Characterization of Acidovorax citrulli strains isolated from solanaceous plants

Acidovorax citrulli is the causal agent of bacterial fruit blotch disease of cucurbits. Strains of this pathogen are distributed into two major groups: Group I strains have been mainly isolated from melon and other non-watermelon cucurbits, while Group II strains have been mainly recovered from watermelon. Here we report the characterization of strains T1 and EP isolated from diseased tomato and eggplant plants, respectively, and further confirmed to belong to A. citrulli species. Based on PCR, PFGE, and rep-PCR, these strains showed high similarity to the Group II strain 7a1. Sequencing and comparative analyses revealed that the genomes of T1 and EP aligned with that of the Group II model strain AAC00-1, over 97.88% and 99.22%, respectively. The virulence of T1, EP, and 7a1 determined on tomato, eggplant, and watermelon was similar and significantly higher than that of Group I strain M6. In contrast, M6 was more virulent on melon. Expression levels of seven virulence genes measured 24 hr after inoculation of tomato, eggplant, watermelon, and melon showed that the expression pattern was generally similar in strains 7a1, T1, and EP, whereas for M6 the expression was high only on melon. Overall, our results indicate that the solanaceous strains belong to Group II. To the best of our knowledge, this is the first study that reports characterization of A. citrulli strains isolated from solanaceous species. The fact that A. citrulli is able to naturally colonize and cause disease in non-cucurbit crops poses additional challenges for management of this important pathogen.     

Genetic diversity and population structure of Acidovorax avenae subsp. avenae isolated from sugarcane in Argentina

A significant increase in the occurrence of red stripe (caused by Acidovorax avenae subsp. avenae) has been observed in the last decade in Argentina. Considering that no extensive sampling of the main sugarcane-producing area in the country has been conducted to characterize the diversity and population structure of A. avenae subsp. avenae, molecular markers were employed to analyse 112 isolates from Tucumán. By using repetitive element polymorphism-based polymerase chain reaction (rep-PCR) almost all isolates were differentiated and grouped into 10 clusters, revealing a high genetic diversity. Using the amplified fragment length polymorphism (AFLP) technique, five pairs of isolates were discriminated that could not be distinguished with rep-PCR. Cluster analysis showed no clear association between isolate clustering, sugarcane host genotype, crop age, type of tissue sampled, fertilization, or year of sampling. Linkage equilibrium analysis by using rep-PCR data indicated that the population has some degree of clonality. Three housekeeping genes were also sequenced: ugpB and pilT sequences were highly similar to A. avenae subsp. avenae sequences from other Argentinian isolates, whereas the lepA sequence did not reveal significant similarity. An additional four housekeeping genes could not be amplified, suggesting the existence of differences in those regions. Subsequently, virulence of 14 A. avenae subsp. avenae isolates was evaluated under controlled conditions. Results showed a differential level of aggressiveness among the isolates on a resistant sugarcane variety. This study confirmed that rep-PCR is an adequate tool for genetic analysis and population structure characterization in bacteria, and revealed both high genetic diversity and clonal population structure of A. avenae subsp. avenae in Tucumán, Argentina.     

High-throughput screening reveals small molecule modulators inhibitory to Acidovorax citrulli

Acidovorax citrulli is a seed-transmitted gram-negative bacterium that can cause substantial economic yield loss in watermelon and melon production worldwide. Four small-molecule libraries containing 4,952 compounds were selected for high-throughput screening against A. citrulli wild-type strain Xu3-14 by evaluation of growth inhibition. One hundred and twenty-seven molecules (2.5% hit rate) were identified as bactericidal or bacteriostatic against A. citrulli at 100 μM. Secondary screens indicated that 27 candidate compounds were more effective against A. citrulli Group II strains than Group I strains (classified using repetitive element PCR). Several compounds were inhibitory to other pathogenic bacteria, including Clavibacter michiganensis subsp. michiganensis and Xanthomonas campestris pv. campestris, but did not affect the growth of plant beneficial bacteria Pseudomonas fluorescens and Bacillus subtilis. More than half of the compounds did not inhibit germination of Arabidopsis or watermelon seeds. The effect of small molecules on A. citrulli seed-to-seedling transmission was evaluated by applying each compound to inoculated watermelon seeds and assessing seedling infection. Nine compounds were chosen for further investigation based on their reduction of percentage seedling infection and compiling scores on their specificity, sensitivity, and phytotoxicity obtained in the secondary screens. The five best compounds were selected (thiamphenicol, nadifloxacin, pipemidic acid, ciclopirox, and zinc pyrithione) for greenhouse tests and were found to effectively reduce the seed-to-seedling transmission of A. citrulli in both artificially and naturally infested seeds. These top five compounds provide a basis for future development of an A. citrulli-specific bactericide.     

Pathways of bacterial invasion and watermelon seed infection by Acidovorax citrulli

This study explored the pathways of ingress of Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbits, into watermelon seeds. Up until 7 days post‐inoculation (DPI), a significantly higher percentage of watermelon seeds was infected with A. citrulli when the bacteria were applied (c. 1 × 10⁶ colony‐forming units) to stigmas versus ovary pericarps of female flowers. Immunofluorescence microscopy showed that, with stigma inoculation, A. citrulli colonized style and ovary tissues by 1 DPI, and the bacteria co‐localized with pollen germ tubes in these tissues. With ovary pericarp inoculation, A. citrulli cells penetrated the epicarp and mesocarp tissues by 1 DPI but did not reach endocarp until 4 DPI. Finally, manual pollination followed by stigma inoculation led to >53% A. citrulli‐infected seed lots, while A. citrulli was not detected in seeds/ovules generated by stigma inoculation without pollination (chemically induced parthenocarpy). These results show that stigma inoculation results in faster colonization of watermelon ovules by A. citrulli than pericarp inoculation, even though there is no difference in the levels of infection in mature seeds. The data also indicate that pollen germ tubes play an important role in A. citrulli ingress into watermelon seeds via stigmas.     

Tn5 transposon mutagenesis in Acidovorax citrulli for identification of genes required for pathogenicity on cucumber

An Acidovorax citrulli–cucumber pathosystem was established through which A. citrulli mutants with altered pathogenicity, generated by transposon mutagenesis, were identified on cucumber cotyledons. The A. citrulli group I strain FC440 was shown to grow faster in cucumber leaf tissues than a group II strain and was used for Tn5 transposon mutagenesis. A total of 2100 Tn5 insertional mutants were generated, and analysis of the mutant library showed that the transposon insertions were single, independent and stable. A conserved non‐flagellar type III secretion system (NF‐T3SS) ATPase gene hrcN was identified and confirmed to be essential for pathogenicity and functionality of NF‐T3SS in A. citrulli. Comparative sequence analysis of the HrcN protein and its homologues in other representative bacterial plant pathogens revealed that the NF‐T3SS of A. citrulli is close to that of Ralstonia solanacearum and Xanthomonas campestris, but distant from that of Pseudomonas syringae and Erwinia amylovora. The generated Tn5 insertional mutant collection is valuable for identification of genes required for A. citrulli pathogenesis, and the established A. citrulli–cucumber pathosystem will facilitate an improved understanding of A. citrulli biology and pathology.     

Bacterial fruit blotch of melon: screens for disease tolerance and role of seed transmission in pathogenicity

Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovorax avenae subsp. citrulli, is a serious threat to the watermelon and melon industries. To date, there are no commercial cultivars of cucurbit crops resistant to the disease. Here we assessed the level of tolerance to bacterial fruit blotch of various commercial cultivars as well as breeding and wild lines of melon, using seed-transmission assays and seedling-inoculation experiments. Selected cultivars were also tested in a greenhouse experiment with mature plants. All tested cultivars/lines were found to be susceptible to the pathogen, and most of them showed different responses (relative tolerance vs. susceptibility) in the different assays; however, some consistent trends were found: cv. ADIR339 was relatively tolerant in all tested assays, and cv. 6407 and wild lines BLB-B and EAD-B were relatively tolerant in seed-transmission assays. We also provide evidence supporting a strong correlation between the level of susceptibility of a cultivar/line and the ability of the pathogen to adhere to or penetrate the seed. To the best of our knowledge, this is the first attempt to assess melon cultivars/lines for bacterial fruit blotch response.     

Screening and identification of Cucurbita germplasm resistant to Xanthomonas cucurbitae,incitant of cucurbit bacterial spot

Bacterial spot of cucurbits, caused by Xanthomonas cucurbitae, is an emerging disease of cucurbits. This study was conducted to identify Cucurbita species that are resistant to X. cucurbitae. We developed a reliable method for inoculating cucurbit plants by spraying plants with X. cucurbitae suspensions containing carborundum. Symptoms of the disease developed within 7 days after inoculation. Subsequently, we evaluated the virulence of six isolates of X. cucurbitae in the greenhouse and observed that X. cucurbitae isolated from cucurbit fields in Illinois, Michigan, Kansas, Ohio, and Wisconsin were more virulent than the reference ATCC 23378 strain. Then, we screened 81 commercial cultivars of gourds, pumpkins, and squashes, as well as 300 Cucurbita spp. accessions, for their resistance to X. cucurbitae under greenhouse and field conditions. In the greenhouse study, all commercial cultivars and some of the accessions developed typical symptoms of bacterial spot disease, while some of the accessions developed fewer lesions. In the field studies, infection of leaves and fruits was caused by both natural inoculum and spray-inoculation of plants with a suspension of the X. cucurbitae isolate from Illinois. Among 300 accessions tested, 9 and 21 accessions were classified as resistant and less resistant, respectively. Resistant and less resistant accessions belong to the species Cucurbita maxima, C. maxima subsp. maxima, C. maxima subsp. andreana, and C. okeechobeensis subsp. martinezii. This is the first report of potential resistance to bacterial spot of cucurbits.     

Bacterial pathogens of rice in the Kingdom of Cambodia and description of a new pathogen causing a serious sheath rot disease

A study of rice diseases in Cambodia from 2005 to 2007 showed widespread occurrence of diseases caused by Acidovorax avenae subsp. avenae, Burkholderia gladioli, B. cepacia and Pantoea ananatis. This is the first report of these pathogens in Cambodia. Additionally, a pseudomonad causing a widespread disease similar to sheath brown rot (caused by Pseudomonas fuscovaginae) was isolated. The studied strains were pathogenic to rice cvs Sen Pidau and IR 66, producing similar, though slightly less severe, symptoms to those observed in the field. Based on comparative 16S rDNA gene sequence analysis, combined with cell wall fatty acid analysis and metabolic profiles, the isolated strains were allocated to the genus Pseudomonas. The novel species were differentiated from Pseudomonas fuscovaginae and P. putida by their inability to metabolize d ‐fructose, d ‐galactose, d ‐galactonic acid lactone, d ‐galacturonic acid, d ‐glucosaminic acid, d ‐glucuronic acid, p‐hydroxy phenylacetic acid, d ‐saccharic acid and urocanic acid. The major fatty acids were C_16:0, summed feature 3 (C_16:1ω7c and C_16:1ω6c) and summed feature 8 (C_18:1ω7c), representing 80% of the total. Partial 16S rRNA gene sequences (1460 bp) were identical, except for two nucleotide changes amongst the six strains. Alignment of the causal strains within type‐culture databases revealed similarities of 99·7% with Pseudomonas parafulva AJ 2129^T, 99·2% with P. fulva IAM 1592^T, 98·9% with P. plecoglossicidia FPC 951^T, and 98·1% with P. fuscovaginae MAFF 301177^T. On the basis of data from this polyphasic study, it is proposed that the unknown strains isolated from rice represent a novel species of the genus Pseudomonas.     

A method to evaluate percentage of cucurbit seeds infested with <Emphasis Type="Italic">Acidovorax avenae</Emphasis> subsp. <Emphasis Type="Italic">citrulli</Emphasis>, pathogen of bacterial fruit blotch

We developed a method to detect Acidovorax avenae subsp. citrulli from cucurbit seeds that is more sensitive than conventional growing-out methods. Seeds were cultivated in test tubes for 2 weeks, and the bacteria grew semi-selectively on the seeds and seedlings. The plants were then vigorously shaken in water to suspend bacterial cells, and the suspension was spread on a selective medium for identifying the bacterium by colony appearance. The method is suitable for research purposes to evaluate the percentage of seeds infested with the bacteria, but not to detect bacterial contamination in commercial lots with thousands of seeds.     

瓜类细菌性果斑病菌hrcN基因的生物学功能分析

以分离自西瓜上的Aac5菌株为例,通过同源重组的方法,构建了hrc N基因插入缺失突变体,通过PCR方法和Southern blot验证突变菌株,对突变体进行致病性、致敏性、生长曲线和运动性测定。为明确hrc N基因与其他基因的关系,通过实时荧光定量PCR法定量检测了hrp A、hrc V、hrc U、Lux I、LuxR 5个基因的表达量。结果显示:与野生型相比,突变体致病力和致敏性明显减弱,致病时间延迟,群体感应信号减弱,生长能力明显下降,运动性减弱,互补菌株只能恢复部分功能;5个基因在突变体中的表达量均上调,hrc N基因与这5个基因之间均为负调控关系。说明hrc N基因在果斑病菌致病能力上发挥重要作用。     

Management of melon bacterial blotch by plant beneficial bacteria

Plant beneficial bacteria (PBB) have shown potential for disease control and are particularly important in the management of bacterial diseases, which are poorly controlled by conventional methods. In melon, bacterial fruit blotch caused by Acidovorax citrulli is a seedborne disease that is particularly destructive under certain conditions. PBB strains were screened for their ability to protect seeds and leaves from bacterial fruit blotch, and their antibiosis activity and plant colonization were studied. When Bacillus sp. RAB9 was applied to infected seeds, it reduced the area under the disease progress curve (AUDPC) by 47% and increased the incubation period (the time between inoculation and the first visible symptoms) by 35%. Three of the selected strains (JM339, MEN2 and PEP91) displayed antibiosis against A. citrulli. The RAB9^Rif-Nal mutant colonized seeds epiphytically and roots and stems endophytically. Paenibacillus lentimorbus MEN2 sprayed on melon seedlings protected leaves, and when challenged with A. citrulli, it reduced the AUDPC (by 88%), disease index (by 81%) and incidence (by 77%). Given that the production of both melon seedlings and commercially grown greenhouse melons is increasing, biocontrol strategies may well be integrated into bacterial blotch management programs.     

Bacterial brown stripe of rice in soil-less culture system caused by <Emphasis Type="Italic">Acidovorax avenae</Emphasis> subsp. <Emphasis Type="Italic">avenae</Emphasis> in China

In 2010, the outbreak of a disease with symptoms similar to bacterial brown stripe was observed in rice seedlings planted in a perlite culture system in China. The causal bacterium was identified as Acidovorax avenae subsp. avenae based on its biochemical and physiological characteristics, cellular fatty acid composition, Biolog data, specific PCR detection and 16S rRNA gene sequence analysis. The bacterial isolates caused similar symptoms after inoculation of rice seedlings. This report is the first of bacterial brown stripe of rice in a soil-less culture system caused by A. avenae subsp. avenae in China.     

Decreased potassium fertilization is associated with increased pathogen growth and disease severity caused by <Emphasis Type="Italic">Acidovorax citrulli</Emphasis> in melon foliage

The gram-negative bacterium Acidovorax citrulli causes bacterial fruit blotch (BFB) disease of cucurbits, which represents a serious threat to melon and watermelon production worldwide. To date, there are no efficient means to manage the disease, and reliable resistance sources for cucurbit germplasm are lacking. Mineral nutrition markedly affects plant diseases. Recently, we reported that disease severity on melon foliage and A. citrulli growth in the leaf tissue were significantly influenced by the form of nitrogen supply. In the present study, we investigated the influence of potassium nutrition on BFB severity and A. citrulli establishment in the foliage of melon plants. Fertilization with relatively low concentrations of potassium increased these variables compared with higher potassium concentrations. Since establishment of A. citrulli during the growing season is assumed to increase the incidence of fruit infection, the fact that mineral nutrition influences BFB incidence in the plant foliage is of particular importance.     

瓠瓜果斑病症状及其病原菌的鉴定

为有效防控瓠瓜果斑病,自浙江省象山县田间采集具有典型果斑病症状的瓠瓜样本,对其进行病原菌分离、形态观察、致病性测定及分子生物学鉴定,并利用特异性引物PL1/PL2 PCR扩增和基质辅助激光解吸电离飞行时间质谱（matrix assisted laser desorption ionization time-of-flight massspectrometry,MALDI-TOF-MS）技术对其病原菌进行亚群鉴定。结果表明：瓠瓜果斑病田间典型症状是发病叶片和果实上病斑由水渍状小斑点逐渐发展为伴有黄色晕圈的褐色不规则病斑,果实腐烂、有臭味。通过菌体形态和培养特性观察、PCR鉴定、16S rDNA序列分析和7个看家基因的系统发育分析将其病原菌鉴定为西瓜噬酸菌Acidovorax citrulli。从瓠瓜上分离的菌株均属于西瓜噬酸菌亚群I,从西瓜上分离的菌株均属于西瓜噬酸菌亚群II。     

Development of a TaqMan probe-based insulated isothermal PCR (TiiPCR) for the detection of <Emphasis Type="Italic">Acidovorax citrulli</Emphasis>, the bacterial pathogen of watermelon fruit blotch

Watermelon (Citrullus lanatus) is an important crop of the Cucurbitaceae family in fruit production worldwide. During its production, bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Acidovorax avenae subsp. citrulli) is an important limiting factor on the volume and value of crops. This pathogen is known as a seed-borne pathogen, and the infested seeds can be a primary source of inoculum. Hence, a rapid and sensitive method for detecting A. citrulli on seeds would be an important tool in the management of BFB. In this study, we sought to develop a method to detect A. citrulli bacterial cells based on a TaqMan probe-based insulated isothermal PCR (TiiPCR) assay. Firstly, the specific primers and probe were designed based on a specific DNA fragment from the genome of A. citrulli. Then, PCR amplification was performed with the plasmid DNA to adjust the components of the PCR reagents, such as the concentrations of primers, magnesium chloride, and Taq DNA polymerase. Results revealed that 10 copies of plasmid DNA were detectable within the modified reagents by TiiPCR. Moreover, 10 bacterial cells in each reaction tube were detectable at a 100 % detection rate in this condition with a fluorescent signal intensification over 1.8. Based on these results, we concluded that a specific, rapid, and sensitive method based on TiiPCR had been successfully developed to detect bacterial cells of A. citrulli.     

Genetic diversity analysis of Acidovorax citrulli in China

Acidovorax citrulli has become quite common in China. A collection of 118 strains of A. citrulli was made from throughout China and other countries to determine their genetic relatedness. Strains were identified as A. citrulli by pathogenicity, phenotypic characterization, and PCR. Genetic diversity was determined using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). PFGE electrophoresis resulted in nine genotypes, which could be typed into two groups based on host; group 1 included strains mainly from melon and group 2 included strains mainly from watermelon. MLST analysis resulted in 73 sequence types (ST) among the 118 A. citrulli strains. All A. citrulli strains were typed into three groups: group 1 with 82 strains (including type strain Fc247), group 2 with 35 strains and group 3 a singleton (Fc380). Similar to PFGE results, group 1 included strains mainly from melon and group 2 included strains mainly from watermelon. The difference was the 10 watermelon strains (pslbtw1-3, 5–11) from Beijing grouped with melon strains of group 1 based on MLST, suggesting that these 10 watermelon strains had a close relationship with melon. Our study indicated that there was genetic differentiation among A. citrulli strains between watermelon and melon. Also, our study was the first attempt to compare PFGE and MLST on analyzing genetic diversity of A. citrulli strains and proved MLST could better distinguish A. citrulli strains.     

Characterization of <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> causing soft-rot disease on <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China

Pinellia ternata is a traditional Chinese herb which has been used in China for over 1,000 years. A soft-rot disease characterized by water-soaked lesions and soft-rot symptoms with a stinking odour was commonly observed in cultivated fields of this plant, and Pectobacterium-like bacteria were consistently isolated from the infected tissues. Two typical strains (SXR1 and ZJR1), isolated from Shanxi and Zhejiang, respectively, were identified. Pathogenicity tests revealed that these strains were virulent to P. ternata and induced the same symptoms as observed in the field. Characterization involving fatty acid profile, metabolic and physiological properties, 16S rDNA sequence and PCR-RFLP identified both isolates as P. carotovorum subsp. carotovorum (Pcc). The 16S rDNA of both isolates shared 97–99% sequence similarity with that of Pcc strains. The phylogenetic trees showed that both isolates were clustered in the group of Pcc and P. carotovorum subsp. odorifera and both PCR-RFLP profiles were consistent with the pattern E produced by the minority of Pcc strains. Thus, isolates SXR1 and ZJR1 were characterized as Pcc in spite of some differences. This is the first report that Pcc has been proven as a causal agent of soft-rot disease on P. ternata.     

Genetic diversity of <Emphasis Type="Italic">Rhizobium vitis</Emphasis> strains in Japan based on multilocus sequence analysis of <Emphasis Type="Italic">pyrG</Emphasis>, <Emphasis Type="Italic">recA</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Forty-one strains of Rhizobium vitis, either tumorigenic (Ti) or nonpathogenic, were characterized using multilocus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD. The strains separated into seven clades. Rhizobium vitis (Ti) strains isolated from Japan were divided into five genetic groups (A to E), and nonpathogenic R. vitis strains were divided into two genetic groups (F and G). This result suggests that there are new genetic groups of R. vitis in Japan. Among these groups, members of A and B groups are widely distributed throughout Japan.     

Characterization of Pectobacterium carotovorum subsp. odoriferum causing soft rot of stored vegetables

Pectobacterium carotovorum subsp. odoriferum has been generally considered to have a narrow host range and has been isolated most often from chicory. Research was conducted to identify 91 Pectobacterium spp. strains isolated from different vegetables in Europe, North and South America, Asia, and Africa, and to compare their ability to cause disease in chicory and potato. Among the 91 strains, 22 strains from Europe were identified as P. c. subsp. odoriferum. Based on phylogenetic analysis of 16S rDNA, recA, and rpoS gene sequences, strains isolated from stored vegetables clustered together with the type strain of P. c. subsp. odoriferum and clustered separately from the P. c. subsp. carotovorum isolates. Eleven strains previously identified as P. c. subsp. carotovorum were reclassified as P. c. subsp. odoriferum. All P. c. subsp. odoriferum isolates were able to cause soft rot symptoms on chicory and potato. Moreover, the symptoms on potatoes were more severe at temperatures from 15 to 37 °C with P. c. subsp. odoriferum isolates than with P. atrosepticum or P. c. subsp. carotovorum isolates. Tissue maceration by P. c. subsp. odoriferum isolates was highest at 28 °C, and at that temperature tissue maceration was two-times greater for P. c. subsp. odoriferum isolates than for P. c. subsp. carotovorum isolates. Symptoms on inoculated chicory leaves were more severe with P. c. subsp. odoriferum (regardless of origin) than with other subspecies or species. To our knowledge, this is the first report that P. c. subsp. odoriferum occurs on a wide range of vegetables and has the ability to cause soft rot during potato storage.     

Genes responsible for coronatine synthesis in <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> present in the genome of soft rot bacteria

Primers for the PCR amplification of homologous genes encoding polyketide coronafacic acid and coronafacic ligase in the cells of Pectobacterium atrosepticum SCRI1043 (BX950851) were developed to study the presence of these genes in the genome of Pectobacterium sp. and Dickeya sp. Coronafacic ligase catalyses the formation of coronatine from polyketide coronafacic acid and coronamic acid. Coronatine is a toxin produced by Pseudomonas syringae and is one of the major virulence factors in this bacterium. This study using several strains of P. atrosepticum, P. carotovorum subsp. carotovorum and Dickeya sp. isolated in different countries, indicated that all strains of P. atrosepticum possess genes coding coronafacic acid (cfa gene cluster) and coronafacic ligase (cfl). However, these genes were present only in the genome of five out of 50 tested P. carotovorum subsp. carotovorum strains and two out of 34 strains of Dickeya sp. tested. The PCR products homologous to the sequence of cfa7 and cfl gene fragments were sequenced in order to check the level of homology between genes of P. atrosepticum, P. carotovorum subsp. carotovorum and Dickeya sp. The sequences of the gene fragments amplified from all P. atrosepticum strains were almost identical (100% and 99.97%, respectively). The homology of the sequences obtained for P. atrosepticum and sequences of five P. carotovorum subsp. carotovorum and two Dickeya sp. was lower, between 89.69% to 95.00% for the cfl gene fragment, and about 94% for the cfa7 gene fragment.