Agrococcus versicolor sp. nov., an actinobacterium associated with the phyllosphere of potato plants

The plant phyllosphere is known to be a typical habitat for several genera of the family Microbacteriaceae. The species of the genera Microbacterium, Leifsonia, Curtobacterium, Clavibacter, Frigoribacterium, Agreia and Plantibacter, in particular, colonize the surfaces of various plant species (Evtushenko & Takeuchi, 2006). In contrast, Agrococcus species have not been isolated from plant surfaces. Strains of this genus have been found in the air, in soil samples of different origins, on a medieval wall painting and on the surfaces of some cheeses (Groth et al., 1996; Wieser et al., 1999; Zlamala et al., 2002; Mayilraj et al., 2006; Bora et al., 2007). Furthermore, the members of the genus are able to thrive under extreme living conditions, such as in permafrost environments (Katayama et al., 2007; Zhang et al., 2007), in extreme alkaline groundwater (Tiago et al., 2004) or in limiting environments (clean rooms; La Duc et al., 2007).

In the context of studying the effects of transgenic fructan-producing potatoes on the diversity of the microbial communities in the phyllosphere, actinobacterial species were found to constitute a significant proportion of the colonizing bacteria. A relatively large number of bacterial taxa belonging to the family Microbacteriaceae were found on an isogenic potato line in comparison with the transgenic lines. Within this group, several strains were isolated that displayed characteristics of the genus Agrococcus. A comparative analysis of the 16S rRNA gene sequences indicated that these strains represent a novel species within this genus. The aim of this study was to describe the novel species on the basis of data collected by using a polyphasic approach.

To study the diversity of the bacterial community, samples from the phyllosphere of the investigated potato lines were processed as described by Heuer & Smalla (1999). Estimation of population densities and screening for bacterial isolates were performed using different culture media. Heterotrophic bacteria were determined on tryptic soy agar (Merck) and nutrient agar (SIFIN) supplemented with cycloheximide (0.4 g l⁻¹) and incubated at 21 °C for 7 days. To determine the abundance of bacterial taxa, some representative colonies were isolated from each medium, purified and then stored at −80 °C in nutrient broth (SIFIN) containing 40 % glycerol until further processing. The isolates were grouped on the basis of morphological and physiological tests, as described previously (Behrendt et al., 1997, 1999). Representative strains selected from groups with uncertain affiliation at the genus level were investigated using partial sequence analysis of the 16S rRNA gene, as described by Ulrich et al. (2008). Strains K 114/01^T and K 35/01 (isolated from nutrient agar) and strain K 42/01 (isolated from tryptic soy agar) were tentatively assigned to the genus Agrococcus and subjected to further taxonomic investigation.

Phylogenetic analysis of a representative strain, K 114/01^T, on the basis of the 16S rRNA gene sequence was performed as described by Behrendt et al. (2003). Phylogenetic trees were based on a 1420 nt alignment (Escherichia coli positions 50–1458) and constructed using the neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, 1981) algorithms (phylip, version 3.6; Felsenstein, 1993). The closest phylogenetic neighbours of strain K 114/01^T, as determined from a pairwise sequence comparison, were the type strains of Microbacterium deminutum and Agrococcus jenensis, with similarities of 97.1 and 96.9 %, respectively. Although the 16S rRNA gene sequence similarity for strain K 114/01^T was slightly greater with respect to the species M. deminutum, it clustered with the established species of the genus Agrococcus (Fig. 1⇓). This phylogenetic position was confirmed by the maximum-likelihood tree and was supported by a bootstrap value that was greater than 50 %. Correspondingly, M. deminutum KV-483^T formed a cluster with species of the genus Microbacterium with both treeing methods (Fig. 1⇓). This position was supported by high bootstrap percentages (86 and 92 %). As reviewed by Stackebrandt et al. (2007), the topology of phylogenetic trees in the family Microbacteriaceae changes depending on the strain set selected for analysis. For this reason, another tree comprising all type strains of the family was constructed in addition to the tree shown in Fig. 1⇓, which is based on the established species of Agrococcus and representative species for the other genera. In the extended tree, the results were similar to those obtained in the tree shown in Fig. 1⇓. Strain K 114/01^T clearly clustered with the species of the genus Agrococcus, and all species of the genus Microbacterium formed a monophyletic branch that clustered next to the genera Okibacterium and Plantibacter (data not shown). These results indicated the affiliation of strain K 114/01^T to the genus Agrococcus. Furthermore, similarity values below 97 % were obtained with respect to the 16S rRNA gene sequences of established species of the genus Agrococcus, suggesting a separate species position for the isolate.

A significant chemotaxonomic characteristic for genus affiliation in the family Microbacteriaceae is the amino acid composition of the cell wall. The amino acids and peptides in cell-wall hydrolysates of the peptidoglycan of strain K 114/01^T were analysed using two-dimensional ascending TLC on cellulose plates with the solvent system described by Schleifer & Kandler (1972). The characteristic cell-wall diamino acid was 2,4-diaminobutyric acid (DAB), which is typical for the genus Agrococcus (Evtushenko & Takeuchi, 2006). In contrast, species of the genus Microbacterium are characterized by the presence of ornithine or lysine as the diagnostic diamino acid (Evtushenko & Takeuchi, 2006). Thus, the cell-wall composition substantiates the affiliation of strain K 114/01^T to the genus Agrococcus. In addition to DAB, the amino acids alanine, glycine, threonine, aspartic acid and glutamic acid were detected. The cell-wall Ala/Gly/Thr/DAB/Asp/Glu ratio was 2.3 : 2.1 : 2.5 : 0.8 : 0.9 : 1.0. This composition is very similar to that of A. jenensis (Groth et al., 1996), its closest phylogenetic neighbour in the genus. The same qualitative amino acid composition was reported for the peptidoglycan of Agrococcus lahaulensis (Mayilraj et al., 2006), while Agrococcus baldri (Zlamala et al., 2002) and Agrococcus citreus (Wieser et al., 1999) lacked threonine and Agrococcus casei (Bora et al., 2007) lacked both threonine and aspartic acid.

Since the phylogenetic analysis and the investigation of the cell-wall composition were only performed for strain K 114/01^T, it was necessary to determine the similarity at the species level for isolates K 35/01 and K 42/01. Recent studies demonstrated the ability of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) to permit the identification of bacteria at the species level in pure culture under carefully controlled culture and analysis conditions (Conway et al., 2001; Fenselau & Demirev, 2001; Valentine et al., 2005). Furthermore, spectra from MALDI-TOF MS reflect the phylogenetic relationship of micro-organisms, corresponding with the results of sequence analyses of conservative genes (Stackebrandt et al., 2005). MALDI-TOF MS analysis was performed according to the method described by Tóth et al. (2008). As shown in Fig. 2⇓, the isolates formed a tight cluster that was separable on the basis of clear spectral dissimilarities with respect to all type strains of the genus Agrococcus. The high level of spectral similarity between the phyllosphere isolates indicated their affiliation to the same species.

The phyllosphere isolates were also subjected to a multitude of phenotypic tests to determine their similarities. Standard tests for morphological and physiological characterization were performed as described by Behrendt et al. (1999). All strains were cultivated on nutrient agar or in the respective broth at 21 °C. Production of l-phenylalanine deaminase was investigated according to the method described by Richard & Kiredjian (1995). Hydrogen sulphide production was tested on Kligler iron agar (Oxoid) and the hydrolysis of l-tyrosine was determined on nutrient agar supplemented with 0.5 % (w/v) l-tyrosine. Oxidation of carbon sources was tested using Biolog GP2 microplates. The instructions of the manufacturer were modified in that the strains were cultivated on tryptic soy agar (Mayilraj et al., 2006). Results were read visually after 24 and 48 h incubation at 30 °C. Oxidative acid production from carbohydrates was investigated using API 50 CH strips (bioMérieux) inoculated with a bacterial suspension in CHB/E medium (bioMérieux). The results were not read until after 72 h, as the strains tested showed delayed reactions. API 20NE (bioMérieux) test strips were applied to determine additional physiological and biochemical features. The phyllosphere isolates displayed high levels of conformity with regard to phenotypic features. On the GP2 microplates, in particular, identical reactions were obtained for all 95 carbohydrates. Furthermore, the API 50 CH results for acid production were very similar. Different reactions were found only for oxidative acid production from d-mannitol, N-acetylglucosamine, cellobiose and maltose. Whilst strain K 114/01^T did not show a reaction for these carbohydrates, acid production for strains K 35/01 and K 42/01 was weak for d-mannitol and N-acetylglucosamine but positive for cellobiose and maltose. These results supported those of the MALDI-TOF MS analysis, i.e. that all three isolates represented one species. Details of the morphological and physiological features of the strains are given in the species description. Characteristics that serve to differentiate the phyllosphere isolates from established species of the genus Agrococcus are presented in Table 1⇓. The isolates were clearly distinguishable from these Agrococcus species on the basis of their ability to use several carbohydrates on the GP2 microplates.

A further prominent taxonomic feature that characterized the isolates and served to differentiate them from related species was their whole-cell fatty acid profiles. Fatty acid methyl esters were extracted from cells cultivated on tryptic soy agar for 1 day at 28 °C and were then analysed with the Sherlock Microbial Identification system (MIDI, version 4.5) as described by Behrendt et al. (1999). As shown in Table 2⇓, the cellular fatty acid profiles of the potato isolates were relatively similar, supporting the suggestion that they represent one species. The major fatty acids were anteiso-C_15 : 0 (39.8±3.3 %), iso-C_15 : 0 (17.1±1.6 %), C_16 : 0 (13.9±1.1 %) and anteiso-C_17 : 1 (7.6±4.3 %) and minor fatty acids were anteiso-C_17 : 0 (5.7±0.6 %), iso-C_16 : 0 (5.6±1.2 %), C_14 : 0 (3.0±0.2 %), iso-C_17 : 0 (1.9±0.4 %) and iso-C_14 : 0 (1.6±0.8 %). Fatty acids typical of the genus, i.e. anteiso-C_15 : 0, iso-C_15 : 0, anteiso-C_17 : 1 and iso-C_16 : 0, were present in the isolates. Nevertheless, significant quantitative and qualitative differences were found in comparison with Agrococcus species. Notable differences were demonstrated in the quantities of the fatty acids C_16 : 0, iso-C_15 : 0 and anteiso-C_17 : 0 (Zlamala et al., 2002; Mayilraj et al., 2006; Bora et al., 2007). The fatty acid anteiso-C_17 : 1 was found only in the potato isolates, whereas C_14 : 0 occurred also in the species A. jenensis, but only in trace amounts (Groth et al., 1996). Thus, a clear demarcation between the potato isolates and established Agrococcus species was possible on the basis of fatty acid profiles. The isoprenoid quinone composition was determined as described by Groth et al. (1996). The quinone system of strain K 114/01^T consisted of the menaquinones MK-10, MK-11, MK-8 and MK-9 (ratio of peak areas 16 : 16 : 4 : 2). MK-12, which is typical of all established species of the genus Agrococcus, could not be detected. MK-10 was found as an additional major component (as in A. lahaulensis; Mayilraj et al., 2006).

In summary, the results of the phenotypic and phylogenetic analyses demonstrated that the isolates of the potato phyllosphere represent a novel species of the genus Agrococcus, for which the name Agrococcus versicolor sp. nov. is proposed.