Rahnella perminowiae sp. nov., Rahnella bonaserana sp. nov., Rahnella rivi sp. nov. and Rahnella ecdela sp. nov., isolated from diverse environmental sources, and emended description of the genus Rahnella.

Bacteria isolated from onion bulbs suffering from bacterial decay in the United States and Norway were previously shown to belong to the genus Rahnella based on partial housekeeping gene sequences and/or fatty acid analysis. However, many strains could not be assigned to any existing Rahnella species. Additionally, strains isolated from creek water and oak as well as a strain with bioremediation properties were assigned to Rahnella based on partial housekeeping gene sequences. The taxonomic status of these 21 strains was investigated using multilocus sequence analysis, whole genome analyses, phenotypic assays and fatty acid analysis. Phylogenetic and phylogenomic analyses separated the strains into five clusters, one of which corresponded to Rahnella aceris. The remaining four clusters could be differentiated both genotypically and phenotypically from each other and existing Rahnella species. Based on these results, we propose the description of four novel species: Rahnella perminowiae sp. nov. (type strain SL6T=LMG 32257T=DSM 112609T), Rahnella bonaserana sp. nov. (H11bT=LMG 32256T=DSM 112610T), Rahnella rivi sp. nov. (FC061912-KT=LMG 32259T=DSM 112611T) and Rahnella ecdela sp. nov. (FRB 231T=LMG 32255T=DSM 112612T).

Complete Genome Sequence Resources for the Onion Pathogen, Pantoea ananatis OC5a.

Here, we report on the genomic sequence and annotation for Pantoea ananatis OC5a, a strain that was isolated from an onion bulb grown in New York and that is pathogenic to onion, causing center rot of onion. OC5a is the first P. ananatis strain pathogenic to onion from New York to be completely assembled and sequenced. Having been assembled using long PacBio reads and high-fidelity Illumina reads, this genome is closed, complete, and of high quality.

HIPM Is a Susceptibility Gene of Malus spp.: Reduced Expression Reduces Susceptibility to Erwinia amylovora.

Fire blight, a devastating disease caused by the bacterium Erwinia amylovora, is a major threat to apple crop production. To improve our understanding of the fire blight disease and to identify potential strategies to control the pathogen, we studied the apple protein HIPM (for HrpN-interacting protein from Malus spp.), which has previously been identified as interacting with the E. amylovora effector protein HrpN. Transgenic apple plants were generated with reduced HIPM expression, using an RNA interference construct, and were subsequently analyzed for susceptibility to E. amylovora infection. Lines exhibiting a greater than 50% silencing of HIPM expression showed a significant decrease in susceptibility to E. amylovora infection. Indeed, a correlation between HIPM expression and E. amylovora infection was identified, demonstrating the crucial role of HIPM during fire blight disease progression. Furthermore, an apple oxygen-evolving enhancer-like protein (MdOEE) was identified via a yeast two-hybrid screen to interact with HIPM. This result was confirmed with bimolecular fluorescence complementation assays and leads to new hypotheses concerning the response mechanism of the plant to E. amylovora as well as the mechanism of infection of the bacterium. These results suggest that MdOEE and, particularly, HIPM are promising targets for further investigations toward the genetic improvement of apple.

Center Rot of Onion (Allium cepa) Caused by Pantoea ananatis Requires pepM, a Predicted Phosphonate-Related Gene.

Pantoea ananatis, a cause of center rot of onion, is problematic in the United States and elsewhere. The bacterium lacks disease determinants common to most other bacterial pathogens of plants. A genomic island containing the gene pepM was detected within many onion-pathogenic strains of P. ananatis of diverse origins. The pepM gene of P. ananatis putatively encodes a protein that converts phosphoenolpyruvate to phosphonopyruvate, the first step in the biosynthesis of phosphonates and related molecules. This gene appears to be essential for center rot disease. Deletion of pepM rendered the mutant strain unable to cause lesions in leaves of growing onions and water-soaking of inoculated yellow onion bulbs. Furthermore, growth of the deletion mutant in onion leaves was significantly diminished compared with wild-type bacteria, and the mutant failed to cause cell death in tobacco. Complementation of the mutated strain with pepM restored the phenotype to wild-type capability. The pepM gene is the first pathogenicity factor identified that affects bacterial fitness as well as symptom development in both leaves and bulbs in a pathogen causing center rot of onion.

Lactic Acid Bacteria Cause a Leaf Blight and Bulb Decay of Onion (Allium cepa).

Several members of the lactic acid bacteria group were isolated from diseased onion plants and bulbs. Based on growth characteristics and sequence analysis of 16S rRNA and rpoA genes, the strains were identified as Lactococcus lactis, Lactobacillus plantarum, and three species of Leuconostoc, i.e., citreum, mesenteroides, and pseudomesenteroides. Pathogenic potential to onion leaves and mature onion bulbs was assessed. L. plantarum and all three Leuconostoc species caused symptoms in both leaves and bulbs. L. lactis caused scale discoloration in bulbs but failed to cause lesions on leaves. Leuconostoc citreum caused bulb decay in 7 days at 18°C as well as 37°C. This is the first report of a group of gram-positive bacteria able to cause disease in onion leaves.

PCR Primers for Detection of Pantoea ananatis, Burkholderia spp., and Enterobacter sp. from Onion.

Bacterial decays of onion bulbs cause sporadic and sometimes serious losses to onion (Allium cepa). In New York, three groups of bacteria were identified as problematic: Burkholderia spp., Pantoea ananatis, and Enterobacter spp. To aid in efficient detection and diagnosis of these pathogens, pairs of specific polymerase chain reaction primers were designed and validated, based on a strategy that utilized various genome sequences now available in public databases. Primer pairs were tested against numerous strains of target bacteria, closely related bacteria, and other onion-pathogenic bacteria. Each primer pair yielded a single, apparently highly specific amplicon from aqueous suspensions of the target bacteria. Minimum sensitivities were approximately 103 CFU per 25-µl reaction mixture for all three primer pairs.

Identification of bacteria pathogenic to or associated with onion (Allium cepa) based on sequence differences in a portion of the conserved gyrase B gene.

We have developed a method for the identification of Gram-negative bacteria, particularly members of the Enterobacteriaceae, based on sequence variation in a portion of the gyrB gene. Thus, we identified, in most cases to species level, over 1000 isolates from onion bulbs and leaves and soil in which onions were grown.

Comparative genomics of 12 strains of Erwinia amylovora identifies a pan-genome with a large conserved core.

The plant pathogen Erwinia amylovora can be divided into two host-specific groupings; strains infecting a broad range of hosts within the Rosaceae subfamily Spiraeoideae (e.g., Malus, Pyrus, Crataegus, Sorbus) and strains infecting Rubus (raspberries and blackberries). Comparative genomic analysis of 12 strains representing distinct populations (e.g., geographic, temporal, host origin) of E. amylovora was used to describe the pan-genome of this major pathogen. The pan-genome contains 5751 coding sequences and is highly conserved relative to other phytopathogenic bacteria comprising on average 89% conserved, core genes. The chromosomes of Spiraeoideae-infecting strains were highly homogeneous, while greater genetic diversity was observed between Spiraeoideae- and Rubus-infecting strains (and among individual Rubus-infecting strains), the majority of which was attributed to variable genomic islands. Based on genomic distance scores and phylogenetic analysis, the Rubus-infecting strain ATCC BAA-2158 was genetically more closely related to the Spiraeoideae-infecting strains of E. amylovora than it was to the other Rubus-infecting strains. Analysis of the accessory genomes of Spiraeoideae- and Rubus-infecting strains has identified putative host-specific determinants including variation in the effector protein HopX1(Ea) and a putative secondary metabolite pathway only present in Rubus-infecting strains.

Infection of Onion Leaves by Pantoea ananatis Leads to Bulb Infection.

Pantoea ananatis has been identified as a cause of center rot of onion. In the field, onion leaves can become infected with P. ananatis and lead to leaf blight. Infected bulbs often are detected only after harvest; however, it has not been demonstrated experimentally that leaf infection by P. ananatis can lead to bulb infection. In this study, onion leaf infection by P. ananatis leading to bulb infection was investigated. Of 18 strains of P. ananatis isolated from symptomatic onion bulbs grown in New York, 14 were pathogenic in bulb and leaf tissue. Pathogenic strains of P. ananatis caused nonmacerated, yellow-brown coloration in fleshy bulb scales following inoculation of bulbs and incubation for 2 days at 28°C. Subepidermal inoculation of onion leaves with pathogenic strains of P. ananatis resulted in gray-white foliar lesions that extended acropetally and basipetally from the points of inoculation. In all, 16% of leaf lesions extended to the onion neck and 11% continued into the bulbs, which developed nonmacerated, yellow-brown scales. Bacteria recovered from the leading edges of lesions had microbiological and molecular characteristics of P. ananatis. This is the first experimental evidence that infection of onion leaves by P. ananatis can lead to bulb infection.

OEM--a new medium for rapid isolation of onion-pathogenic and onion-associated bacteria.

Onions (Allium cepa L.) are plagued by a number of bacterial pathogens including Pantoea ananatis, P. agglomerans, Burkholderia cepacia, Enterobacter cloacae, Pectobacterium carotovorum subsp. carotovorum, Xanthomonas axonopodis pv. axonopodis and several Pseudomonas spp. We developed a semi-selective medium, termed onion extract medium (OEM), to selectively and rapidly isolate bacteria pathogenic to and associated with onions and onion-related samples including bulbs, seeds, sets, transplant seedlings, soil and water. Most strains of interest grow sufficiently on OEM in 24h at 28°C for tentative identification based on colony morphology, facilitating further characterization by microbiological and/or molecular means.

Top 10 plant pathogenic bacteria in molecular plant pathology.

Many plant bacteriologists, if not all, feel that their particular microbe should appear in any list of the most important bacterial plant pathogens. However, to our knowledge, no such list exists. The aim of this review was to survey all bacterial pathologists with an association with the journal Molecular Plant Pathology and ask them to nominate the bacterial pathogens they would place in a 'Top 10' based on scientific/economic importance. The survey generated 458 votes from the international community, and allowed the construction of a Top 10 bacterial plant pathogen list. The list includes, in rank order: (1) Pseudomonas syringae pathovars; (2) Ralstonia solanacearum; (3) Agrobacterium tumefaciens; (4) Xanthomonas oryzae pv. oryzae; (5) Xanthomonas campestris pathovars; (6) Xanthomonas axonopodis pathovars; (7) Erwinia amylovora; (8) Xylella fastidiosa; (9) Dickeya (dadantii and solani); (10) Pectobacterium carotovorum (and Pectobacterium atrosepticum). Bacteria garnering honourable mentions for just missing out on the Top 10 include Clavibacter michiganensis (michiganensis and sepedonicus), Pseudomonas savastanoi and Candidatus Liberibacter asiaticus. This review article presents a short section on each bacterium in the Top 10 list and its importance, with the intention of initiating discussion and debate amongst the plant bacteriology community, as well as laying down a benchmark. It will be interesting to see, in future years, how perceptions change and which bacterial pathogens enter and leave the Top 10.

Comparative analysis of the Hrp pathogenicity island of Rubus- and Spiraeoideae-infecting Erwinia amylovora strains identifies the IT region as a remnant of an integrative conjugative element.

The Hrp pathogenicity island (hrpPAI) of Erwinia amylovora not only encodes a type III secretion system (T3SS) and other genes required for pathogenesis on host plants, but also includes the so-called island transfer (IT) region, a region that originates from an integrative conjugative element (ICE). Comparative genomic analysis of the IT regions of two Spiraeoideae- and three Rubus-infecting strains revealed that the regions in Spiraeoideae-infecting strains were syntenic and highly conserved in length and genetic information, but that the IT regions of the Rubus-infecting strains varied in gene content and length, showing a mosaic structure. None of the ICEs in E. amylovora strains were complete, as conserved ICE genes and the left border were missing, probably due to reductive genome evolution. Comparison of the hrpPAI region of E. amylovora strains to syntenic regions from other Erwinia spp. indicates that the hrpPAI and the IT regions are the result of several insertion and deletion events that have occurred within the ICE. It also suggests that the T3SS was present in a common ancestor of the pathoadapted Erwinia spp. and that insertion and deletion events in the IT region occurred during speciation.

Genome sequence of an Erwinia amylovora strain with pathogenicity restricted to Rubus plants.

Here, we present the genome of a strain of Erwinia amylovora, the fire blight pathogen, with pathogenicity restricted to Rubus spp. Comparative genomics of ATCC BAA-2158 with E. amylovora strains from non-Rubus hosts identified significant genetic differences but support the inclusion of this strain within the species E. amylovora.

Secretion and translocation signals and DspB/F-binding domains in the type III effector DspA/E of Erwinia amylovora.

DspA/E is a type III effector of Erwinia amylovora, the bacterial pathogen that causes fire blight disease in roseaceous plants. This effector is indispensable for disease development, and it is translocated into plant cells. A DspA/E-specific chaperone, DspB/F, is necessary for DspA/E secretion and possibly for its translocation. In this work, DspB/F-binding sites and secretion and translocation signals in the DspA/E protein were determined. Based on yeast two-hybrid assays, DspB/F was found to bind DspA/E within the first 210 amino acids of the protein. Surprisingly, both DspB/F and OrfA, the putative chaperone of Eop1, also interacted with the C-terminal 1059 amino acids of DspA/E; this suggests another chaperone-binding site. Secretion and translocation assays using serial N-terminal lengths of DspA/E fused with the active form of AvrRpt2 revealed that at least the first 109 amino acids, including the first N-terminal chaperone-binding motif and DspB/F, were required for efficient translocation of DspA/E, although the first 35 amino acids were sufficient for its secretion and the presence of DspB/F was not required. These results indicate that secretion and translocation signals are present in the N terminus of DspA/E, and that at least one DspB/F-binding motif is required for efficient translocation into plant cells.

The C-terminal half of the HrpN virulence protein of the fire blight pathogen Erwinia amylovora is essential for its secretion and for its virulence and avirulence activities.

The HrpN (harpin) protein of the fire blight pathogen Erwinia amylovora is an essential virulence factor secreted via the bacterial type III secretion system. HrpN also has avirulence activity when delivered to tobacco by E. amylovora and has defense elicitor activity when applied to plants as a cell-free protein extract. Here, we characterize a series of random mutations in hrpN that altered the predicted amino acid sequence of the protein. Amino acid substitutions and deletions in the highly conserved, C-terminal portion of HrpN disrupted the virulence and avirulence activities of the protein. Several of these mutations produced a dominant-negative effect on E. amylovora avirulence on tobacco. None of the mutations clearly separated the virulence and avirulence activities of HrpN. Some C-terminal mutations abolished secretion of HrpN by E. amylovora. The results indicate that the C-terminal half of HrpN is essential for its secretion by E. amylovora, for its virulence activity on apple and pear, and for its avirulence activity on tobacco. In contrast, the C-terminal half of HrpN was not required for cell-free elicitor activity. This suggests that the N-terminal and C-terminal halves of HrpN mediate cell-free elicitor activity and avirulence activity, respectively.

Identification of specific fragments of HpaG Xooc, a harpin from Xanthomonas oryzae pv. oryzicola, that induce disease resistance and enhance growth in plants.

Harpin proteins from gram-negative plant-pathogenic bacteria can stimulate hypersensitive cell death (HCD) and pathogen defense as well as enhance growth in plants. Two of these diverse activities clearly are beneficial and may depend on particular functional regions of the proteins. Identification of beneficial and deleterious regions might facilitate the beneficial use of harpin-related proteins on crops without causing negative effects like cell death. Here, we report the identification and testing of nine functional fragments of HpaG(Xooc), a 137-amino-acid harpin protein from Xanthomonas oryzae pv. oryzicola, the pathogen that causes bacterial leaf streak of rice. Polymerase chain reaction-based mutagenesis generated nine proteinaceous fragments of HpaG(Xooc); these caused different responses following their application to Nicotiana tabacum (tobacco) and Oryza sativa (rice). Fragment HpaG62-137, which spans the indicated amino acid residues of the HpaG, induced more intense HCD; in contrast, HpaG10-42 did not cause evident cell death in tobacco. However, both fragments stimulated stronger defense responses and enhanced more growth in rice than the full-length parent protein, HpaG(Xooc). Of the nine fragments, the parent protein and one deletion mutant of HpaG(Xooc) tested, HpaG10-42, stimulated higher levels of rice growth and resulted in greater levels of resistance to X. oryzae pv. oryzae and Magnaporthe grisea. These pathogens cause bacterial leaf blight and rice blast, respectively, the two most important diseases of rice world-wide. HpaG10-42 was more active than HpaG(Xooc) in inducing expression of several genes that regulate rice defense and growth processes and activating certain signaling pathways, which may explain the greater beneficial effects observed from treatment with that fragment. Overall, our results suggest that HpaG10-42 holds promise for practical agricultural use to induce disease resistance and enhance growth of rice.

A fragment of the Xanthomonas oryzae pv. oryzicola harpin HpaG Xooc reduces disease and increases yield of rice in extensive grower plantings.

Harpins of phytopathogenic bacteria stimulate defense and plant growth in many types of plants, conferring disease resistance and enhanced yield. In a previous study, we characterized nine fragments of the harpin protein HpaG(Xooc) from Xanthomonas oryzae pv. oryzicola for plant defense elicitation and plant growth stimulation activity relative to the intact protein. In plants grown under controlled conditions, the fragment HpaG10-42 was more active in both regards than HpaG(Xooc). Here, we demonstrate that the activity of HpaG10-42 in rice under field conditions significantly exceeds that of HpaG(Xooc), stimulating resistance to three important diseases and increasing grain yield. We carried out tests in 672 experimental plots with nine cultivars of rice planted at three locations. Application protocols were optimized by testing variations in application rate, frequency, and timing with respect to rice growth stage. Of the concentrations (24, 24, 12, and 6 microg/ml), and number and timing of applications (at one to four different stages of growth) tested, HpaG10-42 at 6 microg/ml applied to plants once at nursery seedling stage and three times in the field was most effective. Bacterial blight, rice blast, and sheath blight were reduced 61.6 and 56.4, 93.6 and 76.0, and 93.2 and 55.0% in indica and japonica cultivars, respectively, relative to controls. Grain yields were 22 to 27% greater. These results are similar to results obtained with typical local management practices, including use of chemicals, to decrease disease severities and increase yield in rice. Our results demonstrate that the HpaG10-42 protein fragment can be used effectively to control diseases and increase yield of this staple food crop.

HrpN of Erwinia amylovora functions in the translocation of DspA/E into plant cells.

The type III secretion system (T3SS) is required by plant pathogenic bacteria for the translocation of certain bacterial proteins to the cytoplasm of plant cells or secretion of some proteins to the apoplast. The T3SS of Erwinia amylovora, which causes fire blight of pear, apple and other rosaceous plants, secretes DspA/E, which is an indispensable pathogenicity factor. Several other proteins, including HrpN, a critical virulence factor, are also secreted by the T3SS. Using a CyaA reporter system, we demonstrated that DspA/E is translocated into the cells of Nicotiana tabacum'Xanthi'. To determine if other T3-secreted proteins are needed for translocation of DspA/E, we examined its translocation in several mutants of E. amylovora strain Ea321. DspA/E was translocated by both hrpW and hrpK mutants, although with some delay, indicating that these two proteins are dispensable in the translocation of DspA/E. Remarkably, translocation of DspA/E was essentially abolished in both hrpN and hrpJ mutants; however, secretion of DspA/E into medium was not affected in any of the mentioned mutants. In contrast to the more virulent strain Ea273, secretion of HrpN was abolished in a hrpJ mutant of strain Ea321. In addition, HrpN was weakly translocated into plant cytoplasm. These results suggest that HrpN plays a significant role in the translocation of DspA/E, and HrpJ affects the translocation of DspA/E by affecting secretion or stability of HrpN. Taken together, these results explain the critical importance of HrpN and HrpJ to the development of fire blight.

AtHIPM, an ortholog of the apple HrpN-interacting protein, is a negative regulator of plant growth and mediates the growth-enhancing effect of HrpN in Arabidopsis.

HrpN (harpin) protein is critical to the virulence of the fire blight pathogen Erwinia amylovora in host plants like apple (Malus x domestica). Moreover, exogenous treatment of Arabidopsis (Arabidopsis thaliana), a nonhost plant, with partially purified HrpN enhances growth. To address the bases of the effects of HrpN in disease, we sought a HrpN-interacting protein(s) in apple, using a yeast two-hybrid assay. A single positive clone, designated HIPM (HrpN-interacting protein from Malus), was found. HIPM, a 6.5-kD protein, interacted with HrpN in yeast and in vitro. Deletion analysis showed that the N-terminal 198 of 403 amino acids of HrpN are required for interaction with HIPM. HIPM orthologs were found in Arabidopsis (AtHIPM) and rice (Oryza sativa; OsHIPM). HrpN also interacted with AtHIPM in yeast and in vitro. In silico analyses revealed that the three plant proteins contain putative signal peptides and putative transmembrane domains. We showed that both HIPM and AtHIPM have functional signal peptides, and green fluorescent protein-tagged HIPM and AtHIPM associated, in clusters, with plasma membranes. Both HIPM and AtHIPM are expressed constitutively; however, they are expressed more strongly in apple and Arabidopsis flowers than in leaves and stems. The size of AtHIPM knockout mutant plants of Arabidopsis was slightly larger than the wild-type plants. Interestingly, the knockout mutant did not exhibit enhanced plant growth in response to treatment with HrpN. Overexpression of AtHIPM conversely resulted in smaller plants. These results indicate that AtHIPM functions as a negative regulator of plant growth and mediates enhanced growth that results from treatment with HrpN.

Analyses of the secretomes of Erwinia amylovora and selected hrp mutants reveal novel type III secreted proteins and an effect of HrpJ on extracellular harpin levels.

SUMMARY Erwinia amylovora is a plant pathogenic enterobacterium that causes fire blight disease of apple, pear and other rosaceous plants. A type III (T3) secretion system, encoded by clustered, chromosomal hrp genes (hypersensitive response and pathogenicity), is essential for infection, but only a few proteins are known that are secreted through this pathway (the T3 'secretome'). We developed an efficient protocol for purification and concentration of extracellular proteins and used it to characterize the T3 secretome of E. amylovora Ea273 by comparing preparations from the wild-type strain with those from mutants defective in hrp secretion, regulation, or in genes encoding putative T3-secreted proteins. Proteins were resolved by gel electrophoresis and identified using mass spectrometry and a draft sequence of the E. amylovora genome. Twelve T3-secreted proteins were identified, including homologues of known effector and helper proteins, and HrpJ, a homologue of YopN of Yersinia pestis. Several previously uncharacterized T3-secreted proteins were designated as Eops for Erwinia outer proteins. Analysis of the secretome of a non-polar hrpJ mutant demonstrated that HrpJ is required for accumulation of wild-type levels of secreted harpins. HrpJ was found to be essential for pathogenesis, and to play a major role in elicitation of the hypersensitive reaction in tobacco.