Small RNA Deep-Sequencing Analyses Reveal a New Regulator of Virulence in Agrobacterium fabrum C58.

Novel ways of regulating Ti plasmid functions were investigated by studying small RNAs (sRNAs) that are known to act as posttranscriptional regulators in plant pathogenic bacteria. sRNA-seq analyses of Agrobacterium fabrum C58 allowed us to identify 1,108 small transcripts expressed in several growth conditions that could be sRNAs. A quarter of them were confirmed by bioinformatics or by biological experiments. Antisense RNAs represent 24% of the candidates and they are over-represented on the pTi (with 62% of pTi sRNAs), suggesting differences in the regulatory mechanisms between the essential and accessory replicons. Moreover, a large number of these pTi antisense RNAs are transcribed opposite to those genes involved in virulence. Others are 5'- and 3'-untranslated region RNAs and trans-encoded RNAs. We have validated, by rapid amplification of cDNA ends polymerase chain reaction, the transcription of 14 trans-encoded RNAs, among which RNA1111 is expressed from the pTiC58. Its deletion decreased the aggressiveness of A. fabrum C58 on tomatoes, tobaccos, and kalanchoe, suggesting that this sRNA activates virulence. The identification of its putative target mRNAs (6b gene, virC2, virD3, and traA) suggests that this sRNA may coordinate two of the major pTi functions, the infection of plants and its dissemination among bacteria.

Rapid and accurate species and genomic species identification and exhaustive population diversity assessment of Agrobacterium spp. using recA-based PCR.

Agrobacteria are common soil bacteria that interact with plants as commensals, plant growth promoting rhizobacteria or alternatively as pathogens. Indigenous agrobacterial populations are composites, generally with several species and/or genomic species and several strains per species. We thus developed a recA-based PCR approach to accurately identify and specifically detect agrobacteria at various taxonomic levels. Specific primers were designed for all species and/or genomic species of Agrobacterium presently known, including 11 genomic species of the Agrobacterium tumefaciens complex (G1-G9, G13 and G14, among which only G2, G4, G8 and G14 still received a Latin epithet: pusense, radiobacter, fabrum and nepotum, respectively), A. larrymoorei, A. rubi, R. skierniewicense, A. sp. 1650, and A. vitis, and for the close relative Allorhizobium undicola. Specific primers were also designed for superior taxa, Agrobacterium spp. and Rhizobiaceace. Primer specificities were assessed with target and non-target pure culture DNAs as well as with DNAs extracted from composite agrobacterial communities. In addition, we showed that the amplicon cloning-sequencing approach used with Agrobacterium-specific or Rhizobiaceae-specific primers is a way to assess the agrobacterial diversity of an indigenous agrobacterial population. Hence, the agrobacterium-specific primers designed in the present study enabled the first accurate and rapid identification of all species and/or genomic species of Agrobacterium, as well as their direct detection in environmental samples.

Genomospecies identification and phylogenomic relevance of AFLP analysis of isolated and non-isolated strains of Frankia spp.

Amplified fragment length polymorphism (AFLP) was tested as an alternative to the DNA-DNA hybridization technique (DDH) to delineate genomospecies and the phylogenetic structure within the genus Frankia. Forty Frankia strains, including representatives of seven DDH genomospecies, were typed in order to infer current genome mispairing (CGM) and evolutionary genomic distance (EGD). The constructed phylogeny revealed the presence of three main clusters corresponding to the previously identified host-infecting groups. In all instances, strains previously assigned to the same genomospecies were grouped in coherent clusters. A highly significant correlation was found between DDH values and CGM computed from AFLP data. The species definition threshold was found to range from 0.071 to 0.098 mismatches per site, according to host-infecting groups, presumably as a result of large genome size differences. Genomic distances allowed new Frankia strains to be assigned to nine genomospecies previously determined by DDH. The applicability of AFLP for the characterization of uncultured endophytic strains was tested on experimentally inoculated plants and then applied to Alnus incana and A. viridis field nodules hosting culture refractory spore-positive (Sp+, that sporulate in planta) strains. Only 1.3% of all AFLP fragments were shown to be generated by the contaminant plant DNA and did not interfere with accurate genomospecies identification of strains. When applied to field nodules, the procedure revealed that Alnus Sp+ strains were bona fide members of the Alnus-Myrica host infecting group. They displayed significant genomic divergence from genomospecies G1 of Alnus infecting strains (i.e. Frankia alni) and thus may belong to another subspecies or genomospecies.

Optimization and biochemical characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. strains.

AIMS: The identification of a new compound active against Agrobacterium tumefaciens. METHODS AND RESULTS: The culture conditions of a newly isolated Bacillus subtilis strain, designed 14B, were optimized, as a first step, to produce its bacteriocin (termed Bac 14B) for the biocontrol of Agrobacterium spp., the causal agents of the crown gall disease. Bac 14B was then partially purified and biochemically characterized. Bacillus subtilis 14B was observed to produce an antibacterial compound having a protinaceous nature. As estimated by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE), the semi-purified bacteriocin substance was found to be a monomeric protein with a molecular weight of 21 kDa. While the latter's antimicrobial activity was completely stable during exposure to a temperature range of up to 100 degrees C for 2 h, its initial activity was totally lost at 121 degrees C for 20 min. The maximum bacteriocin production (4096 AU ml(-1)) was recorded after 96 h-incubation in an optimized Luria Bertani medium supplemented with 10 g l(-1) glucose, 15 g l(-1) K(2)HPO(4) and 5 g l(-1) MgSO(4) 7H(2)O at 30 degrees C in a shaking flask culture. Interestingly, the B. subtilis 14B culture supernatant that contained the bacteriocin under study was proved efficient in reducing both the percentage of galled plants and the number of galls in tomato. CONCLUSION: The findings revealed that B. subtilis 14B and its bacteriocin are efficient in reducing the percentage of infections in plants caused by Ag. tumefaciens. SIGNIFICANCE AND IMPACT OF THE STUDY: The results could be useful for the nurserymen who are particularly interested in the biocontrol of the crown gall disease.

Polyphasic characterization of xanthomonads pathogenic to members of the Anacardiaceae and their relatedness to species of Xanthomonas.

We have used amplified fragment length polymorphism (AFLP), multilocus sequence analysis (MLSA) and DNA-DNA hybridization for genotypic classification of Xanthomonas pathovars associated with the plant family Anacardiaceae. AFLP and MLSA results showed congruent phylogenetic relationships of the pathovar mangiferaeindicae (responsible for mango bacterial canker) with strains of Xanthomonas axonopodis subgroup 9.5. This subgroup includes X. axonopodis pv. citri (synonym Xanthomonas citri). Similarly, the pathovar anacardii, which causes cashew bacterial spot in Brazil, was included in X. axonopodis subgroup 9.6 (synonym Xanthomonas fuscans). Based on the thermal stability of DNA reassociation, consistent with the AFLP and MLSA data, the two pathovars share a level of similarity consistent with their being members of the same species. The recent proposal to elevate X. axonopodis pv. citri to species level as X. citri is supported by our data. Therefore, the causal agents of mango bacterial canker and cashew bacterial spot should be classified as pathovars of X. citri, namely X. citri pv. mangiferaeindicae (pathotype strain CFBP 1716) and X. citri pv. anacardii (pathotype strain CFBP 2913), respectively. Xanthomonas fuscans should be considered to be a later heterotypic synonym of Xanthomonas citri.

Engineering root exudation of Lotus toward the production of two novel carbon compounds leads to the selection of distinct microbial populations in the rhizosphere.

The culture of opine-producing transgenic Lotus plants induces the increase in the rhizosphere of bacterial communities that are able to utilize these molecules as sole carbon source. We used transgenic Lotus plants producing two opines, namely mannopine and nopaline, to characterize the microbial communities directly influenced by the modification of root exudation. We showed that opine-utilizers represent a large community in the rhizosphere of opine-producing transgenic Lotus. This community is composed of at least 12 different bacterial species, one third of which are able to utilize the opine mannopine and two thirds the opine nopaline. Opine utilizers are diverse, belonging to the Gram-positive and -negative bacteria. We described two novel mannopine-utilizing species, Rhizobium and Duganella spp., and five novel nopaline-utilizing species, Duganella, Afipia, Phyllobacterium, Arthrobacter, and Bosea spp. Although opine utilizers mostly belong to the alpha-Proteobacteria, Rhizobiaceae family, there is little overlap between the populations able to utilize each of the two opines produced by the plants. Noticeably, in the rhizosphere of transgenic Lotus, only the opine mannopine favors the growth of Agrobacterium tumefaciens, the bacterium from which opines have been characterized. The diversity of opine utilizers from the rhizosphere of Lotus plants is greater than that observed from any other environment. Therefore, transgenic plants with engineered exudation constitute an excellent tool to isolate and characterize specific microbial populations.

A highly selectable and highly transferable Ti plasmid to study conjugal host range and Ti plasmid dissemination in complex ecosystems.

A conjugal donor system, ST2, was constructed to study the conjugal dissemination of a Ti plasmid to wild-type recipient bacteria in vitro and in situ. The system consisted of a polyauxotrophic derivative of C58 harboring a hyperconjugative and highly selectable Ti plasmid, pSTiEGK, which was constructed by inserting a multiple antibiotic resistance cassette in the traM- mcpA region of pTiC58Delta accR. ST2 transfers pSTiEGK constitutively at frequencies up to 10(-1) to plasmidless Agrobacterium recipients. The host range of pSTiEGK includes all the known genomic species of Agrobacterium, indigenous soil agrobacteria and some Rhizobium and Phyllobacterium spp. All transconjugants became pathogenic upon acquisition of the Ti plasmid and were also able to transfer pSTiEGK by conjugation. This host range was indistinguishable from that of its wild-type parent pTiC58, and therefore pSTiEGK constitute a valid proxy to study the dissemination of Ti plasmids directly in the environment. Transconjugants can be selected on a combination of four antibiotics, which efficiently prevents the growth of the indigenous microbiota present in complex environments. The transfer of pSTiEGK to members of the genus Agrobacterium was affected primarily by the plasmid content of the recipient strain (10(3)- to 10(5)-fold reduction), e.g., the presence of incompatible plasmids. As a consequence, a species should be considered permissive to Ti transfer whenever one permissive isolate is found.

Relationship between spatial and genetic distance in Agrobacterium spp. in 1 cubic centimeter of soil.

The spatial and genetic unit of bacterial population structure is the clone. Surprisingly, very little is known about the spread of a clone (spatial distance between clonally related bacteria) and the relationship between spatial distance and genetic distance, especially at very short scale (microhabitat scale), where cell division takes place. Agrobacterium spp. Biovar 1 was chosen because it is a soil bacterial taxon easy to isolate. A total of 865 microsamples 500 microm in diameter were sampled with spatial coordinates in 1 cm(3) of undisturbed soil. The 55 isolates obtained yielded 42 ribotypes, covering three genomic species based on amplified ribosomal DNA restriction analysis (ARDRA) of the intergenic spacer 16S-23S, seven of which contained two to six isolates. These clonemates (identical ARDRA patterns) could be found in the same microsample or 1 cm apart. The genetic diversity did not change with distance, indicating the same habitat variability across the cube. The mixing of ribotypes, as assessed by the spatial position of clonemates, corresponded to an overlapping of clones. Although the population probably was in a recession stage in the cube (10(3) agrobacteria g(-1)), a high genetic diversity was maintained. In two independent microsamples (500 microm in diameter) at the invasion stage, the average genetic diversity was at the same level as in the cube. Quantification of the microdiversity landscape will help to estimate the probability of encounter between bacteria under realistic natural conditions and to set appropriate sampling strategies for population genetic analysis.

Seasonal fluctuations and long-term persistence of pathogenic populations of Agrobacterium spp. in soils.

Short- and long-term persistence of pathogenic (i.e., tumor forming) agrobacteria in soil was investigated in six nursery plots with a history of high crown gall incidence. No pathogenic Agrobacterium strains were isolated in soil samples taken in fall and winter in any plots, but such strains were isolated from both bulk soils and weed rhizospheres (over 0.5 x 10(5) pathogenic CFU/g of bulk soil or rhizosphere) in three out of six plots in spring and summer. PCR amplifications of a vir sequence from DNA extracted from soil confirmed the presence of Ti plasmids in summer and their absence in fall and winter. The results indicate that strains that harbor a Ti plasmid had an unforeseen positive fitness versus Ti plasmid-free strains in soil and rhizosphere in spring and summer in spite of the apparent absence of tumor, and hence of opines. The gain of fitness occurred during a bloom of all cultivable agrobacteria observed only in conducive soils. An evolution of the pathogenic population was recorded during a 4-year period in one particularly conducive soil. In 1990, the pathogenic population in this soil consisted of only biovar 1 strains harboring both octopine- and nopaline-type Ti plasmids. In 1994, it consisted of only nopaline-type Ti plasmids equally distributed among biovar 1 and 2 strains. These results suggest that nopaline-type Ti plasmids conferred a better survival ability than octopine-type Ti plasmids to biovar 2 agrobacteria under the present field conditions.

Molecular tools for the characterisation of antibiotic-resistant bacteria.

This review will discuss a number of molecular tools which are currently used as well as some innovative approaches for the characterisation of antibiotic-resistant bacterial strains. Various methods involved in the detection and characterisation of genes and mutations associated with antibiotic resistance and that are used for strain typing as part of epidemiological studies, are described. Furthermore, a few examples are discussed in which the results of both gene and strain characterisation are combined to investigate the underlying mechanism of the spread of antibiotic resistance. Some of the available molecular techniques are heavily supported by the existence of databases on the Internet. These databases either contain a fast growing amount of sequence information or a large number of allelic or fingerprint profiles. The current progress in applied DNA technology and the ongoing projects on the elucidation of the whole genomic sequence of bacterial species have lead and will further lead to the development and application of sophisticated new strategies for the analysis of antibiotic resistant bacterial strains.

Novel tellurite-amended media and specific chromosomal and Ti plasmid probes for direct analysis of soil populations of Agrobacterium biovars 1 and 2.

Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K(2)TeO(3) was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K(2)TeO(3) concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K(2)TeO(3)-amended medium. The bona fide agrobacterium colonies growing on media amended with K(2)TeO(3) were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10(3) to 10(4) agrobacteria. g of dry soil(-1) in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.

Plant genome complexity may be a factor limiting in situ the transfer of transgenic plant genes to the phytopathogen Ralstonia solanacearum.

The development of natural competence by bacteria in situ is considered one of the main factors limiting transformation-mediated gene exchanges in the environment. Ralstonia solanacearum is a plant pathogen that is also a naturally transformable bacterium that can develop the competence state during infection of its host. We have attempted to determine whether this bacterium could become the recipient of plant genes. We initially demonstrated that plant DNA was released close to the infecting bacteria. We constructed and tested various combinations of transgenic plants and recipient bacteria to show that the effectiveness of such transfers was directly related to the ratio of the complexity of the plant genome to the number of copies of the transgene.

Comparison between electrochemical and optoelectrochemical impedance measurements for detection of DNA hybridization.

The principles of the electrochemical and optoelectrochemical impedance measurements on bare electrolyte/dielectric/semiconductor structures are described. The analysis of the experimental curves allows access to several indications concerning the electrical behavior of such structures. The application of these techniques to follow the electrical behavior of structures modified with two biological systems was investigated. The antibody/antigen recognition did not change the surface charge and, therefore, did not affect the impedance curves with respect to the applied potential. By contrast, the hybridization of two complementary DNA strands on the surface of the structure induced a variation of flat band potential of the semiconductor leading to a shift of impedance curves along the potential axis. This means that it is possible to detect directly the DNA hybridization without the use of labeled probes. The use of light allows the surface to be probed locally. In the future, the application of this technique for direct detection of hybridization on DNA chips should be possible.

Quantification of bias related to the extraction of DNA directly from soils.

In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed for the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an organism inoculated into the soil. The aim of the present investigation was to improve the extraction, purification, and quantification of DNA derived from as large a portion of the soil microbial community as possible, with special emphasis placed on obtaining DNA from gram-positive bacteria, which form structures that are difficult to disrupt. Furthermore, we wanted to identify and minimize the biases related to each step in the procedure. Six soils, covering a range of pHs, clay contents, and organic matter contents, were studied. Lysis was carried out by soil grinding, sonication, thermal shocks, and chemical treatments. DNA was extracted from the indigenous microflora as well as from inoculated bacterial cells, spores, and hyphae, and the quality and quantity of the DNA were determined by gel electrophoresis and dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield compared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage lambda DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay content strongly influenced the recovery of DNA. The adsorption of DNA to clay particles decreased when the soil was pretreated with RNA in order to saturate the adsorption sites. We also investigated different purification techniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging.

Ti plasmids from Agrobacterium characterize rootstock clones that initiated a spread of crown gall disease in Mediterranean countries.

Crown gall caused by Agrobacterium is one of the predominant diseases encountered in rose cultures. However, our current knowledge of the bacterial strains that invade rose plants and the way in which they spread is limited. Here, we describe the integrated physiological and molecular analyses of 30 Agrobacterium isolates obtained from crown gall tumors and of several reference strains. Characterization was based on the determination of the biovar, analysis of 16S ribosomal DNA restriction fragment length polymorphisms by PCR (PCR-RFLP), elucidation of the opine type, and PCR-RFLP analysis of genes involved in virulence and oncogenesis. This study led to the classification of rose isolates into seven groups with common chromosome characteristics and seven groups with common Ti plasmid characteristics. Altogether, the rose isolates formed 14 independent groups, with no specific association of plasmid- and chromosome-encoded traits. The predominant Ti plasmid characteristic was that 16 of the isolates induced the production of the uncommon opine succinamopine, while the other 14 were nopaline-producing isolates. With the exception of one, all succinamopine Ti plasmids belonged to the same plasmid group. Conversely, the nopaline Ti plasmids belonged to five groups, one of these containing seven isolates. We showed that outbreaks of disease provoked by the succinamopine-producing isolates in different countries and nurseries concurred with a common origin of specific rootstock clones. Similarly, groups of nopaline-producing isolates were associated with particular rootstock clones. These results strongly suggest that the causal agent of crown gall disease in rose plants is transmitted via rootstock material.

Direct conjugal transfers of Ti plasmid to soil microflora.

The bacterial species in soil that can receive a Ti plasmid by conjugation from Agrobacterium spp. were investigated. In order to have direct access to the potential reservoir of Ti plasmid amongst soil microflora, the conjugal system consisting of a multiply auxotrophic derivative of C58 (ST-96-4) and a derivative of pTiC58Delta(acc)R (pSTiEGK) containing a triple antibiotic-resistance cassette in traM was used to transfer the Ti plasmid in a complex soil microflora used as the recipient. Numerous transconjugants were obtained by this method but none was identified as Agrobacterium. This could be explained by the low density of Agrobacterium in the tested soil. As indicated by analysis of the ribosomal gene rrs, transconjugants recovered directly from soil were found to be new bacterial species which appeared to be closely related to Sinorhizobium spp.

Comparison of Randomly Amplified Polymorphic DNA with Amplified Fragment Length Polymorphism To Assess Genetic Diversity and Genetic Relatedness within Genospecies III of Pseudomonas syringae.

Recently, DNA pairing analyses showed that Pseudomonas syringae pv. tomato and related pathovars, including P. syringae pv. maculicola, form a genomic species (Pseudomonas tomato) (L. Gardan, H. L. Shafik, and P. A. D. Grimont, p. 445-448, in K. Rudolph, T. J. Burr, J. W. Mansfield, D. Stead, A. Vivian, and J. von Kietzell, ed., Pseudomonas syringae Pathovars and Related Pathogens, 1997). The genetic diversity of 23 strains belonging to this genomic species and 4 outgroup strains was analyzed with randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphic (AFLP) techniques. Simple boiling of P. syringae cells was suitable for subsequent DNA amplification to obtain reliable patterns in RAPD and AFLP analyses. In general, the grouping of P. syringae strains by both analysis techniques corresponded well with the classification obtained from an RFLP analysis of ribosomal DNA operons, DNA pairing studies, and an analysis of pathogenicity data. However, two strains of P. syringae pv. maculicola produced distinct DNA patterns compared to the DNA patterns of other P. syringae pv. maculicola strains; these patterns led us to assume that horizontal transfer of DNA could occur between bacterial populations. Both techniques used in this study have high discriminating power because strains of P. syringae pv. tomato and P. syringae pv. maculicola which were indistinguishable by other techniques, including pathogenicity tests on tomato, were separated into two groups by both RAPD and AFLP analyses. In addition, data analysis showed that the AFLP method was more efficient for assessing intrapathovar diversity than RAPD analysis and allowed clear delineation between intraspecific and interspecific genetic distances, suggesting that it could be an alternative to DNA pairing studies. However, it was not possible to distinguish the two races of P. syringae pv. tomato on the basis of an analysis of the data provided by either the AFLP or RAPD technique.

Conditions for natural transformation of Ralstonia solanacearum.

The development of competence allowing natural transformation of Ralstonia solanacearum was found to occur during exponential growth and not in response to any excreted factors. Linear DNAs were effectively integrated by recombination requiring a minimum of 50 bp of homologous DNA. Therefore, DNA from other genera and species were ineffective.

IS292: a novel insertion element from Agrobacterium.

A new insertion sequence, IS292, located in the 6b gene of a nopaline-type Agrobacterium strain (X88-292) isolated from poplar was identified and sequenced. IS292 is 2494 bp long, has 21 bp inverted terminal repeats with two mismatches, and generates 10 bp direct repeats upon integration. No sequence similarity was found between IS292 and other insertion elements associated with Agrobacterium, but it shows strong similarity with ISR/1 from Rhizobium leguminosarum bv. viciae. The occurrence of IS292-like sequences in various Agrobacterium isolates, especially different Agrobacterium strains isolated from the same biotope, was demonstrated by DNA hybridization.

Novel Ti plasmids in Agrobacterium strains isolated from fig tree and chrysanthemum tumors and their opinelike molecules.

Galls naturally induced on Fig and chrysanthemum plants by strains of Agrobacterium contained, in addition to other well-characterized opines such as nopaline, three tumor-specific opinelike molecules. These molecules were identified as deoxy-fructosyl-glutamine (dfg), deoxy-fructosyl-5-oxo-proline (dfop), and chrysopine (Chilton et al., unpublished). Strains isolated from Fig tree and chrysanthemum tumors harbored different and unrelated Ti plasmids as judged by hybridization with various vir and T-DNA probes. They also exhibited different opine-catabolic properties. The strains isolated from chrysanthemum plants (Chry strains) and Fig trees degraded chrysopine, but only the Chry strains used dfg and dfop. Remarkably, other strains of Agrobacterium catabolized these two molecules: dfg was degraded by most pathogenic and nonpathogenic Agrobacterium strains, and dfop by all Agrobacterium strains degrading the opine agropinic acid. These results have strong ecological and evolutionary inferences which fit previous speculation on the origin of opine-related functions.