Introduction. Habitat. Erwinia

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1 Prokaryotes (2006) 6: DOI: / x_15 CHAPTER ainiwer dn a detal Re aren Ge Erwinia and Related Genera CLARENCE I. KADO Introduction Members of the genus Erwinia are primarily plant-pathogenic and plant-associated bacteria. As modern approaches have focused on the direct analysis of genes and their gene products, commonly associated phenotypes such as the type of disease that they cause and the relatedness of rdna have prompted splitting of certain Erwinia members into the genus Enterobacter, Pectobacterium, Pantoea or Brenneria. For example, the genus Pectobacterium, which was suggested previously (Brenner et al., 1973), has been resurrected to accommodate those species that profusely produce pectinolytic enzymes in plant pathogenesis and that have related rdna (Hauben et al., 1998). Because of the large difference in the percent G+C and the rdna sequence, the genus Pantoea was added. As the sequences of entire genomes of this group of organisms become available, regrouping of the members of these genera will likely take place. Pairwise 16S rdna sequence comparisons also showed E. alni, E. nigrifluens, E. paradisiaca, E. quercina, E. rubrifaciens and E. salicis grouped into the new genus Brenneria (Hauben et al., 1998). Erwinia The genus Erwinia is classified in the Enterobacter iaceae. Most members of this genus characteristically cause diseases of plants. Recent 16S rdna sequence comparisons have been proposed to delineate species originally classified in the genus Erwinia into the genus Pantoea, Enterobacter, Pectobacterium, or Brenneria. Certain species with the distinct glucose metabolism of oxidizing D-glucose into various forms of gluconate have been removed from the genus Erwinia and moved into the genus Pantoea. All of the genera possess the main phenotypic characteristics of the Enterobacter iaceae. They produce acid from sugars, are Gram-negative rods bearing peritrichous flagella, and can ferment substrates anaerobically. The phylogenetic position of the genus Erwinia, along with other members of the Enterobacter iaceae associated with plants, have been explored using 16S rdna nucleotide sequence comparisons (Kwon et al., 1997; Hauben et al., 1998). Four phylogenetic groups representing a branch of the lineage tree have been proposed. One group comprises E. amylovora, E. rhapontici, E. persicina, E. psidii, E. pyrifoliae, E. mallotivora and E. tracheiphila. The second group consists of E. carotovora subspecies, E. chrysanthemi, and E. cypripedii. The latter group was proposed to be moved to the genus Pectobacterium primarily because it produces pectolytic enzymes for pathogenesis (for history see Brenner et al., 1973). The third group comprises E. alni, E. nigrifluens, E. rubrifaciens, E. paradisiaca, E. quercina, and E. salicis. It was suggested this third group be moved into the proposed genus Brenneria (Hauben et al., 1998). The fourth group comprises members that display unusual oxidative metabolism of D-glucose and produce yellow to mauve colonies. They were reclassified into the genus Pantoea (Gavini et al., 1989; Kageyama et al., 1992; Mergaert et al., 1993). The generic positioning of Enterobacter, Pectobacterium, Pantoea, and Brenneria was essential to avoid further confusion in sorting species previously classified within the genus Erwinia. A more solid positioning will be forthcoming when the entire genome of each of these genera is completed and comparisons of each of the genomic sequences are made. Habitat Erwinia, Enterobacter, Pectobacterium, Pantoea and Brenneria species cause four basic types of plant diseases: 1) rapid necrosis; 2) progressive tissue maceration called soft-rot; 3) occlusion of vessel elements called vascular wilt, and 4) hypertrophy leading to gall or tumor formation. A list of species with these characteristic phenotypes is shown in Table 1.

2 444 C.I. Kado CHAPTER Table 1. Erwinia and subspecific genera and their disease-causing types. Species Disease name Type of infection Natural host plant Necrogenic group emended from Erwinia to Brenneria (Hauben et al., 1998) Brenneria alni Canker Necrogenic Alder Wilson et al., 1957 B. nigrifluens Shallow canker Necrogenic Members of the Juglandaceae B. paradisiaca Brown-black root rot Necrogenic Banana B. quercina Drippy nut and blight Maceration Coast live oak (Quercus agrifolia) Hildebrand and Schroth, 1967 B. rubrifaciens Deep bark canker Necrogenic Members of the Juglandaceae Wilson et al., 1967 B. salicis Bacterial wilt, water mark Vascular wilt Salix species (Day 1924) Chester 1939 Status quo Erwinia group E. amylovora Fire blight Necrogenic Members of the Rosaceae (Burrill, 1882) Winslow et al., 1920 E. billingia Associated with cankers Secondary invader Members of the Rosaceae Mergaert et al., 1999 E. cypripedii (Hori) Brown rot Maceration Cypripedium and other orchids Bergey et al., 1923 E. mallotivora Black leaf spot Necrogenic Mallotus japonicus tree Goto, 1976 E. persicinus Red-brown fruit rot Necrogenic Cucumber, tomato, banana E. psidii Fruit rot Necrogenic Guava E. pyrifoliae Leaf and stem blight Necrogenic Pear, Pyrus pyrifolia cv Shingo Kim et al., 1999 E. rhapontici Crown rot Maceration Rhubarb (Millard) Burkholder 1948 E. tracheiphila (Smith, 1895) Bergey et al., 1923 Cucumber wilt Vascular wilt Cucumis species Storage soft rot Maceration Harvested corms, Carotovora tubers, roots, bulbs (Jones) Bergey et al., 1923 Chrysanthemi Burkholder et al., 1953 atroseptica (van Hall 1902) Dye 1969 betavasculorum odoriferum wasabiae Stem blight, wilt Vascular wilt Corn, carnation, chrysanthemum, tropic plants, e.g., Philodendron, Dieffenbachia Blackleg Vascular wilt Potato roots, lower stems, tubers Soft rot Maceration Beet, Beta vulgaris Soft rot Maceration Endive and chicory Soft rot of excised petioles Maceration Japanese green horse radish Erwinia species emended to Enterobacter species Enterobacter cloacae Internal yellowing, brown-black discoloration Diffuse necrogenic Papaya (Carica papaya L.) Onion (Allium cepa L.) En. nimipressuralis Wet wood Necrogenic Elm En. cancerogenus Canker Necrogenic Poplar En. dissolvens Rot Slow maceration Corn Soft-rotting group proposed emendation from Erwinia to Pectobacterium (Hauben et al., 1998).

3 CHAPTER Erwinia and Related Genera 445 Isolation Erwinia species and species of its subspecific genera can be isolated from diseased tissues most expeditiously from fresh samples. Most Erwiniae are not fastidious with regard to the medium used, but it has been a general practice to employ relatively rich media such as Luria- Bertani (LB) agar, which contains per liter 10g tryptone (Difco), 5g yeast extract, 5g sodium chloride, 15g agar; and medium 523, which contains per liter 10g sucrose, 8g tryptone, 4g yeast extract, 2g dipotassium phosphate, 0.3g magnesium sulfate, and 15g agar. Supplements, such as 1% sorbitol, are added to LB agar medium for E. amylovora and related species. Although LB has been the most popular agar medium for isolation, nutrient agar (NA), which contains per liter 3g beef extract, 5g peptone, 8g sodium chloride and 15g agar, also has been used. Since NA was designed as general purpose medium for the isolation of mammalian bacterial pathogens, its use is not recommended for Erwinia and its subgeneric species that are associated with plants. For example, Pantoea species grow poorly on this medium. Some laboratories routinely have used NA supplemented with glycerol to compensate for poor growth of Erwinia species. This is poor practice. Pantoea species generally will grow more profusely when the medium is supplemented with nicotinic acid. For Erwinia species that produce excessive acid, medium YGC is used. Medium YGC agar contains per liter 20g glucose, 10g yeast extract, 20g ultrafine calcium carbonate and 15g agar. A clear zone surrounding each colony results from acid secretion, which liberates the carbonate as CO 2. The usual temperature for culture of Erwinia and its generic members is 28 C. An incubator with antidesiccation features such as a humidifier (e.g., Kendro HeraCell) is preferable. Agar plates are incubated in the inverted position. Identification Erwinia and its subgeneric members usually are motile rods bearing peritrichous flagella and are able to ferment glucose leading to the formation of acid. Their fermentative pathway yields mixed acids and 2,3-butanediol. They are unable to utilize starch as a carbon source except for E. rhapontici, which produces a weakly diffusible pink pigment called ferrorosamine (Feistner et al., 1983). E. rubrifaciens (B. rubrifaciens) produces a water soluble, diffusible red pigment called rubrifacine A (Fistner et al., 1984) on yeast-glucose calcium carbonate agar. Some strains of Pectobacterium chrysanthemi produce yellow colonies streaked with a green-blue pigment. All are catalase positive and negative for exocellular cytochrome oxidase activity. Except for the Erwinia species that cause soft rot, the necrogenic and vascular wilting group reduce nitrate. On LB or 523 media, their colonies are generally mucoid and domed and can vary in color from white to cream. Members of the genus Pantoea produce colonies that may appear either as yellow to taupe or mauve on LB agar. In contrast to the Erwinia group, Pantoea species oxidize glucose to gluconate. The genus Pantoea includes phytopathogens P. stewartii, P. ananas, P. citrea and P. agglomerans pv. milletiae, P. agglomerans pv. gypsophilae, and P. agglomerans pv. betae. P. ananas (P. ananatis) causes brown patches on internal portions of pineapple fruit [Ananas comosus (L.) Merr.] and honeydew melon (Cucumis melo L.). The disease in pineapple is called marbling. P. uredovora first was described as a epiphyte on uredospores of Ustilago smut of maize and on the panicles of barley, buckwheat and rice. Based on recent 16S rdna sequence data, P. ananas and P. uredovora are homologous and, therefore, the specific name P. ananas takes precedence with P. uredovora as a retired species name. P. ananas, changed to P. ananatis (Hauben et al., 1998) is a Gram-negative, rod-shaped, facultative anaerobe and usually forms yellow, semimucoid colonies on YGC agar. Some strains contain the ice nucleation gene inaa, somewhat similar to inaw and inaz genes of Pseudomonas syringae and P. fluorescens. Pantoea citrea causes pink disease of pineapple (Cha et al., 1998). P. citrea is Gram-negative, rodshaped, facultative anaerobe, and produces pili. It causes the hypersensitivity response in tobacco and, therefore, contains hrp genes. On nutrient agar and trypticase soy agar, the colonies are entire, smooth, glistening, translucent, but not mucoid. As the colonies age, they turn taupe with a slight depression in the center. P. citrea produces two quinoprotein glucose dehydrogenases encoded by gdha and gdhb genes (Pujol and Kado, 1999). The glucose dehydrogenase encoded by gdhb efficiently converts glucose into gluconate, which, in turn, is oxidized to 2-ketogluconate. This latter substrate is further oxidized into 2,5-diketogluconate, a highly unstable compound that is chromogenic. P. punctata and P. terrea can oxidize glucose via the gdha pathway. P. citrea harbors a cryptic plasmid with 5,229 base pairs. The plasmid, designated pucd5000, is required for the full expression of the pink disease (Pujol and Kado, 1998). P. stewartii subsp. stewartii causes Stewart s bacterial wilt and leaf blight disease of corn. This bacterium produces copious amounts of exopolysaccharides, the profuseness of which is believed to cause plugging of vessel elements in the host plant. Beginning symptoms are water-

4 446 C.I. Kado CHAPTER soaked lesions, followed by vascular wilting on seedlings and leaf blight of mature plants. Some strains that cause leaf spots on foxtail millet (Setaria italica) and pearl millet (Penssisetum americanum) have been placed in P. stewartii subsp. indologenes (Mergaert et al., 1993). Multiple numbers of cryptic plasmids are coresidents in P. stewartii subsp. stewartii. The replication and mobilization loci in cryptic plasmids psw100 and psw200 from P. stewartii subsp. stewartii share sequence homologies with pucd5000 from P. citrea. The habitats of members of the genus Pantoea are listed in Table 2. The physiological and biochemical characteristics of Erwinia and its related genera are shown in Table 3. Cultivation P. citrea may be cultured on MGY medium (10g mannitol, 2g of sodium L-glutamate, 0.5g of monobasic potassium phosphate, 0.2g of sodium chloride, 0.2g of magnesium sulfate, 0.25g yeast extract per liter, adjusted to ph 7.0 with 3 N NaOH) at 30 C. The rusty red color produced by P. citrea may be generated in canned pineapple juice. The juice is clarified by centrifugation (12,000 g, 10 min, 4 C) and adjusted to ph 6.0 with 3 N NaOH. The clarified juice is sterilized by filtration (0.4 µm-pore-size polycarbonate membrane filter; Phoretics Corporation, Livermore, California). Table 2. Habitat and disease produced by members of the Pantoea group. Species Disease name Disease type Natural host P. agglomerans pv. agglomerans Stem darkening Secondary Celery P. agglomerans pv. betae Root gall Tumorigenesis Table beets Beta vulgaris L. P. agglomerans pv. gypsophilae Bacterial gall Tumorigenesis by indole-3-acetic acid Baby s breath Gypsophila paniculata L. production P. agglomerans pv. milliteae Bacterial gall Tumorigenesis Japanese wisteria Wisteria floribunda P. ananatis Marbling Soft rot Pineapple, sugarcane, honey dew melon P. citrea Pink disease Transluscent rot Pineapple, mandarin orange P. dispersa Saprophyte None Edaphosphere resident, seeds P. punctata Brown spot Delayed maceration Mandarin orange P. stewartii subsp. indologenes Brown spot Necrogenic Graminae P. stewartii subsp. stewartii Stewart s wilt Vascular wilt Corn P. terrea Saprophyte None Soil inhabitant Table 3. Comparative physiological and biochemical characteristics of Pantoea spp. versus Erwinia spp. Characteristic Erwinia spp. Pantoea spp. Pectobacterium spp. Brenneria spp. Enterobacter spp. Gram-negative rods Motile by peritrichous flagella + +/ Facultative anaerobe Colonies yellow or mauve + Percent genomic G+C content Gluconate dehydrogenase + Cytochrome oxidase released Catalase released Indole production +/ /+ Nitrate reduced to nitrite + +/ + + H 2 S produced from cysteine +/ + + Urease produced /+ Gas produced from glucose + + Ornithine decarboxylase + Lipase + Acid from α-methylglucoside + /+ Gelatin liquefaction /+ + + Acid from sorbitol L-Malate utilized DNase produced +/ Symbols: the +/ symbol indicates that a majority of species are positive; /+ symbol indicates that a majority of species are negative.

5 CHAPTER Erwinia and Related Genera 447 Preservation Erwinia and its related genera can be stored indefinitely in the lyophilized state. Cells in exponential phase of growth are harvested by centrifugation and resuspended in 1% sterile solution of powdered milk (Carnation brand). An appropriate aliquot of the mixture is placed in an ampoule and is quickly frozen in liquid nitrogen or in ethanol containing chunks of solid CO 2. The frozen mixture is lyophilized under vacuum until it is completely desiccated. The ampoule is sealed in vacuo with a torch, appropriately labelled, and stored in a cold room at temperatures between 4 and 16 C. For routine use, it may be convenient to store cultures in the frozen state. In this case, bacterial cells are resuspended in 1% sterile solution of powdered milk prepared in 10% glycerol. The mixture is stored at 70 C. Storing the mixture at 20 C is not recommended. Short-term (1 year maximum) storage of the isolate can rely on specialized media such as Preservation agar (P agar), which contains per liter 5g peptone, 5g sodium chloride, 0.03g cysteine, and 10g agar. The medium is autoclaved in completely filled screw capped tubes. Bacteria are preserved as stab cultures that were allowed to grow for about 4 8 days and then stored at 4 C. Genetics As members of the Enterobacteriaceae, Erwinia, Enterobacter, Pantoea, Pectobacterium and Brenneria are all amenable to genetic manipulation. The molecular genetic tools used in studies dealing with Escherichia coli are directly applicable to the study of members of this group of bacteria. Generally the plasmid replicons that contain the origin of DNA replication of the ColE1 plasmid of E. coli replicate well in these bacteria. Various nonreplicative plasmids, such as those containing the origin of DNA replication of plasmid R6K (which requires an accessory initiator protein like π for replication), have been used to deliver transposable elements to generate mutants. Essentially, the vector harboring the R6K origin is unable to replicate in the recipient bacteria, thus leaving behind the transposon that jumped onto the chromosome of the recipient. Transposon mutagenesis has opened the way to identify genes involved in various metabolic and physiological pathways in these bacteria. When the bacterium of interest is not amenable to transposon mutagenesis, chemical mutagenesis using nitrosamine and alkane sulfonic esters, such as nitrosoguanidine and ethyl methane sulfonate, respectively, have been used. Chemically derived mutants defective in the phenotype of interest would be identified in the affected gene by genetic complementation using a genomic library of the parental strain. Besides the generation of mutants, chromosomal genes can be mobilized by a transducing phage if available. The transducing bacteriophage πm1 was used to transfer chromosomal markers in Erwinia carotovora subsp. atroseptica (Toth et al., 1997). Because of their economic importance, a number of genes involved in pathogenicity and virulence have been identified in Erwinia generic group members. The general approach has been to generate mutants affected in a given phenotype, followed by the isolation of the gene or genes responsible for the phenotype, and characterization of the product or products made by the gene or genes of interest. Hence, this approach has been used to identify the genes (avirulence genes) involved in eliciting the hypersensitivity response in non-host plants. The hypersensitive response-pathogenicity or hrp genes have been identified in several necrogenic strains of this group of bacteria. Epidemiology The disease cycle of this group of bacteria can be classified according to the way they infect and cause disease. For the necrogenic group of pathogens, there are good examples of insect transmission of the pathogen generally infecting at the early blossom stage of the host plant. For example, the fire blight pathogen Erwinia amylovora is transmitted primarily by the honeybee that becomes contaminated or infested by this bacterium during visits to blossoms and oozing sap of an infected tree. Enterobacter cloacae, the cause of the internal yellowing disease of papaya fruit, is transmitted by the oriental fruit fly (Nishijima et al., 1987). The dissemination of pink disease appears to be by fruit flies, since timely applications of insecticides dramatically reduces the incidence of this disease. Although transmission of necrogenic bacteria is vectored efficiently by insects, cultural practices also facilitate the spread of this group of bacteria. For example, the mechanical harvester transmits Erwinia rubrifaciens (emended as Brenneria rubrifaciens) from diseased walnut trees to healthy trees (Kado and Gardner, 1977). The disease cycle of this group of bacteria originates from diseased material in the form of ooze harboring the inoculum that is picked up either by flying insects or harvesting machinery and transmitted to various portals of entry on the host. Bacteria of the macerative group, represented by members of the genus Pectobacterium, have a disease cycle usually associated with the rhizosphere. Infected plant parts tilled into the soil

6 448 C.I. Kado CHAPTER serve as reservoirs for bacteria belonging to this group. For example, Pectobacterium carotovora subsp. carotovora is known to infect various tuber, corn, and bulbs of vegetable and ornamental crops. Replanting in soils containing the infected plant parts exposes the new seedlings, seed pieces, etc., to resident macerative bacteria like carotovora. Wounds caused by harvesting equipment allow entry of the organism into host tissues where infection is initiated. Microaerophilic and anaerobic conditions created by packing the harvested crop into bins or rail cars are most conducive for soft rot. The commercial processing of vegetables that usually are washed in large baths can become contaminated by the bath water used to wash a diseased tuber, root, etc. Dressing water often becomes contaminated with these bacteria and any resulting wounds made during the vegetable washing process are usually sites for postharvest infection that is usually seen at the produce processing station in supermarkets. Members of the vascular wilt group infect their host plant systemically. It is thought that because organisms can multiply quickly and elaborate exopolysaccharide materials in the vascular system, they cause occlusion of the vessel elements culminating in dieback and wilting symptoms. The disease cycle of this group of pathogens, typified by Pantoea stewartii subsp. stewartii, includes the transmission of the bacterium by an insect vector. In the case of P. stewartii subsp. stewartii, the corn flea beetle (Chaetocnema publicaria) disseminates the pathogenic bacterium in cornfields. Erwinia tracheiphila, which causes bacterial wilt of cucumber and related bacteria, is spread via the striped cucumber beetle (Acalymma vittata) and the spotted cucumber beetle (Diabrotica undecimpunctata). Interestingly, these bacteria overwinter in the insect. The tumorigenic group is best represented by Pantoea agglomerans pv. gypsophilae, which induces tumors or galls on Gypsophilum (Baby s Breath). The source of inoculum is primarily from diseased plants bearing galls or nodules. Infected plant stocks, usually originating from a nursery that does not follow sanitation practices, spreads this organism. Disease Members of the genus Erwinia, Enterobacter, Pantoea, Pectobacterium, or Brenneria are associated with plants, and many are pathogens of specific plant species. For example, Erwinia amylovora favors plants of the Rosaceae family (apple, pears, quince), whereas Pantoea stewartii subsp. stewartii infects plants of the maize family specifically Zea mays L.(sweet corn); and Brenneria rubrifaciens infects specifically walnut trees of the species Juglans regia,with J. regia cv. Hartley being the most susceptible commercial cultivated variety. Hence, pathogens of these five genera display host specificity, even though species of the genus Pectobacterium infect a relatively wide range of tuber-, corn- and bulb-crops. Host specificity is conferred by specific residence-establishing genes, defined herein as res genes and is assisted by the products of the hrp genes (for hypersensitive response pathogenicity). Res genes play an important role in maintaining ecological fitness (either in the rhizosphere or phyllosphere) of the pathogen. The importance of res genes in conjunction with hrp (or hrp-equivalent genes) genes is exemplified in other bacterial systems such as with Agrobacterium tumefaciens, where an isoflavonoidinducible efflux pump is essential for competitive colonization of roots (Palumbo et al., 1998); with Pseudomonas syringae pv. syringae, where certain hrp genes (hrpj and hrpc) are needed for epiphytic growth (Hirano et al., 1999; Yu et al., 1999); and with Xanthomonas campestris pv. campestris, where the hrpxc regulatory gene was found essential for colonizing cauliflower plants (Kamoun and Kado, 1990) and is highly conserved among all Xanthomonas pathovars (Oku et al., 1998). The combined activities of the res gene and hrp genes, which are involved in the secretion and injection of pathogenicity factors via a Type III secretion system into the cells of the host plant, are essential for establishing epiphytic fitness. The functional role of the res and hrp genes are not mutually exclusive because the effectors, which are encoded by the res and hrp genes, require the Type III secretion system, which is also encoded by hrp genes and injected into the host cell by the bacterial pathogen. The Type III secretion system is encoded by a cluster of genes contained within a pathogenicity island. More than one Type III secretion system may exist in a pathogen, with each being encoded by separate pathogenicity islands. For example, two distinct virulence-associated Type III secretion systems are encoded within pathogenicity islands, SPI1 and SPI2, of Salmonella spp. (Hensel et al., 1997; Galán and Collmer, 1999). The Type III secretory pathway involves approximately 20 proteins that carry exoproteins directly from the cytoplasm to the cell surface, from where they are then injected into an animal or plant cell upon contact (depending whether the member of the Enterobacteriaceae is a mammalian or plant pathogen). The Type III secretion system and the effector proteins are highly conserved and are related to those in other members of the Enterobacteriaceae such as Yersinia, Salmonella, and Shigella

7 CHAPTER Erwinia and Related Genera 449 (Galán and Collmer, 1999). The conservation of the Type III secretion apparatus and most of the effector proteins reflect the conservation of the genes of the pathogenicity island that was likely acquired by horizontal gene transfer. This is supported by the presence of homologs of genes in the hrp pathogenicity island in Erwinia amylovora to those in Ralstonia solanacearum, Xanthomonas campestris pv. campestris, Pseudomonas syringae pv. syringae, Yersinia enterocolitica, Shigella flexneri, and Salmonella typhimurium (Bogdanove et al., 1996). Thus, Type III secretion systems are functionally conserved as well. For example, the Type III secretion system of X. campestris pv. vesicatoria is able to secrete PopA of the Hrp system in Ralstonia solanacearum; AvirB of Pseudomonas syringae pv. glycinea, and the cytotoxin YopE of Yersinia pseudotuberculosis (Rossier et al., 1999). The overall role of the Type III secretion system is to deliver virulence and pathogenicity effectors that establish and maintain infection and cause disease. Loss of this delivery system s function results in an avirulent phenotype and the inability to compete effectively in the infection court, defined here as the site of infection whether it be on the roots or foliage of the plant. Pathogenicity of the Erwinia group of pathogens, therefore, is highly dependent on the Type III secretion system. Because the genes of the Type III system are highly conserved, these pathogens are likely to have originated through gene acquisition via a horizontal gene transfer system. Literature Cited Abe, K., S. Watabe, Y. Emori, M. Watanabe, and S. Arai An ice nucleation active gene of Erwinia ananas. Sequence similarity to those of Pseudomonas species and regions required for ice nucleation activity. FEBS Lett. 258: Bogdanove, A. J., Z.-M. Wei, L. Zhao, and S. V. Beer Erwinia amylovora secretes harpin via a type III pathway and contains a homolog of yopn of Yersinia spp. J. Bacteriol. 178: Brenner, D. J., G. R. Fanning, and A. G. Steigerwalt Deoxyribonucleic acid relatedness among Erwiniae and other Enterobacteriaceae: the gall, wilt and dry-necrosis organism (Genus Erwinia Winslow et al. sensu stricto). Intl. J. Syst. Bacteriol. 24: Cha, J-S., C. Pujol, A. R. Ducusin, E. A. Macion, C. H. Hubbard, and C. I. Kado Studies on Pantoea citrea, the causal agent of pink disease of pineapple. J. Phytopath. 145: Cha, J.-S., C. Pujol, and C. I. Kado Identification and characterization of a Pantoea citrea gene encoding glucose dehydrogenase that is essential for causing pink disease of pineapple. Appl. Environ. Microbiol. 63: Feistner, G., H. Korth, H. Ko, G. Pulverer, and H. Budzikiewicz Ferrorosamine A from Erwinia rhapontici. Curr. Microbiol. 8: Feistner, G., H. Korth, H. Budzikiewicz, and G. Pulverer Rubrifacine from Erwinia rubrifaciens. Curr. Microbiol. 10: Galán, J., and A. Collmer Type III secretion machines: bacterial devices for protein delivery into host cells. Science 284: Gavini, F., J. Mergaert, A. Beji, C. Mielcarek, D. Izard, K. Kersters, and J. DeLey Transfer of Enterobacter agglomerans (Beijerinck 1888) Ewing and Fife 1972 to Pantoea gen. Nov. as Pantoea agglomerans comb. Nov. and description of Pantoea dispersa sp. nov. Intl. J. Syst. Bacteriol. 39: Hauben, L., E. R. B. Moore, L. Vauterin, M. Steenackers, J. Mergaert, L. Verdonck, and J. Swings Phylogenetic position of phytopathogens within the Enterobacteriaceae. System. Appl. Microbiol. 21: Hensel, M., J. E. Shea, A. J. Bäumler, C. Gleeson, F. Blattner, and D. W. Holden Analysis of the boundaries of Salmonella pathogenicity island 2 and the corresponding chromosomal region of Escherichia coli K-12. J. Bacteriol. 179: Hirano, S. S., A. O. Charkowski, A. Collmer, D. K. Willis, and C. D. Upper Role of the Hrp type III protein secretion system in growth of Pseudomonas syringae pv. syringae B728a on host plants in the field. Proc. Natl. Acad. Sci. USA 96: Kado, C. I., and J. M. Gardner Transmission of deep bark canker of walnuts by the mechanical harvester. Plant Dis. Reptr. 61: Kageyama, B., M. Nakae, S. Yagi, and T. Sonoyama Pantoea punctata sp. nov., Pantoea citrea sp. nov., and Pantoea terrea sp. nov. isolated from fruit and soil samples. Intl. J. Syst. Bacteriol. 42: Kamoun, S., and C. I. Kado A plant-inducible gene of Xanthomonas campestris pv. campestris encodes an exocellular component required for growth in the host and hypersensitivity on nonhosts. J. Bacteriol. 172: Kwon, S. W., S. J. go, H.W. Kang, J. C. Ryu, and J. K. Jo Phylogenetic analysis of Erwinia species based on 16S rrna gene sequences. Int. J. Syst. Bacteriol. 47: Mergaert, J., L. Verdonck, and K. Kersters Transfer of Erwinia ananas (synonym, Erwinia uredovora) and Erwinia stewartii to the genus Pantoea emend. As Pantoea ananas (Serrano 1928) comb. Nov. and Pantoea stewartii (Smithe 1898) comb. nov., respectively, and description of Pantoea stewartii subsp. indologenes subsp. nov. Intl. J. Syst. Bacteriol. 43: Nishijima, K. A., H. M. Couey, and A. M. Alvarez Internal yellowing, a bacterial disease of papaya fruits caused by Enterobacter cloacae. Plant Dis. 71: Oku, T., Y. Wakasaki, N. Adachi, C. I. Kado, K. Tsuchiya, and T. Hibi Pathogenicity, non-host hypersensitivity and host defence non-permissibility regulatory gene hrpx is highly conserved in Xanthomonas pathovars. J. Phytopathol. 146: Palumbo, J. D., C. I. Kado, and D. A. Phillips An isoflavonoid-inducible efflux pump in Agrobacterium tumefaciens is involved in competitive colonization of roots. J. Bacteriol. 180:

8 450 C.I. Kado CHAPTER Pujol, C. J., and C. I. Kado Characterization of pucd5000 involved in pink disease color formation by Pantoea citrea. Plasmid 40: Pujol, C. J., and C. I. Kado gdhb, a gene encoding a second quinoprotein glucose dehydrogenase in Pantoea citrea, is required for pink disease of pineapple. Microbiology 145: Rossier, O., K. Wengelnik, K. Hahn, and U. Bonas The Xanthomonas Hrp type III system secretes proteins from plant and mammalian bacterial pathogens. Proc. Natl. Acad. Sci. USA 96: Toth, I. K., V. Mulholland, V. Cooper, S. Bentley, Y.-L. Shih., M. C. M. Perombelon, and G. P. C. Salmond Generalized transduction in the potato blackleg pathogen Erwinia carotovora subsp. atroseptica by bacteriophage πm1. Microbiology 143: Wells, J. M., W.-S. Sheng, M. J. Ceponis, and T. A. Chen Isolation and characterization of strains of Erwinia ananas from honeydew melons. Phytopathol. 77: Yu, J., A. Panaloza-Vazquez, A. M. Chakrabarty, and C. L. Bender Involvement of the exopolysaccharide alginate in the virulence and epiphytic fitness of Pseudomonas syringae pv. syringae. Mol. Microbiol. 33: