Reference Work Entry

The Prokaryotes

pp 747-750

The Genera Phyllobacterium and Ochrobactrum

  • Jean Swings
  • , Bart Lambert
  • , Karel Kersters
  • , Barry Holmes

Abstract

The study of leaf nodulation in several tropical plant families dates back to the beginning of the 20th century. Various genera of bacteria, e.g., Phyllobacterium, were thought to be associated with leaf nodules (see below). Phyllobacterium was described by Knösel (1962) but the genus only found wide taxonomic recognition with the publication of Bergey’s Manual of Systematic Bacteriology (Knösel, 1984). DNA-rRNA hybridizations revealed a close relationship between Phyllobacterium and bacteria from the Centers for Disease Control (CDC) group Vd within the “alpha” subclass (rRNA superfamily IV) of the proteobacteria together with those throughout of the genera Brucella, Rhizobium, Mycoplana, and Agrobacterium (De Ley et al., 1987; Stackebrandt et al., 1988). Holmes et al. 1988 proposed the name Ochrobactrum anthropi for CDC group Vd. They also demonstrated that Phyllobacterium showed closest phenotypic similarity to Ochrobactrum anthropi. In the present chapter, the genera Phyllobacterium and Ochrobactrum are treated together because of their close phylogenetic relatedness. Nevertheless, the ecological niches from which these bacteria have been isolated are quite different: Phyllobacterium strains occur in leaf nodules and in the rhizosphere, whereas Ochrobactrum strains have been isolated almost exclusively from human clinical material. In clinical microbiology, Ochrobactrum, more recently described as Achromobacter group Vd until formally named, was usually treated together with Achromobacter xylosoxidans (more recently known as Alcaligenes xylosoxidans subsp. xylosoxidans) (Gilardi, 1978; Rubin et al., 1985). It has now become apparent that Ochrobactrum and Phyllobacterium are not related phylogenetically to Achromobacter xylosoxidans, which belongs to the Alcaligenaceae in the “beta” subclass (rRNA superfamily III) of the proteobacteria (De Ley et al., 1986).

Abstract

The study of leaf nodulation in several tropical plant families dates back to the beginning of the 20th century. Various genera of bacteria, e.g., Phyllobacterium, were thought to be associated with leaf nodules (see below). Phyllobacterium was described by Knösel (1962) but the genus only found wide taxonomic recognition with the publication of Bergey’s Manual of Systematic Bacteriology (Knösel, 1984). DNA-rRNA hybridizations revealed a close relationship between Phyllobacterium and bacteria from the Centers for Disease Control (CDC) group Vd within the “alpha” subclass (rRNA superfamily IV) of the proteobacteria together with those throughout of the genera Brucella, Rhizobium, Mycoplana, and Agrobacterium (De Ley et al., 1987; Stackebrandt et al., 1988). Holmes et al. 1988 proposed the name Ochrobactrum anthropi for CDC group Vd. They also demonstrated that Phyllobacterium showed closest phenotypic similarity to Ochrobactrum anthropi. In the present chapter, the genera Phyllobacterium and Ochrobactrum are treated together because of their close phylogenetic relatedness. Nevertheless, the ecological niches from which these bacteria have been isolated are quite different: Phyllobacterium strains occur in leaf nodules and in the rhizosphere, whereas Ochrobactrum strains have been isolated almost exclusively from human clinical material. In clinical microbiology, Ochrobactrum, more recently described as Achromobacter group Vd until formally named, was usually treated together with Achromobacter xylosoxidans (more recently known as Alcaligenes xylosoxidans subsp. xylosoxidans) (Gilardi, 1978; Rubin et al., 1985). It has now become apparent that Ochrobactrum and Phyllobacterium are not related phylogenetically to Achromobacter xylosoxidans, which belongs to the Alcaligenaceae in the “beta” subclass (rRNA superfamily III) of the proteobacteria (De Ley et al., 1986).

1

Habitats

The Occurrence of Phyllobacterium in Leaf Nodules

Plants of over 400 species of the families Rubiaceae and Myrsinaceae are reported to produce leaf nodules. Short-lived bacterial colo-nies become established intercellularly and die before full leaf expansion (Lersten and Horner, 1976). Bacterial leaf nodules were defined by Horner and Lersten (1972) as:

Internal cavities in the leaf lamina, open to the exterior by way of stomatal pores only in early stages of nodule development. The bacteria inhabiting such nodules are part of a population maintained by the host plant in the vegetative and floral buds and passed to the succeeding generations through the seeds.

This definition rules out all casual bacteria-plant associations.

Despite more than a century of research on leaf nodulation (for a review, see Lersten and Horner, 1976) it is not yet clear which bacteria are involved and how they participate in the nodulation process. In the past, the bacterial symbionts in the nodules were recognized as belonging to the genera Chromobacterium, Xanthomonas, Klebsiella and Phyllobacterium (Horner and Lersten, 1972). Knösel (1962) isolated and described Phyllobacterium and stated that it is able to induce characteristic nodules on leaves (Knösel, 1984), basing his conclusions on earlier investigations by Miehe (1911), von Faber (1912), and de Jongh (1938). However, it is not clear whether the bacteria previously studied by these workers are identical to those described by Knösel. There is no proof for the leaf nodulation capacity of Phyllobacterium.

The Occurrence of Phyllobacterium in the Rhizosphere

Pseudomonas fluorescens and Phyllobacterium were found to be the two most frequently occurring bacteria on root surfaces during a large-scale assessment of the rhizobacterial communities of young sugar beet plants (between the second and 10th leaf stage) (Lambert et al., 1990). These bacteria were found in 198 out of 1,100 plants investigated in Belgium and Spain at densities up to 2 × 108 per gram of root. This was the first record of the occurrence of Phyllobacterium in the rhizosphere. Extensive analyses of the microflora from the rhizoplane of other crop plants have not revealed the presence of Phyllobacterium.

Occurrence of Ochrobactrum (CDC group Vd) in Clinical Samples and the Environment

Ochrobactrum anthropi strains have been isolated predominantly from human blood, urogenital tracts, urine, respiratory tracts, ears, and wounds (Tatum et al., 1974; Holmes et al., 1988). Only a few strains were isolated from feces, spinal fluid, eye, hospital apparatus, or the environment (arsenical cattle-dipping fluid, soil, sewage, and from thin-layer Sephadex plates) (Holmes et al., 1988). The clinical significance of O. anthropi remains largely unknown; strains of this species are considered to be opportunistic pathogens. One strain has been reported in association with a pancreatic abscess (Appelbaum and Campbell, 1980) and another has been associated with a puncture wound leading to osteochondritis of a foot (Barson et al., 1987).

Isolation

Isolation of Phyllobacterium from the Sugar Beet Rhizosphere (Lambert et al., 1990)

The entire root system of a plant is carefully washed to remove adhering soil, then vigorously shaken for 15 min using a flask shaker in a phosphate buffered saline solution containing 0.025% Tween 20. Serial dilutions of the resulting suspensions are plated on 10% trypticase soy broth plus 2% agar (TSBA). After incubation at 28°C for 2 days, Phyllobacterium colonies are beige-colored, large (±5 mm), and convex with an entire edge. They are very mucoid and glistening. Several subcultures are necessary in order to obtain pure isolates. Phyllobacterium readily grows on other nonselective media for aerobic, heterotrophic bacteria (nutrient agar, trypticase soy agar, Luria-Bertani agar, etc.).

Isolation of Phyllobacterium from Leaf Nodules (Knösel, 1984)

Washed leaf pieces containing nodules are macerated by rubbing and placed in saline. After shaking, serial dilutions of the suspension are plated onto carrot juice agar containing yeast extract. After incubation at 28°C, a variety of pigmented and nonpigmented colonies develops. Typical colonies are transferred into liquid carrot juice medium which is then used to inoculate the tests used for identification. The cultures should be observed after 24 to 48 h to confirm the presence of star clusters.

Isolation of Ochrobactrum anthropi from Clinical Material

Samples from human blood, urine and urogenital tract, sputum, wound, throat, stool, rectum, and other material are plated on blood agar or on MacConkey agar (Rubin et al., 1985; Holmes et al., 1988). Two colony types were recognized on blood agar at 35°C after 48 h: 1) an extremely mucoid type, 0.1 to 4 mm in diameter, opaque and gray-white, showing beta-hemolysis; and 2) a pinpoint type, opaque and semiglossy, gray or white, showing beta-hemolysis after prolonged incubation (Chester and Cooper, 1979). On MacConkey agar after continued incubation, colonies from 3 to 5 mm diameter were formed, opaque and pink to purple. Half the strains produced

colonies with such notably gummy consistency that it was difficult to remove them from the agar surface (Chester and Cooper, 1979). On MacConkey agar, Ochrobactrum can be easily overlooked due to the small size of the colonies after overnight incubation (Chester and Cooper, 1979).

Preservation of Cultures

Well-grown cultures may be kept at 4°C for 2 to 3 months on nutrient agar slants in screw-capped bottles. Isolates can be stored freeze-dried or as a suspension in 25% glycerol at −70°C.

Identification

Phyllobacterium and Ochrobactrum are both Gram-negative, strictly aerobic bacteria with a respiratory type of metabolism. They show oxidase and catalase activity and both grow on nutrient agar. Indole is not produced. They oxidize glucose and xylose but not lactose (Clark et al., 1984; G. L. Gilardi, personal communication, Lambert et al., 1990). They do not produce extracellular enzymes for the hydrolysis of Tween 80, DNA, gelatin, starch, or casein (Gilardi, 1978). The API 20NE system will identify them as Achromobacter sp., which is still a “dumping ground” (Kersters and De Ley, 1984) for various aerobic peritrichously flagellated rods. Recent work on Phyllobacterium and Ochrobactrum has led to the differentiation scheme given in Table 1. An extensive description of Phyllobacterium and Ochrobactrum can be found in Lambert et al. (1990) and Holmes et al. (1988), respectively. A comment should be made on the flagellar arrangement of strains of both genera. Phyllobacterium and Ochrobactrum strains both show 1 to 3 polar, subpolar, or lateral flagella (Lambert et al., 1990). According to Holmes et al. 1988 and Gilardi (1978)Ochrobactrum strains are characterized by peritrichous flagella, but other workers have found only polar, subpolar, or lateral flagella but no definitely peritrichous flagella (Clark et al., 1984; Chester and Cooper, 1979).

Table 1.

Features that differentiate Phyllobacterium and Ochrobactrum (CDC group Vd).

Property

Phyllobacterium c

Ochrobactrum d

Utilizationa of:

L-Citrulline

+

Glutarate

+

Erythritol

+

Glycine

+

L-Tryptophan

+

Hydrolysisb of paranitrophenol-α-maltoside

+

Hydrolysisb of paranitrophenol-α-xylopyranoside

+

Using API 50CH and 50AO, 50AA strips (API System, La Balme-les-Grottes, Montalieu-Vercieu, France).

Using API ZYM strips.

15 strains total. From Lambert et al. (1990).

56 strains total. From Holmes et al. (1988).

An unambigous identification of Phyllobacterium or Ochrobactrum is possible by application of carbon assimilation tests (API 50CH, 50AO, or 50AA) or various conventional tests (Tatum et al., 1974; Rubin et al., 1985; Holmes et al., 1988; Lambert et al., 1990) or by protein electrophoretic fingerprinting. Variability in Gram reaction, the presence of curved cells with swollen-ends, and the formation of extremely small colonies after overnight incubation, which develop into mucoid colonies upon continued incubation, may lead to incorrect identification as Corynebacterium or Klebsiella (Chester and Cooper, 1979).

Knösel (1984) used the criterium of nitrate reduction to differentiate Phyllobacterium myrsinacearum (+) from P. rubiacearum (−). Lambert et al. (1990), however, found that P. rubiacearum LMG 1t1T reduced nitrates. Using the API 2ONE system, they found that all Phyllobacterium strains showed a slight NO3 reduction. These observations cast serious doubt on the usefulness of this criterion for species differentiation within the genus Phyllobacterium. All Ochrobactrum strains are capable of nitrate reduction (Gilardi, 1978; Clark et al., 1984; Holmes et al., 1988).

Physiological Properties

Cellular fatty acids of Ochrobactrum anthropi (Dees and Moss, 1978) as well as antimicrobial susceptibilities have been determined (von Graevenitz, 1985; Gilardi, 1989). Growth of Phyllobacterium strains was inhibited by doxycycline, novobiocin, framycetin, and tetracycline (Lambert et al., 1990). The most intriguing question that remains unanswered is whether Phyllobacterium has a real symbiotic cyclical relationship with its hosts. Reinfection studies did not allow unambiguous conclusions to be made. It has also been suggested that leaf nodule bacteria produce plant growth hormones, particularly cytokinins, which are necessary for the normal functioning of the plant (Fletcher and Rhodes-Roberts, 1976; reference is not an exact match Rodrigues-Pereira et al., 1972). Other authors claimed that leaf nodule bacteria can fix nitrogen, but this was contested by Van Hove (1976) and Lersten and Horner (1976). The lack of ability to fix nitrogen is also indicated by the absence of the nif HDK-like genes (Lambert et al., 1990), which occur in all the classical nitrogen-fixing bacteria.

Phyllobacterium is able to interact with plant tissues, as demonstrated by the tumor induction by Ti-plasmid-carrying Phyllobacterium strains on Kalanchoe plants (Lambert et al., 1990). Tumor induction involves the attachment of bacterial cell wall sites to plant cell wall sites prior to the induction of T-DNA transfer. The chromosomal genes chvA, chvB, exoC, and att are the only ones found to be involved in attachment but could not be detected in Phyllobacterium strains, suggesting that other genes are present with similar functions. There are no data indicating that Phyllobacterium is pathogenic or deleterious to plants.

A striking feature, common to both Phyllobacterium and Ochrobactrum, is nutritional versatility, which makes their rapid growth and proliferation possible in rich environments such as the root surface and clinical sites.

A number of Phyllobacterium isolates showed antifungal and antibacterial activities (Lambert et al., 1990).

The oxidation of lactose to 3-ketolactose is a unique feature of many Agrobacterium strains (Kersters and De Ley, 1984) and is negative for all Phyllobacterium and Ochrobactrum strains.

Applications

No applications of the genera Phyllobacterium or Ochrobactrum are yet known. However, the fact that Phyllobacterium is a predominant bacterium on the root surface of sugar beet plants, its capacity to “communicate” with plant tissues, and its non-pathogenic status make this bacterium an interesting new candidate for use in plant growth promotion or biological control of soil-borne diseases. Indeed, some of the sugar beet isolates exert a broad-spectrum antifungal activity against major phytopathogenic fungi.

Footnotes
1

This chapter was taken unchanged from the second edition.

 

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