Deletion derivatives of pAgK84 and their use in the analysis of Agrobacterium plasmid functions.

The 47.7-kb plasmid pAgK84, present in Agrobacterium radiobacter strain K84, confers production of a novel, highly specific, antiagrobacterial antibiotic called agrocin 84. Strain K84 is used commercially to biocontrol crown gall caused by agrocin 84-susceptible strains of Agrobacterium tumefaciens. Efficient biocontrol is dependent upon production of agrocin 84 by strain K84. Starting with a derivative of pAgK84 containing a Tn5 insertion, a series of deletion derivatives of the plasmid were isolated. The smallest of these, pJS500, contains about 8 kb of the original agrocin plasmid and localized the replication functions to between 4 and 6 o'clock on the physical map. A smaller derivative, produced by clonal rescue of a Tn5 insertion in the 4 o'clock region, further localized the minimal replication functions to a 1.5-kb region mapping between coordinates 18.1 and 19.6. Analysis of plasmid stability indicated that functions required for maintenance of the plasmid under nonselective conditions are tightly linked to the minimal replication region. This region also encodes incompatibility functions; the deletion derivatives were all incompatible with the wild-type pAgK84. The stability/replication locus of pAgK84 maps just anticlockwise from the Tra region. This region is retained fully in pAgK1026, the directed Tra- derivative of pAgK84 which is now in use as the primary crown gall biocontrol agent in Australia. One of the deletion derivatives, the 15-kb pJS400, was used as a vector to clone the KpnI fragments of an octopine-type Ti plasmid. Traits known to be encoded on these fragments were expressed and properly regulated in Agrobacterium hosts. One clone, encoding the Ti plasmid replication/incompatibility region, was used to cure IncRh1 Ti plasmids from their hosts. This clone also was found to be incompatible with pAtK84b, a large plasmid encoding opine catabolism present in A. radiobacter strain K84. This indicates that the opine catabolic plasmid is closely related to the IncRh1 Ti plasmids.     

Construction of a Tra? deletion mutant of pAgK84 to safeguard the biological control of crown gall

Summary Agrobacterium radiobacter strain K84 is used commercially for the biological control of crown gall. It contains the conjugative plasmid pAgK84, which encodes the synthesis of agrocin 84, an antibiotic that inhibits many pathogenic agrobacteria. A breakdown of control is threatened by the transfer of pAgK84 to pathogens, which then become resistant to agrocin 84. A mutant of pAgK84 with a 5.9-kb deletion overlapping the transfer (Tra) region was constructed using recombinant DNA techniques. The BamHI fragment B1 which covers most of the Tra region was cloned in pBR325 and its internal EcoRI fragments D1 and H, which overlap the Tra region, were removed, leaving 3.7 kb and 0.5 kb of pAgK84 on either side of the deletion. The latter was increased to 3.3 kb by adding EcoRI fragment D2 from a BamHI fragment C clone. The modified pBR325 clone was mobilized into Agrobacterium strain NT1 harbouring pAgK84 with a Tn5 insertion just outside the Tra region but covered by the deletion. A Tra⁺ cointegrate was formed between the Tn5-insertion derivative and the pBR325-based deletion construct by homologous recombination. The cointegrate was transferred by conjugation to a derivative of strain K84 lacking pAgK84, in which a second recombination event generated a stable deletion-mutant by deletion-marker exchange. The resultant new strain of A. radiobacter, designated K1026, shows normal agrocin 84 production. Mating experiments show that the mutant plasmid, designated pAgK1026, is incapable of conjugal transfer at a detectable frequency.     

Genetic isolation and physical characterization of pAgK84, the plasmid responsible for agrocin 84 production

The plasmid responsible for agrocin 84 biosynthesis by Agrobacterium radiobacter strain K84 has been genetically isolated free from any opine-catabolic plasmids. This was accomplished by mobilizing the agrocin plasmid, pAgK84, into a Ti plasmid-free A. tumefaciens strain, A136. The mobilizing element, pAt84a, was then cured from such a transconjugant by cultivation at 37 °C. Derivatives of strain A136 harboring both plasmids or pAgK84 only produce agrocin 84. The agrocin plasmid isolated from these strains is indistinguishable by restriction endonuclease analysis from that in strain K84. A physical map of pAgK84 has been constructed with respect to six restriction endonucleases. The plasmid is cut only once by XbaI and twice by HpaI. Hybridization analysis shows that pAgK84 is closely related to pAtBo542a, a 25-Mdal plasmid from a virulent, agrocinogenic A. tumefaciens strain of European origin. Similar analyses indicate, however, that pAgK84 shows no detectable homology to octopine or nopaline-type Agrobacterium plasmids.     

Tn5 insertions in the agrocin 84 plasmid: the conjugal nature of pAgK84 and the locations of determinants for transfer and agrocin 84 production

The kanamycin-resistance transposon Tn5 was randomly introduced into pAgK84, a 47.7-kb plasmid coding for agrocin 84 production in Agrobacterium. Using such marked plasmids, pAgK84 was found to be conjugal. It could be transferred to several Agrobacterium strains including those harboring octopine- or nopaline-type Ti plasmids. Its presence has no effect on Ti plasmid functions such as opine utilization and tumorigenicity, but it does confer agrocin 84 immunity upon previously sensitive strains. The plasmid could also be conjugally transferred to a Nod+ Fix+ strain of Rhizobium meliloti. The production of agrocin 84 is expressed in all Agrobacterium and Rhizobium transconjugants tested. The agrocin plasmid could not be introduced into restrictionless Escherichia coli or Pseudomonas aeruginosa recipients by conjugation or transformation. The sites of 92 independent Tn5 insertions were mapped on pAgK84. These insertions are dispersed over the entire length of the plasmid. Analysis of the sites and effects of the Tn5 insertions has allowed us to construct a functional map of pAgK84. Forty-three of these insertions, spanning a 20-kb segment of the plasmid, abolished or greatly reduced the production of agrocin 84. The presence of two insertions within this segment having an effect on agrocin production suggests that at least three regions of the plasmid are involved in agrocin 84 biosynthesis. Fourteen of the Tn5 insertion derivatives are no longer conjugally transferable. These insertions all map to a single region of the plasmid and define about 3.5-kb as being associated with transfer functions.     

Cocolonization of the Rhizosphere by Pathogenic Agrobacterium Strains and Nonpathogenic Strains K84 and K1026, Used for Crown Gall Biocontrol

The crown gall biocontrol agent strain K84 and three mutants derived from it, K1026 (Tra⁻ deletion mutant of pAgK84), K84 Agr⁻ (lacking pAgK84), and K1143 (lacking pAgK84 and pNoc), significantly reduced gall formation caused by two pathogenic strains resistant to agrocin 84 in peach × almond seedlings planted in infested soil. Cocolonization of roots by pathogenic and nonpathogenic strains was observed in these biocontrol experiments under field conditions. In spite of the efficient biocontrol observed, average populations consisting of 10² and 10⁶ pathogenic agrobacteria per g of root were found 8 months after planting. The total numbers of pathogenic bacteria on roots were similar for plants treated with the biocontrol strains and for the untreated plants. Strain K84 and the genetically engineered organism K1026 survived at a level of 10⁶ agrocin 84-producing bacteria per g of root. The population size of genetically engineered strain K1026 was not significantly different than the population size of wild-type strain K84 8 months after root inoculation. Strains K84 and K1026 controlled two pathogens resistant to agrocin 84 without reducing the total number of pathogenic bacteria in the root system. In addition, this study shows that some biological control activity of strain K84 against agrocin 84-resistant pathogens is independent of plasmids pAgK84 and pNoc.     

Agrobacterium plasmids encode structurally and functionally different loci for catabolism of agrocinopine-type opines

Summary Agrobacterium tumefaciens strains C58, T37, K827 and J73, A. rhizogenes strains A4 and 15834, and A. radiobacter strain K299 were all susceptible to agrocin 84 and this sensitivity was enhanced in each case by addition of agrocinopines A and B. Analysis of transconjugants showed that sensitivity of strain A4 to agrocin 84 was encoded by pArA4a and not by the rhizogenic plasmid, pRiA4. The acc region of the A. tumefaciens nopaline-type Ti plasmid pTiC58, contained on the recombinant plasmid pTHH206, hybridized strongly to restriction fragments of plasmids from strains T37, K827, J73 and K299. Hybridizing fragment patterns generated with BamHI and EcoRI were identical among the four Ti plasmids while pAtK299 showed restriction fragment length polymorphisms at acc with the two enzymes. At moderate stringency, the pTiC58 acc region hybridized weakly to a single restriction fragment from the Ar plasmid of A. rhizogenes strain A4, but not to pTiBo542, which encodes catabolism of the closely related opines agrocinopines C and D. Plasmid pAtK84b of A. radiobacter strain K84 is induced for conjugal transfer by agrocinopines A and B. However, no hybridization was detected between this plasmid and acc from pTiC58 under conditions of moderate stringency. Like pTiC58, pAtK84b conferred transport of agrocinopines A and B on its host bacteria despite the absence of detectable sequence homology with the pTiC58-derived acc probe. However, unlike pTiC58, pAtK84b failed to confer sensitivity to or uptake of agrocin 84 on its bacterial host. These results indicate that at least four distinguishable systems exist for catabolism of the two agrocinopine opine families with the prototype locus, exemplified by acc from pTiC58, being strongly conserved among nopaline-type Ti plasmids.     

Biological Control of Agrobacterium tumefaciens, Colonization, and pAgK84 Transfer with Agrobacterium radiobacter K84 and the Tra- Mutant Strain K1026

The efficacies of Agrobacterium radiobacter K84 and K1026 in root colonization, crown gall control, and plasmid transfer were compared. Levels of root colonization by K84 and K1026 of Montclar and Nemaguard peach seedlings were similar during the 21 days of the experiment. Four strains of A. tumefaciens bv. 1 were used for soil inoculations in biological control experiments on GF677 and Adafuel peach × almond rootstocks; two were sensitive and two were resistant to agrocin 84. Both strains K84 and K1026 were very efficient in controlling the sensitive strains, but some tumors appeared with both treatments. In the biocontrol of resistant strains, no galls were observed in K1026-treated plants, but some K84-treated plants had galls. Recovery of agrobacteria from galls in experiments with sensitive and resistant strains showed that all of the isolates from the controls or K1026-treated plants and most of the isolates from K84-treated plants had the same characteristics as the inoculated strains. Nine isolates from the K84-treated plants growing in soil inoculated with one resistant strain were virulent and produced agrocin 84. These isolates had a plasmid that hybridized with a probe prepared with the BamHI C fragment from pAgK84. These results show the efficiency of K1026 in biocontrol of agrocin 84-sensitive and -resistant strains of A. tumefaciens and suggest the use of K1026 as a safer organism than K84 for biological control of crown gall.     

Conditions for the production of Agrocin 84 byAgrobacterium radiobacter K84

Summary A chemically defined medium was developed that supported the growth ofAgrobacterium radiobacter K84 and the production of agrocin 84. Various supplements were investigated for their effect on growth rate and production of agrocin 84 using a well-diffusion assay method. Mannitol was found to be a better substrate for growth ofA. radiobacter K84 compared to the other sugar alcohols and sugars tested. By contrast,d-fructose was the better substrate for the production of agrocin 84. Biotin supplementation stimulated production of agrocin 84 but did not eliminate the diauxic lag seen with the basal medium. The opine, octopine, inhibited growth ofA. radiobacter K84 and production of agrocin 84 as did coenzyme B₁₂. By contrast, the cytokinin, isopentenyl adenosine, was marginally stimulatory to production, as was vitamin B₁₂. Acetosyringone supplementation had a negligible effect on growth rate and production of agrocin 84.     

Construction of a Range of Derivatives of the Biological Control Strain Agrobacterium rhizogenes K84: a Study of Factors Involved in Biological Control of Crown Gall Disease

The biological control strain Agrobacterium rhizogenes K84 is an effective agent in the control of Agrobacterium pathogens, the causative agents of crown gall disease. A number of factors are thought to play a role in the control process, including production of the specific agrocins 84 and 434, which differ in the spectra of pathogenic strains that they inhibit in vitro. A range of derivatives of strain K84 has been developed with every combination of the three resident plasmids, pAgK84, pAgK434, and pAtK84b, including a plasmid-free strain. These derivatives produced either both, one, or neither of the characterized agrocins 84 and 434 and were isolated by plasmid curing, conjugation, and Tn5 transposon mutagenesis. The ability of the derivative strains to inhibit gall formation on almond roots was compared to that of the wild-type K84 parent. Treatment with the plasmid-free derivative did not result in a significant level of control of an A. rhizogenes pathogen based on numbers or dry weight of galls formed on injured almond roots. The presence of plasmid pAgK84, pAgK434, or pAtK84b significantly enhanced the biological control efficacy of K84 derivatives, and the highest level of control was observed with strains harboring two or more plasmids. The results observed with strains deficient in agrocin 434 production suggest that this product may play an important role in the biological control of A. rhizogenes pathogens. The involvement of plasmid pAgK84b in biological control has not previously been reported. This study supports the conclusion that multiple factors are involved in the success of strain K84 as a biological control agent.     

Bacteriocin production inAgrobacterium tumefaciens

Agrobacterium tumefaciens C58 forms “plaques” during layer cultivation. The “plaques” were shown not to be caused by the presence of a temperate bacteriophage or by random contamination. The “plaques” and their central microcolonies were used to repeatedly isolate cultures producing an antibiotic substance against the original strainA. tumefaciens C58, other nopaline strains, some octopine strains ofA. tumefaciens and some strains of the related genusRhizobium. The substance is thus a bacteriocin; in analogy to agrocins 84 and D286 it was named agrocin C58. The agrocin is not inactivated by trypsin. Its production by strain C58 was found only on cultivation on solid but not liquid media. The producing isolate ofA. tumefaciens C58 (strain C58i2) contains neither plasmid pTiC58 nor the plasmid analogous to pAgK84 which controls the production of agrocin 84 inA. radiobacter K84.     

Genetic analysis of an Agrobacterium tumefaciens strain producing an agrocin active against biotype 3 pathogens

Summary The successful biocontrol agent for crown gall disease, Agrobacterium radiobacter strain K84, is unable to protect grapevines from infection. We have identified a strain of Agrobacterium tumefaciens, J73, which produces an agrocin active both in vitro and in vivo against grapevine pathogens (Webster et al. 1986). We now report on the curing of this strain of its nopaline-type Ti plasmid and the location, by transposon mutagenesis, of the genes involved in the production of the agrocin. The Ti plasmid was cured by the introduction of selectable plasmids carrying the origins of replication of either the nopaline Ti plasmid, pTiC58, or the octopine Ti plasmid, pTi15955. Tn5 mutagenesis indicated that the genes responsible for agrocin production and/or export are located both on the chromosome and on a plasmid, pAgJ73, which co-migrated in agarose gels with pTiJ73. As the two plasmids were separable after transposon mutagenesis, we postulate that during or after mutagenesis of the agrocin plasmid, DNA rearrangements occurred between it and pTiJ73, resulting in an increase in size of pAgJ73. We provide evidence that the rearrangements involved the duplication of nopaline catabolism genes from pTiJ73 and their insertion into pAgJ73, which facilitated the resolution of the two plasmids. As expected pTiJ73 has homology with the nopaline Ti plasmid, pTiC58.     

Negative effects of agrocin 84-encoding Agrobacterium plasmids on symbiotic properties of Rhizobium meliloti

Two plasmids, pAgK84::Tn5-Mob from Agrobacterium radiobacter carrying genes for the production of agrocin 84, and RP4-4 from E. coli were inserted either separately or together into a strain of Rhizobium meliloti. Each of these plasmid-containing R. meliloti transconjugants was less effective than the wild type strain in their ability to fix nitrogen in Medicago tornata. The pAgK84::Tn5-Mob-containing transconjugant was significantly less effective than that containing RP4-4. The transconjugant strains were inferior to the wild type strain in their ability to nodulate seedlings and to compete for nodulation.     

Biological Control of Agrobacterium tumefaciens, Colonization, and pAgK84 Transfer with Agrobacterium radiobacter K84 and the Tra Mutant Strain K1026

The efficacies of Agrobacterium radiobacter K84 and K1026 in root colonization, crown gall control, and plasmid transfer were compared. Levels of root colonization by K84 and K1026 of Montclar and Nemaguard peach seedlings were similar during the 21 days of the experiment. Four strains of A. tumefaciens bv. 1 were used for soil inoculations in biological control experiments on GF677 and Adafuel peach x almond rootstocks; two were sensitive and two were resistant to agrocin 84. Both strains K84 and K1026 were very efficient in controlling the sensitive strains, but some tumors appeared with both treatments. In the biocontrol of resistant strains, no galls were observed in K1026-treated plants, but some K84-treated plants had galls. Recovery of agrobacteria from galls in experiments with sensitive and resistant strains showed that all of the isolates from the controls or K1026-treated plants and most of the isolates from K84-treated plants had the same characteristics as the inoculated strains. Nine isolates from the K84-treated plants growing in soil inoculated with one resistant strain were virulent and produced agrocin 84. These isolates had a plasmid that hybridized with a probe prepared with the BamHI C fragment from pAgK84. These results show the efficiency of K1026 in biocontrol of agrocin 84-sensitive and -resistant strains of A. tumefaciens and suggest the use of K1026 as a safer organism than K84 for biological control of crown gall.     

Agrobacterium radiobacter strains K84, K1026 and K84 Agr‐ produce an antibiotic‐like substance,active in vitro against A. tumefaciens and phytopathogenic Erwinia and Pseudomonas spp.

Agrobacterium radiobacter strains K84, K1026 and K84 Agr^‐ produced in vitro an antibiotic‐like substance (ALS 84), different from agrocin 84 and observed in mannitol‐glutamate medium. Twenty five out of 39 A. tumefaciens strains of biovars 1, 2 and 3 were sensitive to ALS 84 regardless of their sensitivity to agrocin 84. Sensitivity in A. tumefaciens strain C58 was not encoded by the Ti‐plasmid. Most isolates tested of Erwinia carotovora subsp. carotovora E. carotovora subsp. atroseptica, Pseudomonas corrugata P. cichorii and unidentified isolates from galls were also sensitive to this substance. ALS 84 was not affected by the proteases studied, nor by treatment at 62°C for 30 min and had a bacteriostatic effect. The production of ALS 84 might play a role in the complex mechanism of biological control of crown gall, especially in strains resistant to agrocin 84 and sensitive to ALS 84, and by the creation of an ecological niche favourable to A. radiobacter strains K84, K1026 or K84 Agr^‐.     

Site-specific mutagenesis of the Ti plasmid by transformation of Agrobacterium tumefaciens with mutagenized T-DNA fragments cloned in E. coli plasmids

Summary A DNA fragment covering the complete T-region of the Ti plasmid from Agrobacterium tumefaciens strain C58 was cloned in the Escherichia coli cosmid pHC79. This fragment was mutagenized by insertion of transposon Tn5. The isolated DNA from hybrid plasmids was used to transform cells of A. tumefaciens strain C58 applying the freeze-thaw method. Although the E. coli plasmids with the mutagenized Ti plasmid fragment cannot replicate in these cells, they can be rescued by recombination with the homologous region of the Ti plasmid. The cointegrates formed were resolved in a second recobination event, which was detected by loss of the drug resistance marker of the E. coli plasmid. Subcloning of the Ti plasmid fragments labeled with Tn5 showed that the frequency of rescue of the hybrid plasmid as a cointegrate and its segregation in agrobacteria depend on the degree of homology with the Ti plasmid. We also applied the strategy for site-directed Tn5 mutagenesis to insert specifically the replication origin of bacteriophage fd and the thymidine kinase gene from Herpes virus into the T-DNA of Ti plasmid-C58.     

Iron-Binding Compounds from Agrobacterium spp.: Biological Control Strain Agrobacterium rhizogenes K84 Produces a Hydroxamate Siderophore

Iron-binding compounds were produced in various amounts in response to iron starvation by a collection of Agrobacterium strains belonging to the species A. tumefaciens, A. rhizogenes, and A. vitis. The crown gall biocontrol agent A. rhizogenes strain K84 produced a hydroxamate iron chelator in large amounts. Production of this compound, and also of a previously described antibiotic-like substance called ALS84, occurred only in cultures of strain K84 grown in iron-deficient medium. Similarly, sensitivity to ALS84 was expressed only when susceptible cells were tested in low-iron media. Five independent Tn5-induced mutants of strain K84 affected in the production of the hydroxamate iron chelator showed a similar reduction in the production of ALS84. One of these mutants, M8-10, was completely deficient in the production of both agents and grew poorly compared to the wild type under iron-limiting conditions. Thus, the hydroxamate compound has siderophore activity. A 9.1-kb fragment of chromosomal DNA containing the Tn5 insertion from this mutant was cloned and marker exchanged into wild-type strain K84. The homogenote lost the ability to produce the hydroxamate siderophore and also ALS84. A cosmid clone was isolated from a genomic library of strain K84 that restored to strain M8-10 the ability to produce of the siderophore and ALS84, as well as growth in iron-deficient medium. This cosmid clone contained the region in which Tn5 was located in the mutant. Sequence analysis showed that the Tn5 insert in this mutant was located in an open reading frame coding for a protein that has similarity to those of the gramicidin S synthetase repeat superfamily. Some such proteins are required for synthesis of hydroxamate siderophores by other bacteria. Southern analysis revealed that the biosynthetic gene from strain K84 is present only in isolates of A. rhizogenes that produce hydroxamate-type compounds under low-iron conditions. Based on physiological and genetic analyses showing a correlation between production of a hydroxamate siderophore and ALS84 by strain K84, we conclude that the two activities share a biosynthetic route and may be the same compound.     

Evidence of Biological Control of Agrobacterium tumefaciens Strains Sensitive and Resistant to Agrocin 84 by Different Agrobacterium radiobacter Strains on Stone Fruit Trees

The effectiveness of Agrobacterium radiobacter K84, 0341, and a K84 non-agrocin-producing mutant (K84 Agr^-) in biological control of crown gall on rootstocks of stone fruit trees was determined in three experiments. In experiment 1, K84 and 0341 controlled crown gall on plum plants in soil inoculated with two strains of Agrobacterium tumefaciens resistant to agrocin 84. In experiment 2, K84 controlled crown gall on peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84 or with a mixture of both. However, the effectiveness of K84 was higher against the sensitive strain than against the resistant strain. There was a residual effect of K84 from one year to another in soil inoculated with the sensitive strains. In experiment 3, K84 and K84 Agr^- controlled crown gall on plum and peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84. The control afforded by K84 was higher than that provided by K84 Agr^- against the sensitive strain but was similar against the resistant strain.     

Genetic analysis of a transposon carrying toluene degrading genes on a TOL plasmid pWWO

Summary Toluene degrading (xyl) genes on a Pseudomonas TOL plasmid pWWO are located within a 39-kb DNA portion. The 56-kb region including these xyl genes and its 17-kb derivative with a deletion of the internal 39-kb portion transposed to various sites on target replicons such as pACYC184 and R388 in escherichia coli recA strains. Thus the 56- and 17-kb regions were designated Tn4651 and Tn4652, respectively. Genetic analysis of Tn4652 demonstrated that its transposition occurs by a two-step process, namely, cointegrate formation and its subsequent resolution. The presence in cis of DNA sequences of no more than 150 bp at both ends of Tn4652 was prerequisite for cointegrate formation, and this step was mediated by a trans-acting factor, transposase, which was encoded in a 3.0-kb segment at one end of the transposon. Cointegrate resolution took place site-specifically within a 200-bp fragment, which was situated 10 kb away from the transposase gene. Based on the stability of cointegrates formed by various mini-Tn4652 derivatives, it was shown that the cointergrate resolution requires two trans-acting factors encoded within 1.0- and 1.2-kb fragments that encompass the recombination site involved in the resolution.     

Correlative Association between Resident Plasmids and the Host Chromosome in a Diverse Agrobacterium Soil Population

Soil samples collected from a fallow field which had not been cultivated for 5 years harbored a population of Agrobacterium spp. estimated at 3 × 10⁷ CFU/g. Characterization of 72 strains selected from four different isolation media showed the presence of biovar 1 (56%) and bv. 2 (44%) strains. Pathogenicity assays on five different test plants revealed a high proportion (33%) of tumorigenic strains in the resident population. All tumorigenic strains belonged to bv. 1. Differentiation of the strains by restriction fragment length polymorphism analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cellular proteins, and utilization patterns of 95 carbon substrates (Biolog GN microplate) revealed a diversified bv. 1 population, composed of five distinct chromosomal backgrounds (chr A, C, D, E, and F), and a homogeneous bv. 2 population (chr B). chr A, B, C, and D were detected at similar levels throughout the study site. According to opine metabolism, pathogenicity, and agrocin sensitivity, chr A strains carried a nopaline Ti plasmid (pTi), whereas chr C strains had an octopine pTi. In addition, four of six nontumorigenic bv. 1 strains (two chr D, one chr E, and one chr F) had distinct and unusual opine catabolism patterns. chr B (bv. 2) strains were nonpathogenic and catabolized nopaline. Although agrocin sensitivity is a pTi-borne trait, 14 chr B strains were sensitive to agrocin 84, apparently harboring a defective nopaline pTi similar to pAtK84b. The other two chr B strains were agrocin resistant. The present analysis of chromosomal and plasmid phenotypes suggests that in this Agrobacterium soil population, there is a preferential association between the resident plasmids and their bacterial host.