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Group 5 Facultatively Anaerobic Gram-Negative Rods

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Group 5 Facultatively Anaerobic Gram-Negative Rods

    The Erwinia revolution 1

    Group 5 Facultatively Anaerobic Gram-Negative Rods

    The Erwinia revolution

Alphaproteobacteria Rhizobiales Rhizobiaceae (Agrobacterium)

    Sphingomonadales Sphingomonadaceae

    (Rhizomonas)

Betaproteobacteria Burkholderiales Burkholderiaceae (Burkholderia,

    Ralstonia)

     Comamonadaceae (Acidovorax)

     Oxalobacteraceae (Herbaspirillum)

     Genera incertae sedis - (Xylophilus)

    Gammaproteobacteria Pseudomonadales - Pseudomonadaceae

    (Pseudomonas, Rhizobacter)

    Xanthomonadales Xanthomonadaceae

    (Xanthomonas, Xylella)

    Enterobacteriales - Enterobacteriaceae

    (Erwinia, Brenneria,

    Pectobacterium, Pantoea)

Enterobacteriales Gram-negative straight rods. Motile by peritrichous

    flagella or nonmotile. Do not form endospores. Grow in presence or absence

    fo oxygen.

Type genus - Escherichia

    Much of information in this section extracted from:

S. H. De Boer, D. L. Coplin, and A. L. Jones. Erwinia and Pantoea. in: Laboratory rdguide for identification of plant pathogenic bacteria, 3 edition.

Erwinia, Brenneria, Pantoea, and Pectobacterium

Diseases caused by this group

     Soft rot

     Necrotic diseases

    The Erwinia revolution 2

     Wilt diseases

     Galls

     The taxonomic position, nomenclature, and interrelationships of the members of the genus Erwinia have been the subject of diverse proposals. The broadest

    classification is that of Dye (1968, 1969a, 1969b, 1969c), which separates Erwinia into

    the pectolytic soft rot carotovora” group, the yellow pigmented “herbicola” group,

    the white nonpectolytic wilt-causing “amylovora” group, and the “atypical” Erwinia.

    These have turned out to be valid clusterings based on DNA-DNA homology studies and 16S sequence homology (Hauben et al, 1998; Kwon 1997), but do not completely agree with purely phenetic groupings (Verdonck et al, 1987). Controversy remains on whether or not the differences are great enough to constitute new genera. On the basis of 16S rRNA sequence homologies, Hauben et al. (1998) recommended that these four groups be split into the genera Pectobacterium emend, Pantoea gen. nov., Erwinia emend., and

    Brenneria gen. nov., respectively. For now the genus will remain intact with the exception of the group being placed in Pantoea.

     The species and subspecies currently included in the pectolytic soft rot or

    carotovora” group have now been placed in Pectobacterium and include:

    Pectobacterium carotovorum subsp. carotovorum, P. carotovorum subsp. atrosepticma, P.

    carotovorum subsp. betavasculorum, P. carotovorum subsp. wasabiae, P. carotovorum

    subsp. odoriferum, P. chrysanthemi, P. cypripedii, P. rhapontici and P. cacticida.

     The species in the “amylovora” group will be considered together with the atypical erwinias and will include E. amylovora, E. persicina, E. pyrifoliae, E.

    mallotivora, E. psidii, and E. tracheiphila.

E. alni, E. nigrifluens, E. paradisiaca, E. quercina, E. rubrifaciens, and E. salicis (24).

    comprises the species included in the genus Brenneria.

It should be noted that G+C ratios do not entirely support the division of the Erwinia

    species according to the presence or absence of pectolytic abilities, since they suggest a closer similarity between E. amylovora and E. chrysanthemi than between E. amylovora

    and the other species of the “amylovora” group (21). Nevertheless, division of the

    Erwinia genus on the basis of phenotypic characteristics is useful for grouping together those species that require similar protocols for their manipulation within the laboratory.

    The “herbicola” group of yellow pigmented strains which consists of epiphytic

    as well as plant pathogenic bacteria, has now been classified as Pantoea together with

    some species of the genus Enterobacter (12, 16, 18). Initially, Ewing and Fife (1972)

    using DNA-DNA homology data, recommended that Enterobacter agglomerans, E.

    herbicola and E. milletiae be place in E. agglomerans. Gavini et al. in 1989

    recommended that E. agglomerans (including the type species of E. herbicola and E.

    milletiae) be transferred to the new genus Pantoea. Later, E. ananas and E. stewartii

    The Erwinia revolution 3

    were placed into Pantoea. The present genus Pantoea includes the phytopathogens P.

    stewartii, P. ananas, P. citrea and P. agglomerans pvs. milletiae, gypsophilae and

    betae.

     Due to the heterogenity of the genus, a general description of Erwinia is

    necessarily limited. As other members of the family Enterobacteriaceae, the erwinia occur

    as straight rods (0.5-1.0 X 1-3 m) singly, in pairs, or sometimes in short chains, are

    Gram negative and are motile by peritrichous flagella. They are facultatively

    anaerobic, chemoorganotrophic, and grow optimally at 23-30?C. All species are

    oxidase negative but catalase positive; nitrates are not reduced by most species. All

    species catabolize glucose and various other carbohydrates with the production of acid but

    usually without gas formation.

    Much of information in this section extracted from:

De Boer, S. H. and A. Kelman.Erwinia soft rot group. in: Laboratory guide for rdidentification of plant pathogenic bacteria, 3 edition.

Pectobacterium

    The species of the genus Pectobacterium comprise a distinct phylogenetic group as determined by 16S rRNA gene sequence comparisons

A. Erwinia Soft Rot Group

    In 1945 Waldee stated that the soft-rot Erwinia species be placed in a separate genus, Pectobacterium although to date this has not been accepted. Other recommendations to place them in a

    separate genus have lacked support (Mergaert et al, 1984). The soft rot or “carotovora” group

    comprises those species which incite soft rot diseases of plants.

    The following designations are used for the “carotovora” group:

    - Pectobacterium carotovorum subsp. carotovorum * Widely distributed and most

    common

    - P. carotovorum subsp. atrosepticum * Primary cause of blackleg of potatoes.

    - P. carotovora subsp. betavasculorum

    - P. carotovorum subsp. wasabiae

    - P. carotovorum subsp. odorifera

    - P. chrysanthemi *

    - P. cypripedii Nonpectolytic

    - P. rhapontici Nonpectolytic

    - P. cacticida

    * Most important species

    DIFFERENTIATION OF COMMONLY ISOLATED SPECIES AND SUBSPECIES

    The soft rot Erwinia, (Pectobacterium) members of the Enterobacteriaceae are facultatively

    anaerobic, peritrichously flagellated, Gram-negative rods. All strains of the five species considered in this chapter are catalase positive, oxidase negative, ferment glucose, reduce nitrate, produce β-

    galactosidase and HS, and utilize L-arabinose, D-galactose, D-glucose, glycerol, D-mannose, D-ribose, 2

    and sucrose, but do not produce urease, or acid from adonitol. Most strains utilize L-rhamnose and D-

    The Erwinia revolution 4

    mannitol but not dextrin. Holt et al. (1994) noted that a meaningful comparison of Erwinia with other

    genera of Enterobacteriaceae has not been made so the true relationships are up in the air.

    For the pectolytic Erwinia (Pectobacterium) presumptive identification can be made simply on

    the basis of pectolytic activity and colony characteristics on CVP (Crystal violet pectate) medium

    observed with oblique illumination. Further identification to genus can be made on the basis of Gram negative stain reaction, rod shape, facultative anaerobic metabolism, and peritrichous flagellation.

    The non-pectolytic species, E. cypripedii and E. rhapontici, require further tests to establish species identity. Hyman et al. (1995) suggest that pectolytic bacteria can be confirmed as Pectobacterium on

    the basis of being facultatively anaerobic, catalase positive and oxidase negative.

    All the pectolytic Pectobacterium species and subspecies can be differentiated from one another on the basis of a limited number of tests (Table 1). Although there are several different ways in which

    many of these tests can be completed, only one procedure is described for each test. The methods have

    been selected as the most convenient and simplest for testing a number of isolates at one time. It is

    essential that positive and negative control strains be included in each test.

Table 1. Biochemical and physiological tests that differentiate pectolytic species and subspecies of the aErwinia soft rot group

    Pcc Pca Pcb Pco Pcw Pch Pct Test

    + - + + - + + Growth at 37C

Reducing sugars from sucrose - + + + - - -

     bPhosphatase activity - - - - - + V

Sensitivity to erythromycin - - - ND - + -

Indole production - - - - - + -

Acid produced from :

     sorbitol - - - + ND - -

     Melibiose + + - + - + -

     Citrate + + - + + + ND

     Raffinose + + - ND - + -

     Arabitol - - - + ND - ND

     Lactose + + + + + + -

Utilization of Keto-methyl glucoside - + + + - - -

    +, 80% or more strains positive; V, between 21-79% of strains positive; -, 80% or more strains negative; ND, not

    determined. a Pcc = P. carotovorum subsp. carotovorum

     Pca = P. carotovorum subsp. atroseptica

     Pcb = P. carotovora subsp. betavasculorum

     Pco = P. carotovorum subsp. odorifera

     Pcw = P. carotovorum subsp. wasabiae

     Pch = P. chrysanthemi

     Pct = P. cacticida b Some strains of P. carotovorum subsp. carotovorum may be weakly positive

    The Erwinia revolution 5

    Serological techniques

    Serological procedures (eg., indirect immunofluorescence and triple antibody sandwich

    ELISA) are useful for preliminary identification of purified cultures or bacteria in plant and soil

    samples if specific antibodies to commonly occurring serogroups are available.

    Lipopolysaccharides (LPSs) are important antigenic determinants of most Gram

    negative bacteria including the pectolytic erwiniae. The usefulness of serology for identification

    of pectolytic Erwinia to species and subspecies is limited. Antibodies to lipopolysaccharides of P.

    carotovorum subsp. carotovorum and atrosepticum are highly specific (De Boer, 1979) the

    existence of many P. carotovorum subsp. carotovorum serogroups makes identification of this

    subspecies by serology impractical. Most P. carotovorum subsp. atrosepticum strains belong to

    serogroup I and several serogroup-specific monoclonal antibodies are useful for identification of

    the subspecies (De Boer, 1985); however, at least three other serogroups occur among P.

    carotovorum subsp. atrosepticum strains, and although they occur infrequently, it limits

    serological identification of all strains in the subspecies.

    Serological techniques using “O” antigens, purified membrane protein complex, or

    fimbrial-specific monoclonal antibodies are very useful for rapid presumptive identification of

    suspected cultures of P. chrysanthemi. Several serological typing schemes of three or four

    serovars or serogroups have been suggested but these have not been reconciled with one another

    or adopted for further studies. Polyclonal and monoclonal antibodies produced to the

    lipopolysaccharide of P. chrysanthemi do react with many strains of the species but their use

    for identification and detection is greatly hampered by their cross-reactivity with a

    lipopolysaccharide epitope of some common soil PSEUDOMONADS (van der Wolf, 1993). A

    monoclonal antibody to a fimbrial epitope of P. chrysanthemi has potential for identification and

    perhaps detection of strains isolated from potato and some other hosts in Europe (48).

    Much of information in this section extracted from:

    Jones, A. L. and K. Geider. Erwinia amylovora Group. in: Laboratory guide for rdidentification of plant pathogenic bacteria, 3 edition. N. W. Schaad, J. B. Jones, and W.

    Chun (eds.) (In Press)

    Amylovora Group

     The non-soft rot group in the genus Erwinia corresponds to the “amylovora”

    group and to a second genus, Brenneria. The species of Erwinia included in this group

    are:

    - E. amylovora Fire blight of apple and pear. Also affects other

    rosaceaous crops

    - E. mallotivora

    - E. paradisicina

    - E. persicina

    - E. psidii

    - E. pyrifoliae

    - E. tracheiphila (synonym: E. amylovora var. tracheiphila (Smith

    1895) Dye 1968). Bacterial wilt of cucumber

    The Erwinia revolution 6

     Although most species in this group are found on different hosts, they occur

    primarily on trees causing various blights, cankers, wilts. E. amylovora, the fire blight pathogen, is not only the type species of the genus but it also the most important

    economically.

Erwinia amylovora

Fire blight restricted to plants belonging to Rosaceae

Burrilll 1882 - United States

North America, part of Central America, Europe, New Zealand

Characteristics

     Weak growth under anaerobic conditions

     Inability to reduce nitrate to nitrite

     Does not produce cell wall degrading enzymes

    The species is serologically homogeneous

Produce two types of EPS

     Levan (polyfructan)

     Amylovoran (acidic exopolysaccharide)

Brenneria Cause diseases on trees

    Type species B. salicis

    - B. alni

    - B. nigrifluens

    - B. quercina

    - B. rubrifacinens

    - B. salicis

Members within this group are Gram-negative plant-associated bacteria with the following

    characteristics: facultative anaerobes, peritrichous flagella, rod shaped, and acid produced from

    fructose, glucose, galactose, and sucrose. All species exhibit motility. Identification to species is based on the tests listed below in Table 1. Only one species, E. persicinus reduces nitrate while the

    other species are nitrate reduction negative. None of the bacteria.l species cause a bacterial soft rot.

    The Erwinia revolution 7

    Table 1. Major diagnostic tests differentiating species in the non-soft rot group of Brenneris and aErIwinia.

     Test B. alni bTobacco hypersensitivity - + + - ND ND - + - - - ND cPectate degradation E. amylovora - - - - + + - - + + + - d Growth factors required - + + - ND ND - + - - + E. ND mallotivora Pink pigment on YDC - - - - ND + - - + - - ND E. nigrifluens Growth at 36;C D + - - + ND + - - + + - -

    B.paradisiaca Growth at 39;C - - - - + - - - - - - - HS from cysteine 2E. persicinus + - - + + + + - + + + + DUrease + - - + - - - - - - - - E.psidii Indole test - - - - + - - - - - - - E. pyrifoliae Nitrate reduction - - - - ND + - - - - - - B. quercina Gelatin liquification - + - - - - - - - - - - eAcid production from: B. rubrifaciens Salicin + - - + + + + + - + - ND B. salicis α-methyl glucoside - - - - + - + + - - ND ND E. Melibiose trachei- phila- - + + ND ND ND ND ND ND ND

     Inositol - - - + ND ND + ND ND - + ND L-Arabinose + v - + + + + + - + - ND a Data taken from Hao et al. (10), Hauben et al. (11), Neto et al. (21), Schroth and Hildebrand (28), Surico et al. (30). b D+, 80% or more strains positive; +, 80% or more strains delayed positive; -, 80% or more strains negative; ND, not determined; v, variable. c Tests were made on Paton;s media (22). Pitting after 3 days represents a positive test. d All positive species required yeast extract to grow in basal media with glucose. E. amylovora requires nicotinic

    acid for good growth in some minimal media. e After 7 days growth at 27;C in unshaken aqueous solution of 1% organic compound and 1% peptone with bromocresol purple as indicator.

Much of information in this section extracted from:

    The Erwinia revolution 8

Coplin, D. L. and C. I. Kado. Erwinia amylovora Group. in: Laboratory guide for identification of plant rdpathogenic bacteria, 3 edition.

Disease cycle

Pantoea

Type species: P. agglomerans

    Comparison of rrs gene (encoding 16S rRNA) sequences showed the genus to constitute a

    monophyletic cluster distinct from clusters corresponding to Erwinia, Pectobacterium, and

    Brenneria

    Herbicola group

    The present genus Pantoea corresponds to the Erwinia herbicola/Enterobacter agglomerans group and it includes the phytopathogens:

    - P. stewartii subsp. stewartii and indologenes

    The Erwinia revolution 9

    - P. ananas

    - P. citrea

    - P. agglomerans pvs. milletiae, gypsophilae and betae.

    Note: Other species in this genus represent non-pathogenic plant epiphytes and related

    soil bacteria, such as P. agglomerans, P. punctata and P. terrea.

The major pathogen in this genus is P. stewartii subsp. stewartii, which incites Stewart’s bacterial

    wilt and leaf blight of corn. P. ananas (synonomous with E. uredovora) causes brown colored

    coalescing spots leading to rots (“marbling”) of pineapple fruits and honeydew melons. It also survives as

    a epiphyte on uredospores of Ustilago smut of maize and the panicles of barley, buckwheat and rice. P.

    citrea causes the pink-disease of pineapple fruits. P. agglomerans comprises a diverse group of common plant epiphytes. The phytopathogenic strains in this species cause abnormal growth; P. agglomerans pv.

    milletiae causes galls on Wisteria spp. and pvs. gypsophilae and betae cause galls on Gypsophilia

    paniculata and beets, respectively.

     Pantoea spp. share many characteristics with other members of the original genus Erwinia. They

    are Gram-negative, rod-shaped, facultatively anaerobic bacteria that are negative for nitrate

    reduction, oxidase negative and catalase positive. Most strains produce pili. Except for P. stewartii and

    P. citrea, they are motile by peritrichous flagella. Acid is produced from arabinose, fructose, galactose, glucose, trehalose and N-acetylglucosamine. Gas is not produced from glucose. They utilize acetate,

    formate, fumarate, gluconate, lactate, malate and succinate, but not a-methyl glucoside, melezitose,

    adonitol, dulcitol, benzoate, oxalate or propionate.

    The group is commonly differentiated from the other erwiniae by:

    - Production of a yellow pigment., (P. citrea and a few P. agglomerans strains

    produce no yellow pigment.)

    - They do not degrade pectate

    - Do not require growth factors

    - Do not produce urease, lysine decarboxylase or ornithine decarboxylase

    - Do not produce HS on Triple Sugar Iron agar. 2

    Pantoea spp. Isolated from plant surfaces, seeds, soil,

    water, as well as animals (opportunistic human pathogen)

Characteristics

     Peritrichous flagella except P. stewartii

     M ost produce water insoluble, yellow water pigment

     Reduce nitrates

     Grows well anaerobically

    Types of Diseases caused by members of this genus

     Crown gall (Pantoea agglomerans (E. herbicola))

    The Erwinia revolution 10

     Wilt P. stewartii (Stewart’s wilt of corn)

     Rot (P. ananas on pineapple)

Ice nucleation-active (INA) bacteria : P. ananas, P. agglomerans, and P.

    stewartii.

Differentiation of three groups of facultative anaerobe,

    Gram-negative rods associated with plants

    PectobacterPantoea Test E. amylovora/

    ium Brenneria Spp.

    Pectate degradation + - -

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