expression of PR1a::GUS on tobacco and elicited systemic resistance against SCC1 when compared to individual application. However, there was low expression of PR1a::GUS in the water-treated control. Hence, the integrated use of BTH and B4 strain might be one of the strategies for biological control of soft rot disease through induced systemic resistance (ISR) in tobacco plants.     

Host-induced gene silencing of Pectobacterium carotovorum quorum sensing gene enhances soft rot disease resistance in potato plants

Quorum sensing is a regulatory mechanism of gene expression in bacteria which regulated many physiological processes such as production of virulence factors in Pectobacterium carotovorum. In this research, the plant expression vector, pBI121, harbouring the anti-quorum sensing gene, aiiA, was transformed into Potato (Solanum tuberosum cv. Agria) explants by Agrobacterium mediated transformation procedure and putative transformants containing pBI121/aiiA were obtained. The results of bioassay test revealed that the expression of aiiA gene in potato plant induces the resistance to the early stage of bacterium pathogenesis. The soft rot symptoms such as wilting, yellowing and tissue maceration were not observed until 48?hr after inoculation of transgenic potatoes with P. carotovorum. Although in non-transgenic plants, the disease symptoms were appeared after 24?hr of plants infection. Hence, this study proves that the heterologous expression of aiiA gene enhances the resistance against potato soft rot disease.     

Characterization of Pectobacterium carotovorum subsp. carotovorum Causing Watermelon Soft Rot Disease in Iran

The aim of this study was characterized Pectobacterium carotovorum subsp. carotovorum (Pcc) the causal pathogen of watermelon soft rot disease in Iran. Of fifty bacterial isolates with white grey and convex colonies on nutrient agar obtained from symptomatic watermelon, ten isolates were selected for further tests. Pathogenicity tests results showed that all test isolates developed typical water‐soak symptoms after 2 days and signs of soft rot began 4 days after inoculation on watermelon fruits. Based on the phenotypic properties, the isolates were identified as Pectobacterium carotovorum subsp. carotovorum. The 16S rDNA sequences of isolates were 99% similar to the corresponding 16S rDNA sequence of the reference Pcc isolate. BOX and ERIC‐PCR analysis indicated that genetic diversity was present among the isolated Pcc isolates did not relate to the geographic location isolated from. To the best of our knowledge, this is the first study of biochemical and genotypic characterization of Pcc isolates the causal agents of soft rot disease on watermelon, in Iran.     

Single gene enables plant pathogenic Pectobacterium to overcome host-specific chemical defence

Plants of the Brassicales order, including Arabidopsis and many common vegetables, produce toxic isothiocyanates to defend themselves against pathogens. Despite this defence, plant pathogenic microorganisms like Pectobacterium cause large yield losses in fields and during storage of crops. The bacterial gene saxA was previously found to encode isothiocyanate hydrolase that degrades isothiocyanates in vitro. Here we demonstrate in planta that saxA is a virulence factor that can overcome the chemical defence system of Brassicales plants. Analysis of the distribution of saxA genes in Pectobacterium suggests that saxA from three different phylogenetic origins are present within this genus. Deletion of saxA genes representing two of the most common classes from P. odoriferum and P. versatile resulted in significantly reduced virulence on Arabidopsis thaliana and Brassica oleracea. Furthermore, expressing saxA from a plasmid in a potato-specific P. parmentieri strain that does not naturally harbour this gene significantly increased the ability of the strain to macerate Arabidopsis. These findings suggest that a single gene may have a significant role in defining the host range of a plant pathogen.