Agrobacterium Radiobacter Strain K84 Research Articles

Control of Agrobacterium tumefaciens with Essential Oils Compared to Antagonistic Agrobacterium radiobacter Strain K84

Control of Agrobacterium tumefaciens with Essential Oils Compared to Antagonistic Agrobacterium radiobacter Strain K84

Efficacy of the Non-Pathogenic Agrobacterium Strains K84 and K1026 against Crown Gall in Tunisia

The non-pathogenic Agrobacterium radiobacter strain K84 and its genetically modified (GEM) strain K1026 were tested for their effectiveness against local Tunisian strains and two reference strains (C58 and B6) of the crown gall bacterium Agrobacterium tumefaciens. Tests in planta were carried out on herbaceous plants (tomato and tagetes) and on some sensitive rootstocks (bitter almond, peach almond hybrid GF677 and quince BA29). In vitro tests showed that both K84 and K1026 were effective and that the difference between these strains was not statistically significant. On tomato and tagetes, strain K84 was effective against all crown gall isolates with the exception of the A. tumefaciens reference strain B6. GEM strain K1026 was very effective against all isolates from Tunisia and against the reference strains. Both antagonistic strains significantly reduced the percentage of galled plants as well as the number of galls per plant. Under field conditions, both antagonists controlled crown gall effectively. Best results were obtained on the bitter almond-tree rootstock. Antagonist effectiveness was less evident on quince BA29 and peach almond GF677 rootstocks. The genetically modified strain K1026 is of interest in controlling crown gall disease in Tunisia.

Effect of Antibiosis on Antagonist Dose-Plant Disease Response Relationships for the Biological Control of Crown Gall of Tomato and Cherry

ABSTRACT The crown gall pathosystem was used to evaluate a model that describes the dose-response relationship between biological control agents and plant pathogens. The model predicts that this relationship can become asymptotic, such that increased antagonist doses cannot compensate for deficiencies in disease suppression. Wounded roots of tomato (Lycopersicon esculentum) and cherry (Prunus mahaleb) plants were dipped into different concentrations of the biological control organism Agrobacterium radiobacter strain K84 prior to inoculation with the pathogen A. tumefaciens. Pathogen strains sensitive or resistant to the antibiotic agrocin 84 were used, and for tomato experiments, a derivative of A. radiobacter strain K84 that does not produce agrocin 84 also was included as an experimental treatment. As predicted by the dose-response model, the amount of disease suppression per unit of antagonist decreased with increasing antagonist dose and became asymptotic at high antagonist densities. Control of crown gall of tomato was nearly complete with the combination of A. radiobacter K84 and an agrocin 84-sensitive strain of A. tumefaciens. Pathogen resistance to agrocin 84 or lack of agrocin 84 production by A. radiobacter resulted in antagonist dose-crown gall incidence relationships that were apparently asymptotic at levels of control significantly less than 100%. For field-grown cherry, similar dose-response relationships were observed with higher asymptotic levels of disease suppression obtained when trees were inoculated with an agrocin 84-sensitive A. tumefaciens strain compared with an agrocin 84-resistant pathogen strain. The differences among bacterial strain combinations in the magnitude of the asymptote defined by the dose-response relationships suggest that A. radiobacter impacts a smaller proportion of the pathogen population when the activity of agrocin 84 is muted.

Behavior of a Virulent Strain Derived from Agrobacterium radiobacter Strain K84 After Spontaneous Ti Plasmid Acquisition

ABSTRACT The behavior of the virulent transconjugant K84N6 derived from Agrobacterium radiobacter strain K84 after spontaneous Ti plasmid transfer in crown gall tissue in a biocontrol experiment was studied and compared with the behavior of the wild-type A. tumefaciens donor of the Ti plasmid. The main difference between the strains was a greatly reduced ability of the transconjugant to catabolize nopaline. Host range, ability to induce tumors in several fruit trees, and stability of the pathogenic determinants in isolates from tumors did not differ between the strains. Nevertheless, in a biocontrol experiment, the transconjugant was not controlled by strain K84 or K1026 in peach x almond hybrids and survived in the plant rhizo-sphere for 9 months with larger population densities than the wild strain. The appearance and persistence in soil of strains harboring a Ti plasmid in the K84 chromosomal background could represent a risk in the medium term, if they show good competitive ability.

Attachment of Agrobacterium tumefaciens B6 and A. radiobacter K84 to Tomato Root Tips

Agrobacterium tumefaciens B6 and the avirulent Agrobacterium radiobacter strain K84 attached to in vitro-cultured tomato root tips, but the binding of strain B6 to root tips was greater than the binding of strain K84. Strain K84 was not able to block the attachment of A. tumefaciens B6 to in vitro-cultured tomato root tips.

Transfer of pAgK84 from the Biocontrol Agent Agrobacterium radiobacter K84 to A. tumefaciens Under Field Conditions

Agrobacterium radiobacter strain K84 effects biological control of crown gall disease caused by A. tumefaciens in part by the plasmid-conferred production of agrocin 84, an antibiotic with specific activity against certain strains of the pathogen. A transmissible plasmid (pAgK84) resident in the biocontrol bacterium contains genes for agrocin 84 biosynthesis and immunity. The frequency of pAgK84 transfer from K84 to A. tumefaciens strain B49c, a naturally occurring strain that is insensitive to agrocin 84, in gall tissue of cherry seedlings was evaluated under field conditions. Putative transconjugants were detected by colony hybridization in 4 of 13 galls evaluated. The identity of transconjugants recovered from one of those galls was confirmed by serology, agrocin production, pathogenicity, and Southern blot analysis using agrocin 84 biosynthesis, T-DNA, and nopaline synthase gene probes. In the four galls in which transconjugants were detected, the estimated frequency of plasmid transfer from K84 to B49c was approximately 10 -4 transconjugants per recipient. A transconjugant strain and B49c did not differ in their capacities to colonize the rhizosphere of cherry or to cause crown gall under field conditions. The transconjugant retained pAgK84 for up to 7 months in the rhizosphere of field-grown plants. These results confirm that Agrobacterium strains harboring both a tumor-inducing plasmid and pAgK84 can develop from plasmid transfer in the field and that such newly derived strains can persist under field conditions.

Evaluation of Biological and Chemical Treatments for Control of Crown Gall on Young Apple Trees in the Kootenay Valley of British Columbia

AbstractField trials at Creston in 1988, 1989, and 1990 on silty loam soil naturally infested with Agrobacterium tumefaciens showed that Agrobacterium radiobacter strain K84 did not control crown gall on young Antonovka apple trees when apphed as a root‐dip treatment. Copper oxychloride applied as a root‐dip treatment at 2.5 and 5.0 g ai/1 and sewage sludge applied at 260 g per tree as broadcast were effective in reducing crown gall infection but these treatments were toxic to young apple trees in 1989. Lower rates of copper oxychloride, 0.3, 0.6 and 0.9 g ai/1, and sewage sludge at 130 g per tree, did not control crown gall in the 1990 field trial. The biological treatment with the strain AB8 of Bacillus subtilis applied as a root‐dip effectively controlled crown gall and was not phytotoxic to young Antonovka apple trees. These results suggest that strain AB8 of B. subtilis has the potential to control crown gall on young apple trees under field conditions.

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 Tn 5 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 Tn 5 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.

Biological Control of Crown Gall Using Using Agrobacterium Strains K84 and K1026

Crown gall has been successfully controlled on a commercial scale for over 15 years by the use of Agrobacterium radiobacter strain K84. The continued success of strain K84 has been jeoparised by the possibility of transfer of the agrocin plasmid, pAgK84, to pathogenic agrobacteria, making them resistant to control. The construction of a transfer-deficient deletion mutant of pAgK84 has resulted in a strain (K1026) from which the agrocin plasmid (pAgK1026) can no longer be transferred to other agrobacteria. Strain K1026 controls crown gall as effectively as strain K84 and may be used to ensure that the danger of breakdown of biological control is minimised.

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

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.

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

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.

Etiology of grape crown gall in Ontario

Crown gall was found mostly on vinifera grape (Vitis vinifera L.) cultivars in Ontario vineyards. The galls were usually small and aggregated along the trunk and cane; large woody galls were less common. Vines affected by crown gall died back in 3–4 years after planting. All the nine strains of Agrobacterium tumefaciens (Smith and Townsend) Conn isolated from galls corresponded to biotype 3 in determinative tests and were pathogenic on Datura, tobacco, tomato, and sunflower plants. Eight strains were sensitive to agrocin 84 on amended Stonier's medium. They were suppressed if inoculation was preceded 6 h earlier by Agrobacterium radiobacter strain K84 at wound sites on Datura and tomato plants.

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.