First report on the whole genome sequence of Pseudomonas cichorii strain JBC1 and comparison with other Pseudomonas species

Pseudomonas cichorii, a plant pathogen that infects a wide range of host plants worldwide, causes several diseases in economically important vegetable crops. Availability of the genome sequences of pathogens can greatly enhance research necessary for the advancement of disease management programmes. Despite the significance of P. cichorii, its whole genome sequence has not been reported previously. The genome sequence of P. cichorii JBC1, described for the first time in this study, is 5 986 012 bp with an average GC content of 58·1% and has 5174 coding sequences (CDS). The genes related to virulence, transport mechanisms, phytotoxic compounds, and secondary metabolite products were analysed and the genome was compared to eight other Pseudomonas species to understand the diversity at species level. Despite the high similarity (up to 80·85%), significant diversity was found among the different Pseudomonas species at the genome level. A comparison of JBC1 pathogenicity island (PAI) regions indicated that the P. viridiflava UASWS0038 PAI has more similarity than the P. syringae PAI region, and the analysis revealed significant divergence at PAI regions among the Pseudomonas species, providing an insight into the differences in host specificity and degree of virulence. In addition, JBC1 encodes antibiotic resistance and tolerance to heavy metals, and two different prophage segments were inserted at three different regions. The genome sequence of JBC1, which was deposited into the NCBI GenBank (accession no. CP007039 ), will be a reference sequence for other P. cichorii strains and a useful resource for further research.     

Development of a real‐time PCR assay for Pseudomonas cichorii,the causal agent of midrib rot in greenhouse‐grown lettuce,and its detection in irrigating water

Midrib rot is an emerging disease in greenhouse production of lettuce caused by Pseudomonas cichorii, and probably introduced through contaminated irrigation water. Concentrations of 100 CFU mL^?1 are enough to induce the typical midrib rot symptoms. A sensitive real‐time PCR assay was developed, based on a 90‐bp amplicon from the pathogenicity gene cluster hrcRST and a Taqman Minor Groove Binding probe. Specificity of the assay was tested with 39 P. cichorii strains, including the type strain, and 89 strains from 83 other Pseudomonas species. The relationship between detection signals and P. cichorii DNA concentrations was linear over 6‐logs. Detection threshold with excellent reproducibility was 500 fg of DNA or about 70 genome copies. Sample preparation and DNA isolation were optimized to allow detection in 1 L water samples. The assay was first evaluated with greenhouse irrigation water spiked with serial dilutions of P. cichorii. The calculated cell numbers obtained with real‐time PCR were 10‐fold lower than plate counts of actual spiked cells. However, the assay consistently detected 100 CFU per reaction, corresponding to the detection of 1 CFU mL^?1 of irrigation water, which is well below the concentration needed for midrib rot infection. Finally, the assay proved to be valuable for detecting infective P. cichorii concentrations in the irrigation water of a commercial lettuce production greenhouse.     

Motility-mediated regulation of virulence in Pseudomonas syringae

We have investigated Pseudomonas syringae pv. tabaci–plant interactions using a large variety of virulence-related mutants. A flagellin-defective mutant, ΔfliC, lost flagellar motility and the ability to produce N-acyl homoserine lactones; it had reduced ability to cause disease symptoms, but the expression of genes encoding a multidrug efflux pump transporter, mexEFoprN, was activated. A type IV pili (T4P)-defective mutant, ΔpilA, lost swarming motility, had reduced expression of hrp-related genes and virulence toward the host tobacco plant, but expression of the genes encoding another multidrug efflux pump transporter, mexABoprM, was activated. These results suggest that the genes regulating flagella- and T4P-mediated motilities also regulate expression of other virulence-related genes.     

Comprehensive prediction of plant cytoplasmic and apoplastic effectors underlying Erwinia psidii pathogenicity

Erwinia psidii (Eps) is the causal agent of emerging diseases of eucalypt and guava; however, the mechanisms underlying its pathogenicity are not fully understood. Here, we predicted factors involved in the ability of Eps to cause disease on its host plants. For that, the genomes of four Eps strains exhibiting different virulence on eucalypt were sequenced, and hrp/hrc genes coding for the type III secretion system (T3SS), effectors injected into the plant cell cytoplasm through the T3SS (T3SEs) and their plant subcellular localizations, as well as proteins deployed to the host apoplast, were predicted. It was found that Eps possesses a complete hrp/hrc gene cluster based on comparison with Erwinia amylovora. A total of 18 T3SEs were predicted, 11 of which were shared among all strains, none were exclusive to any strain and seven were absent in at least one strain. No sequence variation among strains was found for five T3SE candidates whereas extensive variation was found for six, suggesting the latter may be determinants of virulence differences. The T3SE candidates are predicted to target the plant cell nucleus, cytoplasm, mitochondrion, chloroplast and peroxisome. The predicted apoplastic effector repertoire common to all four strains was over-represented in proteins of unknown functions or predicted to possess enzymatic activities, among which the most abundant were oxidoreductases and peptidases. Proteins with lytic transglycosylase activity were predicted in strain-specific apoplastic effector repertoires. These results provide an important framework for future research aimed at uncovering the factors underlying Eps pathogenicity.     

Genomic variability and plasticity of Pseudomonas causing coffee leaf spots in Minas Gerais state,Brazil

Foliar spots caused by Pseudomonas coronafaciens pv. garcae (Pcg), Pseudomonas amygdali pv. tabaci (Pat) and Pseudomonas cichorii (Pch) are major bacterial diseases that can reduce coffee production. However, little is known about the genetic diversity and molecular mechanisms underlying the pathogenicity to coffee plants of these bacteria. In this study, genome sequences of Pcg, Pat and Pch strains isolated from coffee plants in Minas Gerais state, Brazil, were used to assess their variability and plasticity, and compare their type III secretion system (T3SS) and apoplastic effector repertoires as well as tabtoxin biosynthetic/detoxification genes. Genomic diversity was found for all three phytopathogens, among which Pch possesses the highest number of exclusive proteins. The Pcg genome is the most stable whereas that of Pch is the most plastic, which is related to their host ranges. When compared with those of Pseudomonas syringae pv. tomato DC3000, hrp/hrc gene sequences are more conserved in Pcg and Pat than in Pch, which also possesses the smallest T3SS and the largest apoplastic effector repertoires. The only T3SS effector family common to all three pathogens is AvrE, suggesting that, as for other plant–Pseudomonas interactions, it may play a crucial role for pathogenicity towards coffee plants. Apoplastic proteins associated with maintaining the redox balance and degrading proteins/peptides not previously described as important in plant–bacteria interactions were found. Gain/loss of the tabtoxin biosynthetic cluster with retention of the antitoxin gene was observed, indicating that tabtoxin production is not a limiting factor for the occurrence of mixed infections.     

Variation and distribution of virulence phenotypes of Bremia lactucae in natural populations of Lactuca serriola

During 1997–2000 the virulence variation and distribution of virulence phenotypes of Bremia lactucae (lettuce downy mildew) in natural populations of Lactuca serriola (prickly lettuce) were studied. Altogether 139 isolates of B. lactucae originating from the Czech Republic, France and Germany were examined for the presence of 27 virulence factors (v-factors) and their combinations. In the Czech population, 37 different v-phenotypes (P1–P37) of B. lactucae were found to occur on L. serriola . Most v-phenotypes were characterized by v-factors that match resistance ( Dm genes/R-factors) carried by L. serriola . A wide diversity of v-phenotypes was recorded every year, but most were rare and did not reappear in other populations of B. lactucae . The three v-phenotypes P21, P28 and P29 were most frequent and widely distributed. There was variation both between spatially isolated populations and within populations of the pathogen. Geographic differences in virulence were found for the southern parts of Moravia, where the phenotypic composition of pathogen populations was completely different from the remaining part of the area investigated. However, some populations had v-phenotypes similar to those of spatially distant populations in Moravia.     

丁香假单胞菌III型分泌系统组分HrpH酶学功能解析

 III型分泌系统(T3SS)是植物病原细菌的主要致病系统,负责分泌和转运效应蛋白。T3SS结构的形成需要跨越由肽聚糖包被的细菌细胞壁周质层。HrpH是丁香假单胞菌T3SS特化的裂解性转糖基酶。本研究通过原核表达纯化了HrpH及其相关结构域蛋白,发现HrpH具有结合肽聚糖的能力,其中SLT结构域是肽聚糖结合的关键功能域,第148位的谷氨酸是结合的关键位点。进一步研究发现HrpH能够抑制丁香假单胞菌番茄致病变种DC3000(Pst DC3000)的生长,透射电镜显示,HrpH蛋白处理Pst DC3000使其细胞壁的完整性受损。以上研究结果表明HrpH通过裂解细胞壁、结合肽聚糖从而协助T3SS穿透细菌细胞壁形成超分子复合体。     

Genetic diversity, presence of the syrB gene, host preference and virulence of Pseudomonas syringae pv. syringae strains from woody and herbaceous host plants

A total of 101 Pseudomonas syringae pv. syringae strains, obtained from international culture collections or isolated from diseased tissues of herbaceous and woody plant species, were assessed by repetitive PCR using the BOX primer, and for the presence of the syrB gene. Representative strains were also tested for pathogenicity to lilac, pear, peach, corn and bean, as well as for virulence to lemon and zucchini fruits. The unweighted pair-group method using arithmethic averages analysis (UPGMA) of genomic fingerprints revealed 17 different patterns which grouped into three major clusters, A, B and C. Most of the strains (52·4%) were included in patterns 1–4 of group A. These patterns comprised strains obtained from either herbaceous or woody species, and showed four fragments of similar mobility. Genetic variability was ascertained for strains isolated from apple, pear, apricot, Citrus spp. and cereals. No clear relationship was observed between host plant and bacterial genomic fingerprint. Variability was also observed in pathogenicity and virulence tests. The inoculation of pear leaves discriminated strains isolated from pear as well as the very aggressive strains, whereas inoculation of lilac, peach and corn did not discriminate the host plant from which the strains were originally isolated. Lemon fruit inoculation proved very effective for P. syringae pv. syringae virulence assessment. The syrB gene was present in almost all strains.     

Biology of Pseudomonas cichorii in chrysanthemum1

Since 1982, outbreaks of stem blight caused by Pseudomonas cichorii are sometimes found in greenhouse-grown chrysanthemums in The Netherlands. In this study, 13 Dutch chrysanthemum isolates and, for comparison, six from other origins were biochemically, serologically and pathologically characterized. All isolates were biochemically homogeneous in most of some 40 tests. Different serotypes were observed, which were not host or virulence-correlated. After inoculation into cut and potted chrysanthemum, endive and chicory, P. cichorii isolates were found to be non-host-specific. Isolates showed differences in virulence after inoculation into potted chrysanthemum. For five isolates, disease development at high humidity (85–95% RH, 18°C) was more than 100% greater than at low humidity (55–70% RH, 18°C). Using a selective medium and immunosorbent dilution plating (ISDP), P. cichorii could be isolated from symptomless leaves and soil from nurseries where the disease was observed and from soil of a disease-free nursery. ISDP did not significantly increase the recovery of the pathogen. It was concluded that: (a) P. cichorii is an opportunistic pathogen; (b) epidemic infections of stem blight are due to a combination of high humidity, higher temperatures, high plant density and use of very susceptible cultivars; (c) control measures should concentrate on preventing this combination.     

The tomato Pto gene confers resistance to Pseudomonas floridensis,an emergent plant pathogen with just nine type III effectors

A newly discovered bacterial species, Pseudomonas floridensis, has emerged as a pathogen of tomato in Florida. This study compares the virulence and other attributes of P. floridensis to Pseudomonas syringae pv. tomato, which causes bacterial speck disease of tomato. Pseudomonas floridensis reached lower population levels in leaves of tomato as compared to the P. syringae pv. tomato strains DC3000 and NYT1. Analysis of the genome sequence of the P. floridensis type strain GEV388 revealed that it has just nine type III effectors including AvrPtoB_GEV388, which is 66% identical to AvrPtoB in DC3000. Five of these effectors have been previously reported to be members of a ‘minimal effector repertoire’ required for full DC3000 virulence on Nicotiana benthamiana; however, GEV388 grew poorly on leaves of this plant species compared to the DC3000 minimal effector strain. The tomato Pto gene recognizes AvrPtoB in race 0 P. syringae pv. tomato strains, thereby conferring resistance to bacterial speck disease. Pto was also found to confer resistance to P. floridensis, indicating this gene will be useful in the protection of tomato against this newly emerged pathogen.     

具有III型分泌系统的Pseudomonas moraviensis FP1761植物益生功能及其全基因组分析

 本研究从节瓜根际分离到一株具有防病促生功能的荧光类假单胞菌FP1761。该菌株对部分植物病原真菌和细菌具有拮抗能力,能够解钾、解有机磷和无机磷,可产生氨、蛋白酶、嗜铁素、吲哚乙酸。生物测定表明菌株FP1761可显著促进小麦生长。生理生化、平均核苷酸相似度、16S rDNA和多基因分析将FP1761鉴定为摩拉维亚假单胞菌(Pseudomonas moraviensis)。菌株FP1761基因组草图全长6.12 Mb,(G+C)含量为59.9%,共编码5467个基因序列。将该菌株与种内3个代表性菌株进行泛基因组和核心基因组分析,共产生 4 357个共有基因,菌株FP1761特有基因327个。利用antiSMASH对菌株次生代谢基因簇进行预测,发现其含有8个潜在的次生代谢产物基因簇。其中两个基因簇与嗜铁素pyoverdine合成相关,未见聚酮类合成基因。基因组分析发现,该菌株具有与病原性假单胞菌相似的III型分泌系统,但丢失了效应蛋白调控因子hrpS和转运相关的hrpH和hrpK1基因。对全基因组扫描,菌株FP1761仅保留了病原性假单胞菌的保守效应蛋白AvrE和HopAA1-1。FP1761是目前已发现的唯一具有III型分泌系统的摩拉维亚假单胞菌。本研究表明摩拉维亚假单胞菌FP1761具有潜在的植物防病促生功能,但其III型分泌系统与植物益生互作机制有待进一步解析。     

The N-acetyltransferase gene-implicated iron acquisition contributes to host specificity of Pseudomonas cichorii strain SPC9018 and its virulence

A pathogenicity island within the genome of a multi-host plant bacterium, Pseudomonas cichorii strain SPC9018, comprises the hrp genes encoding a type III secretion system and the pat gene encoding an N-acetyltransferase proposed to play a role in virulence. However, the function of the N-acetyltransferase remains poorly characterized. Interestingly, limiting the iron condition using a phytosiderophore, mugineic acid, resulted in reduced virulence of strain SPC9018 on respective host plants, including eggplant, similar to the reduced virulence observed with a pat gene-deletion mutant. Spectroscopic analyses showed that the pat deletion reduced the concentration of pyoverdine, which is the main siderophore produced by strain SPC9018, leading to a reduction in pyoverdine-mediated iron acquisition. Furthermore, the pat gene deletion mutant showed enhanced expression of the fecA, pvdL, and pvdR genes, whose expression is induced under deficient siderophore-mediated iron uptake. The pat-deletion mutant showed a hyper-swarming phenotype, and the addition of iron decreased this swarming motility. The pat deletion also reduced the adhesion ability of the bacteria, similar to the effect of iron-limited conditions. Furthermore, deletion of the pat gene enhanced expression of the hrp genes. These findings suggested that the pat gene encoding the N-acetyltransferase may be implicated in iron acquisition, contributing to host specificity of P. cichorii strain SPC9018 and its virulence.     

Participation of Xanthomonas axonopodis pv. citri hrp cluster in citrus canker and nonhost plant responses

The participation of the Xanthomonas axonopodis pv. citri hypersensitive response and pathogenicity ( hrp ) cluster in interactions with host and nonhost plants was characterized in pathogenicity and avirulence models. The hrp cluster encodes the type III secretion system indispensable for trafficking of proteins to the plant cell. Mutations in operons hrpB and hrpD and the hrpF gene failed to produce canker in citrus plants or hypersensitive response in cotton plants. The interaction of the phytopathogen with various nonhost plants has been characterized . The results showed that the hypersensitive response is activated in leaves of cotton, bean, tobacco, tomato, pepper and Nicotiana benthamiana , and that genes present in operons hrpB and hrpD and the hrpF gene are required for pathogenicity in hosts and induction of the hypersensitive response in nonhost plants.     

青枯菌Po82菌株Ⅵ型分泌系统基因簇功能研究

Ⅵ型分泌系统(typeⅥsecretion system,T6SS)是革兰氏阴性细菌中新近发现的分泌系统,控制细菌的毒性和蛋白泌出。本试验构建了植物青枯菌Po82菌株的T6SS基因簇完全缺失菌株,从全局水平初步分析了T6SS的功能。与野生型菌株相比,T6SS基因簇的缺失导致了突变菌株运动能力显著增强,在接种前期突变株病情指数明显下降;通过qRT-PCR分析Ⅲ型分泌系统效应子基因,其中popA、popB和popP基因表达量上调,而popC表达水平下调。T6SS基因簇的缺失影响了Po82菌株的运动能力和Ⅲ型效应子基因的表达,使得Po82病程延长。这些结果说明,T6SS参与青枯菌的致病过程,且T6SS与T3SS之间有复杂的未知调控关系。     

花生网斑病不同病斑类型及其病原菌致病力差异

本文描述了河南省发生日趋严重的花生网斑病在田间出现的一种新的病斑类型,并结合已报道的两种病斑类型,对3种病斑类型的病原菌开展致病力差异研究。研究结果表明:田间新发现的花生网斑病病斑特征明显,综合分析后将花生网斑病病斑类型分别定为网纹型、类褐斑型和污斑型;同一花生品种上3种病斑类型病原菌菌株间存在明显的致病力差异,同一病斑类型菌株对不同花生品种的致病力也表现出显著差异。     

Hrp Pilus – Reaching Through the Plant Cell Wall

A number of Gram negative pathogenic bacteria are equipped with a macromolecule translocation machinery that enables them to inject virulence determinants directly into the cells of their eukaryotic hosts. For plant pathogens, the plant cell wall represents an extra challenge for such injection. The ability to overcome this barrier has turned out to be so beneficial that two non-homologous injection systems, type III and type IV secretion machineries, have evolved. A morphologically similar pilus structure is associated with both of these systems. In this review, the direct protein translocation by type III (Hrp) secretion machinery is discussed with focus on the function of Hrp pilus. Comparisons between the Hrp system and the analogous translocation of T-DNA by Agrobacterium type IV secretion machinery are made and the differences between type III systems of plant and animal pathogens are discussed. Alternative hypothetical models for the mechanistic mode of action of the pilus are presented.