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1.
Pectobacterium species are enterobacterial plant-pathogenic bacteria that cause soft rot disease in diverse plant species. Previous epidemiological studies of Pectobacterium species have suffered from an inability to identify most isolates to the species or subspecies level. We used three previously described DNA-based methods, 16S-23S intergenic transcribed spacer PCR-restriction fragment length polymorphism analysis, multilocus sequence analysis (MLSA), and pulsed-field gel electrophoresis, to examine isolates from diseased stems and tubers and found that MLSA provided the most reliable classification of isolates. We found that strains belonging to at least two Pectobacterium clades were present in each field examined, although representatives of only three of five Pectobacterium clades were isolated. Hypersensitive response and DNA hybridization assays revealed that strains of both Pectobacterium carotovorum and Pectobacterium wasabiae lack a type III secretion system (T3SS). Two of the T3SS-deficient strains assayed lack genes adjacent to the T3SS gene cluster, suggesting that multiple deletions occurred in Pectobacterium strains in this locus, and all strains appear to have only six rRNA operons instead of the seven operons typically found in Pectobacterium strains. The virulence of most of the T3SS-deficient strains was similar to that of T3SS-encoding strains in stems and tubers.The genus Pectobacterium (formerly Erwinia) contains both narrow- and broad-host-range bacterial plant pathogens that cause soft rot, stem rot, wilt, and blackleg in species belonging to over 35% of plant orders (20). Four Pectobacterium species have been described: Pectobacterium atrosepticum, Pectobacterium betavasculorum, Pectobacterium carotovorum, and Pectobacterium wasabiae (9). The recently described organism P. carotovorum subsp. brasiliensis is genetically distinct from previously described Pectobacterium taxa; approximately 82% of its genes are shared with P. atrosepticum, and 84% of its genes are shared with P. carotovorum subsp. carotovorum, while 13% of its genes are found in neither P. atrosepticum nor P. carotovorum subsp. carotovorum (7, 10, 20). To date, only P. carotovorum subsp. carotovorum and P. atrosepticum have been reported to occur in the same field (14, 21). P. carotovorum subsp. carotovorum is found worldwide, and P. atrosepticum is found in cool climates; while P. carotovorum subsp. brasiliensis has been found only in Brazil, Israel, and the United States, it is likely to have a wider distribution (20). Compared to the ecology and genetics of P. carotovorum subsp. carotovorum and P. atrosepticum, little is known about the ecology and genetics of P. betavasculorum, P. wasabiae, or P. carotovorum subsp. brasiliensis.Pectobacterium strains isolated from potato are diverse based on serology, genome structure, and fatty acid composition (5, 35). Previous epidemiological studies of pectolytic Enterobacteriaceae were complicated by the diversity of this group and the lack of tools capable of placing all isolates into clades. For example, Gross et al. (14) were unable to classify over 50% of Pectobacterium isolates obtained from potato, and Pitman et al. (23) were unable to type 13% of their isolates. Novel PCR-based methods potentially capable of classifying all Pectobacterium isolates have been described, but they were developed prior to the recognition of P. carotovorum subsp. brasiliensis (1, 34).The main virulence determinants of Pectobacterium are the pectolytic enzymes secreted through the type II secretion system. Although these enzymes are required for development of symptoms, many other virulence genes have been shown to contribute to Pectobacterium pathogenicity, including the type III secretion system (T3SS) genes, the cfa gene cluster, and the type IV secretion system genes (3, 15, 19). Recent genomic analysis showed that some of these gene clusters, such as the cfa and type IV secretion system cluster genes, as well as genes important for interactions with insects, are present in only some Pectobacterium species (10). Thus, Pectobacterium species appear to use different genetic tools to overcome plant host barriers and to interact with insect vectors.Many gram-negative pathogenic bacteria secrete virulence proteins, known as effectors, through the T3SS into host cells. Once inside host cells, the effectors manipulate host defenses and promote bacterial growth (13). Unlike many other gram-negative plant pathogens, Pectobacterium does not require the T3SS for pathogenicity. Rather, this secretion system makes a small, but measurable, contribution to the early stages of P. carotovorum growth in leaves of the model plant Arabidopsis thaliana (26) and contributes to the virulence of P. atrosepticum on potato (15). Recently, we isolated Pectobacterium strains that lack the T3SS from potatoes and also found P. wasabiae and P. carotovorum subsp. brasiliensis on potatoes in Wisconsin (35). The first goal of this study was to determine if P. wasabiae and P. carotovorum subsp. brasiliensis are common in agricultural fields or if soft rot disease is typically caused by P. carotovorum subsp. carotovorum and P. atrosepticum, which have been the focus of nearly all previous studies of potato soft rot, stem rot, and blackleg disease. Second, since we recently isolated a strain lacking the T3SS (35), we also aimed to determine if strains lacking the T3SS are common in infected potatoes and if these strains tend to be less virulent on potato stems and tubers than strains encoding a T3SS.  相似文献   

2.
To identify bacteria causing soft rot and blackleg in potato in Finland, pectinolytic enterobacteria were isolated from diseased potato stems and tubers. In addition to isolates identified as Pectobacterium atrosepticum and Dickeya sp., many of the isolated strains were identified as Pectobacterium carotovorum subsp. carotovorum. Phylogenetic analysis and biochemical tests indicated that one of the isolates from potato stems resembled Pectobacterium wasabiae. Furthermore, two blackleg‐causing P. carotovorum strains recently isolated in Europe clustered with P. wasabiae, suggesting that at least some of these isolates were originally misidentified. All the other Finnish P. carotovorum isolates resembled the subsp. carotovorum type strain in biochemical tests but could be clustered into two distinct groups in the phylogenetic analysis. One of the groups mainly contained strains isolated from diseased tubers, whereas the other mainly included isolates from potato stems. In contrast to the tuber isolates, the stem isolates lacked genes in Type III secretion genes, were not able to elicit a hypersensitive response in tobacco leaves and produced only small amounts of autoinducers in the stationary phase in vitro. P. wasabiae isolate was able to cause similar amount of blackleg‐like symptoms as P. atrosepticum in a field experiment with vacuum‐infiltrated tubers, whereas both P. atrosepticum and P. carotovorum isolates reduced emergence and delayed growth more than P. wasabiae. Our findings confirm the presence of P. wasabiae in Finland and show that the Finnish P. carotovorum subsp. carotovorum isolates can be divided into two groups with specific characteristics and possibly also different ecologies.  相似文献   

3.
Soft rot disease of Amorphophallus konjac is caused by Pectobacterium species. Infected corms are considered a primary and important source of inocula. Based on the 16S rDNA sequences of the soft rot pathogens, one pair of specific primers was designed to identify the soft rot disease by real-time PCR and the other two were used to identify the pathogens of Pectobacterium carotovorum subsp. carotovorum. and P. chrysanthemi respectively. According to the results, a single cell of Pectobacterium could be detected by real-time PCR with the designed primer pair, while at least 100 bacteria were required for conventional PCR. Moreover, the two special primers can directly and accurately authenticate to Pectobacterium carotovorum subsp. carotovorum and P. chrysanthemi by the conventional PCR system without testing the pathogenicity, biochemical and phenotypic characterizations and so on. In conclusion, the PCR-based techniques showed several significant advantages in identifying the soft rot pathogens from konjac, such as higher sensitivity, rapidness and precision, and it could be widely used in seed quarantine.  相似文献   

4.
Pectobacterium carotovorum subsp. carotovorum (Pcc) is a gram-negative, broad host range bacterial pathogen which causes soft rot disease in potatoes as well as other vegetables worldwide. While Pectobacterium infection relies on the production of major cell wall degrading enzymes, other virulence factors and the mechanism of genetic adaptation of this pathogen is not yet clear. In the present study, we have performed an in-depth genome-wide characterization of Pcc strain ICMP5702 isolated from potato and compared it with other pathogenic bacteria from the Pectobacterium genus to identify key virulent determinants. The draft genome of Pcc ICMP5702 contains 4,774,457 bp with a G + C content of 51.90% and 4,520 open reading frames. Genome annotation revealed prominent genes encoding key virulence factors such as plant cell wall degrading enzymes, flagella-based motility, phage proteins, cell membrane structures, and secretion systems. Whereas, a majority of determinants were conserved among the Pectobacterium strains, few notable genes encoding AvrE-family type III secretion system effectors, pectate lyase and metalloprotease in addition to the CRISPR-Cas based adaptive immune system were uniquely represented. Overall, the information generated through this study will contribute to decipher the mechanism of infection and adaptive immunity in Pcc.  相似文献   

5.
A multiplex polymerase chain reaction (PCR) assay for simultaneous, fast and reliable detection of the main soft rot and blackleg potato pathogens in Europe has been developed. It utilises three pairs of primers and enables detection of three groups of pectinolytic bacteria frequently found in potato, namely: Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum together with Pectobacterium wasabiae and Dickeya spp. in a multiplex PCR assay. In studies with axenic cultures of bacteria, the multiplex assay was specific as it gave positive results only with strains of the target species and negative results with 18 non‐target species of bacteria that can possibly coexist with pectinolytic bacteria in a potato ecosystem. The developed assay could detect as little as 0.01 ng µL–1 of Dickeya sp. genomic DNA, and down to 0.1 ng µL–1 of P. atrosepticum and P. carotovorum subsp. carotovorum genomic DNA in vitro. In the presence of competitor genomic DNA, isolated from Pseudomonas fluorescens cells, the sensitivity of the multiplex PCR decreased tenfold for P. atrosepticum and Dickeya sp., while no change was observed for P. carotovorum subsp. carotovorum and P. wasabiae. In spiked potato haulm and tuber samples, the threshold level for target bacteria was 101 cfu mL–1 plant extract (102 cfu g–1 plant tissue), 102 cfu mL–1 plant extract (103 cfu g–1 plant tissue), 103 cfu mL–1 plant extract (104 cfu g–1 plant tissue), for Dickeya spp., P. atrosepticum and P. carotovorum subsp. carotovorum/P. wasabiae, respectively. Most of all, this assay allowed reliable detection and identification of soft rot and blackleg pathogens in naturally infected symptomatic and asymptomatic potato stem and progeny tuber samples collected from potato fields all over Poland.  相似文献   

6.
Plant cell wall degrading enzymes (PCWDE) are the major virulence determinants in phytopathogenic Pectobacterium, and their production is controlled by many regulatory factors. In this study, we focus on the role of the AepA protein, which was previously described to be a global regulator of PCWDE production in Pectobacterium carotovorum (Murata et al. in Mol Plant Microbe Interact 4:239–246, 1991). Our results show that neither inactivation nor overexpression of aepA affects PCWDE production in either Pectobacterium atrosepticum SCRI1043 or Pectobacterium carotovorum subsp. carotovorum SCC3193. The previously published observation based on the overexpression of aepA could be explained by the presence of the adjacent regulatory rsmB gene in the constructs used. Our database searches indicated that AepA belongs to the YtcJ subfamily of amidohydrolases. YtcJ-like amidohydrolases are present in bacteria, archaea, plants and some fungi. Although AepA has 28% identity with the formamide deformylase NfdA in Arthrobacter pascens F164, AepA was unable to catalyze the degradation of NdfA-specific N-substituted formamides. We conclude that AepA is a putative aminohydrolase not involved in regulation of PCWDE production.  相似文献   

7.
8.
Pectobacterium species are enterobacterial plant-pathogens that cause soft rot disease in diverse plant species. Unlike hemi-biotrophic plant pathogenic bacteria, the type III secretion system (T3SS) of Pectobacterium carotovorum subsp. carotovorum (P. carotovorum) appears to secrete only one effector protein, DspE. Previously, we found that the T3SS regulator HrpL and the effector DspE are required for P. carotovorum pathogenesis on leaves. Here, we identified genes up-regulated by HrpL, visualized expression of dspE in leaves, and established that DspE causes host cell death. DspE required its full length and WxxxE-like motifs, which are characteristic of the AvrE-family effectors, for host cell death. We also examined expression in plant leaves and showed that hrpL is required for the expression of dspE and hrpN, and that the loss of a functional T3SS had unexpected effects on expression of other genes during leaf infection. These data support a model where P. carotovorum uses the T3SS early in leaf infection to initiate pathogenesis through elicitation of DspE-mediated host cell death.  相似文献   

9.
Pectobacterium carotovorum and Pectobacterium atrosepticum are dreadful causal agents of potato soft rot. Actually, there are no efficient bactericides used to protect potato against Pectobacterium spp. Biological control using actinobacteria could be an interesting approach to manage this disease. Thus, two hundred actinobacteria isolated from Moroccan habitats were tested for their ability to inhibit in vitro 4 environmental Pectobacterium strains and the two reference strains (P. carotovorum CFBP 5890 and P. atrosepticum CFBP 5889). Eight percent of these isolates were active against at least one of the tested pathogens and only 2% exhibited an antimicrobial activity against all tested Pectobacterium strains. Four bioactive isolates having the greatest pathogen inhibitory capabilities and classified as belonging to the genus Streptomyces species through 16S rDNA analysis were subsequently tested for their ability to reduce in vivo soft rot symptoms on potato slices of Bintje, Yukon Gold, Russet and Norland cultivars caused by the two pathogens P. carotovorum and P. atrosepticum. This test was carried out by using biomass inoculums and culture filtrate of the isolates as treatment. Among these, strain Streptomyces sp. OE7, reduced by 65–94% symptom severity caused by the two pathogens on potato slices. Streptomyces OE7 showed a potential for controlling soft rot on potato slices and could be useful in an integrated control program against potato soft rot pathogens in the objective to reduce treatments with chemical compounds.  相似文献   

10.
Seven Gram-negative, rod-shaped pectinolytic bacteria strains designated as IFB5227, IFB5228, IFB5229, IFB5230, IFB5231, IFB5232, IFB5636, isolated from potato tubers cultivated in Peru at high altitude (2400–3800 m) were subjected to polyphasic analyses that revealed their distinctiveness from the other Pectobacterium species. Phylogenetic analyses based on five housekeeping genes (gyrA, recA, recN, rpoA and rpoS) clearly showed strains separateness, simultaneously indicating Pectobacterium atrosepticum, Pectobacterium wasabiae, Pectobacterium parmentieri and Pectobacterium betavasculorum as the closest relatives. In silico DNA–DNA hybridization of strain IFB5232T with other Pectobacterium type strains revealed significant drop in DDH value below 70%, which is a prerequisite to distinguish Pectobacterium peruviense. The ANI values supported the proposition of delineation of the P. peruviense. Genetic REP-PCR fingerprint and detailed MALDI-TOF MS proteomic profile sealed the individuality of the studied strains. However, phenotypic assays do not indicate immense differences.Provided results of analyses performed for seven Peruvian strains are the basis for novel species distinction and reclassification of the strains IFB5227-5232 and IFB5636, previously classified as Pectobacterium carotovorum subsp. carotovorum. Here, we propose to establish the IFB5232 isolate as a type strain (=PCM2893T = LMG30269T = SCRI179T) with the name Pectobacterium peruviense sp. nov.  相似文献   

11.
Soft rot is the most important disease on calla lily in Poland. The isolation of the presumptive pathogen from symptomatic tubers on nutrient agar yielded bacteria with different colony morphology. Of 41 isolates collected, 10 showed pectolytic activity on crystal violet pectate medium and caused soft rot on potato slices. All pectolytic bacteria appeared to be Gram‐negative rods producing typical soft rot on inoculated leaf petioles of calla lily. Bacteria with colonies which morphologically resembled those used for inoculation were re‐isolated from diseased petioles. Their identification was based on phenotypic characters and sequence of the gene fragment coding 16S rRNA. It was found that, in addition to Pectobacterium carotovorum subsp. carotovorum, soft rot of calla lily can be caused by Pectobacterium carotovorum subsp. atrosepticum, Pseudomonas marginalis, Pseudomonas veronii and Chryseobacterium indologenes. The latter two are described for the first time as plant pathogens. The pectolytic activity of all identified bacteria, except that of P. carotovorum subsp. atrosepticum, was lower than that of P. carotovorum subsp. carotovorum, but strains of P. veronii showed a higher activity than P. marginalisand C. indologenes species.  相似文献   

12.
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.  相似文献   

13.
Potatoes are an important agroeconomic crop worldwide and maceration diseases caused by pectolytic bacterial pathogens result in significant pre‐ and post‐harvest losses. Pectobacterium carotovorum shares a common host range with other Pectobacterium spp. and other members of the Enterobacteriaceae, such as Dickeya spp. As these pathogens cannot be clearly differentiated on the basis of the symptoms they cause, improved methods of identification are critical for the determination of sources of contamination. Current standardized methods for the differentiation of pectolytic species are time consuming and require trained personnel, as they rely on traditional bacteriological practices that do not always produce conclusive results. In this growing world market, there is a need for rapid diagnostic tests that can differentiate between pectolytic pathogens, as well as separate them from non‐pectolytic enteric bacteria associated with soft rots of potato. An assay has been designed previously to detect the temperate pathogen Pectobacterium atrosepticum, but there is currently no recognized rapid assay for the detection of the tropical/subtropical counterpart, Pectobacterium carotovorum. This report describes the development of a loop‐mediated isothermal amplification (LAMP) assay that detects P. carotovorum with high specificity. The assay was evaluated using all known species of Pectobacterium and only showed positive reactions for P. carotovorum. This assay was also tested against 15 non‐target genera of plant‐associated bacteria and did not produce any false positives. The LAMP assay described here can be used as a rapid test for the differentiation of P. carotovorum from other pectolytic pathogens, and its gene target can be the basis for the development of other molecular‐based detection assays.  相似文献   

14.
15.
Soft rot disease can be found worldwide on fleshy storage tissues of fruits, vegetables and ornamentals. The soft rot Pectobacterium carotovorum subsp. carotovorum (Pcc) is an important pathogen of Kalanchoe spp. and other ornamental plants. The disease occurs on crops in the field, greenhouses and during transit, resulting great economic damages. The economic importance of crop loss by soft rot bacteria varies by severity of the disease and value of the crop. A destructive disease on Kalanchoe gastonis-bonnierii was observed in commercial ornamental plant greenhouses in Cameron highland and Melaka, Malaysia in 2011. Samples suspected to be infested with Pectobacterium spp. were brought to the laboratory. In pathogenicity test, a suspension of 106?CFU/ml of strains was able to cause soft rot on leaves and stems. A 434?bp banding pattern on 1% agarose gel was produced in polymerase chain reaction (PCR) amplification of pectate lyase encoding gene (Pel gene). PCR amplification of the intergenic transcribed spacer (ITS) (16S–23S rRNA) ITS region with G1 and L1 primers produced two main bands at about 540 and 570?bp. The ITS-PCR products were digested with RsaI restriction enzyme. For discrimination of the P. carotovorum subsp. carotovorum (Pcc) from P. carotovorum subsp. odoriferum (Pco), all isolates subjected to α-methyl glucoside test. All isolates were identified as Pcc based on phenotypic and molecular methods. This is the first report of soft rot disease caused by P. carotovorum subsp. carotovorum on K. gastonis-bonnierii, in Malaysia.  相似文献   

16.
17.
We report the complete and annotated genome sequence of the plant-pathogenic enterobacterium Pectobacterium sp. strain SCC3193, a model strain isolated from potato in Finland. The Pectobacterium sp. SCC3193 genome consists of a 516,411-bp chromosome, with no plasmids.  相似文献   

18.
The study of plant parasitic nematodes such as Meloidogyne spp. and their interactions with phytopathogenic bacteria remains underexplored. One of the challenges towards establishing such interactions is the dependence on symptom development as a measure of interaction. In this study, mCherry was employed as a reporter protein to investigate the interaction between the soft rot Enterobacteriaceae (SRE) Pectobacterium carotovorum subsp. brasiliensis (Pcb) and root‐knot nematode (Mincognita). Pectobacterium carotovorum subsp. brasiliensis was transformed with pMP7604 generating Pcb_mCherry strain. This strain was shown to attach to the surface coat of M.incognita J2 at the optimum temperature of 28°C. This suggests that RKN juveniles may play a role in disseminating Pcb in soils that are heavily infested with Pcb. The presence of RKN juveniles was shown to play a role in introducing Pcb_mCherry into potato tubers potentially acting as a source of latent tuber infections.

Significance and Impact of the Study

This study uses fluorescent reporter protein tagging as a tool to demonstrate the interaction between root‐knot nematode (Meloidogyne incognita) and the soft rot Enterobacteriacea (Pectobacterium carotovorum subsp. brasiliensis). Introduction of Pectobacterium through wounds generated by second‐stage juveniles (J2) into potato tubers was demonstrated. These results suggest that RKN juveniles can facilitate latent infection of potato tubers in the soil. These findings have important implications in the management of RKN and SRE in seed potato production. Furthermore, this tool can be used to study other nematode–bacteria interactions that have not been previously studied.  相似文献   

19.
Four Gram-negative, rod-shaped pectinolytic bacterial strains designated as 2M, 9M, DPMP599 and DPMP600 were subjected to polyphasic analyses that revealed their distinctiveness from the other Pectobacterium species. Strains 2M and 9M were isolated from Calla lily bulbs cultivated in Central Poland. DPMP599 and DPMP600 strains were isolated from Calla lily leaves from plants grown in Serbia. Phylogenetic analyses based on nine housekeeping genes (gapA, gyrA, icdA, pgi, proA, recA, recN, rpoA, and rpoS), as well as phylogeny based on the 381 most conserved universal proteins confirmed that Pectobacterium zantedeschiae strains were distantly related to the other Pectobacterium, and indicated Pectobacterium atrosepticum, Pectobacterium betavasculorum, Pectobacterium parmentieri and Pectobacterium wasabiae as the closest relatives. Moreover, the analysis revealed that Pectobacterium zantedeschiae strains are not akin to Pectobacterium aroidearum strains, which were likewise isolated from Calla lily.The genome sequencing of the strains 2M, 9M and DPMP600 and their comparison with whole genome sequences of other Pectobacterium type strains confirmed their distinctiveness and separate species status within the genus based on parameters of in silico DNA–DNA hybridization and average nucleotide identity (ANI) values. The MALDI-TOF MS proteomic profile supported the proposition of delineation of the P. zantedeschiae and additionally confirmed the individuality of the studied strains. Based on of all of these data, it is proposed that the strains 2M, 9M, DPMP599, and DPMP600 isolated from Calla lily, previously assigned as P. atrosepticum should be reclassified as Pectobacterium zantedeschiae sp. nov. with the strain 9MT (PCM2893 = DSM105717 = IFB9009) as the type strain.  相似文献   

20.
Pectobacterium carotovorum is a plant-pathogenic enterobacterium responsible for soft rot in various commercially important plants. Here we report the complete genome sequence and automatic annotation of strain PCC21.  相似文献   

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