Agrobacterium Vitis Research Articles

The effect of resistance of rootstocks to crown gall (Agrobacterium spp.) on the susceptibility of scions in grape vine cultivars

The effects of two grapevine rootstocks on susceptibility of grafted scions to crown gall (Agrobacterium vitis and Agrobacterium tumefaciens) was investigated in greenhouse and field experiments. The incidence of crown gall on a susceptible grape scion cultivar (Vitis vinifera, cv. Kiralyleanyka) was not affected by grafting it on a crown gall resistant rootstock (Vitis riparia, cv. Gloire) or on a susceptible rootstock (V. berlandieri × V. riparia, cv. Teleki 5C) when the inoculated vines were observed over a 2‐month period in the greenhouse. Weights of galls that were induced by six of eight strains of Agrobacterium (six A. vitis and two A. tumefaciens) were not significantly different, but A. tumefaciens strain 16/6 and A. vitis strain 2/3 produced significantly larger galls on Kiralyleanyka vines that were growing on their own roots or were grafted on 5C than on those grafted on Gloire. When the same vines were planted in the field and observed over a 4‐year period, no differences in incidence of crown gall were appa_rent until the third and fourth years, when vines grafted on Gloire had less crown gall than vines on 5C. By that time, the natural symptoms (galls along the trunk) of the disease appeared, coinciding with the decline of whole vines. In another field experiment, with naturally infected scions of V. vinifera, cv. Blau Frankisch (susceptible), crown gall was appa_rent on vines grafted on both rootstocks after 2 years, but in the fifth year 79% of the vines on 5C had crown gall whereas only 9% on Gloire showed symptoms. After the appearance of typical natural symptoms, vines showed reduced vigor with substantial loss of trunks and entire vines. During this period, many vines on 5C died as compared with only a few on Gloire. By the end of the sixth year, 13% of the originally planted vines on 5C and 82% on Gloire were alive, and although many pathogenic strains of Agrobacterium spp. were isolated from the roots of 5C, none was isolated from Gloire.

Identification and mutation of a gene required for glycerate kinase activity from a facultative methylotroph, Methylobacterium extorquens AM1.

A gene (gckA) responsible for the activity of glycerate kinase has been identified within a chromosomal fragment of the serine cycle methylotroph Methylobacterium extorquens AM1. A mutation in gckA leads to a specific C1-negative phenotype. The polypeptide sequence derived from gckA showed high similarity to a product of ttuD essential for tartrate metabolism in Agrobacterium vitis. Our data suggest that gckA and ttuD might be structural genes for glycerate kinase and that the serine cycle and the tartrate utilization pathway share a series of reactions.

Biological Control of Grape Crown Gall by Strain F2/5 Is Not Associated with Agrocin Production or Competition for Attachment Sites on Grape Cells

ABSTRACT Agrocin-minus mutants of nontumorigenic Agrobacterium vitis strain F2/5 controlled grape crown gall as well as the wild-type strain, indicating that agrocin is not a major factor in the mechanism of biological control. Relative levels of attachment to grape cells by tumorigenic and biocontrol strains were also measured. Attachment of tumorigenic strains (CG49 and K306) and biological control strains (F2/5 and agrocin-minus mutant 1077) was often reduced when mixtures of the strains were applied. However, high populations (10(3) to 10(5) CFU/ml) of all strains attached following mixed inoculations, suggesting that competition for attachment sites is also not a factor in the mechanism of biological control. Transfer of T-DNA to grape by CG49 was prevented or greatly inhibited in the presence of F2/5 or 1077 as measured by expression of the GUS reporter gene. The Ti plasmid virulence genes, however, were induced by exudates from grape shoots that had been inoculated with F2/5. Sonicated and autoclaved preparations of F2/5 and 1077 did not control crown gall or inhibit T-DNA transfer. Control by F2/5 is specific to grape, since gall formation on tomato, sunflower, and Kalanchoe daigremontiana were not inhibited.

Biochemical and Molecular Characterization of 18 Agrobacterium vitis Isolates

Agrobacterium vitis is the causal organism of crown gall in grapevine. Infection is particularly severe in areas that experience winter damage to vines. Improving resistance to A. vitis will require a detailed knowledge about this organism. In this study, 18 grapevine isolates of A. vitis were collected from different locations near Lubbock, Texas. Isolates were subjected to a phenotypic characterization using 12 biochemical tests, including production of alkali from L-tartrate, production of 3-ketolactose, utilization of citrate, and others. Previously characterized isolates of A. vitis and A. tumefaciens obtained from the American Type Culture Collection served as positive and negative controls in these assays. Isolates were also evaluated for host range, tumor morphology, and opine utilization, and were compared at the molecular level by restriction fragment length polymorphism analysis of the oncogenic regions of the T-DNA plasmid. Although all isolates were able to metabolize tartrate and grow on Roy–Sasser media, there was much variability based on other tests. Twelve of 18 isolates were able to utilize octopine as a sole carbon source. All isolates tested thus far have been pathogenic on tomato and tests on grapevines are underway.

Characterization of Erwinia amylovora strains using random amplified polymorphic DNA fragments (RAPDs).

The genetic diversity among 16 strains of Erwinia amylovora, chosen to represent different host plant origins and geographical regions, was investigated by RAPD analysis. One strain of Erwinia herbicola and one of Agrobacterium vitis were used as outgroups. Ninety-eight different RAPD fragments were produced by polymerase chain reaction amplification with six different 10-mer primers. RAPD banding profiles were found that enabled the Erw. amylovora strains to be distinguished from one another. Cluster analysis based on the number of RAPD fragments shared between strains showed that strains of Erw. amylovora isolated from subfamily Pomoideae formed a single group, whereas two strains from Rubus (subfamily Rosoideae) formed a second group. Two strains isolated from Asian pear on Hokkaido, Japan, formed a third group. Sets of RAPD fragments were identified that enabled each of the two host-range groups and one geographical region (Hokkaido) of Erw. amylovora strains to be unambiguously distinguished from one another and from the outgroups. This study shows that strains of Erw. amylovora exhibit genetic diversity detectable by RAPD analysis, and that molecular and statistical analysis of RAPD fragments can be used both to distinguish between strains and to determine relatedness between them.

Characterization and distribution of tartrate utilization genes in the grapevine pathogen Agrobacterium vitis.

Agrobacterium vitis is a common pathogen of grapevine. Most strains utilize tartrate, an abundant compound in grapevine. Strain AB3 carries two tartrate utilization (or TAR) regions: TAR-I (on the large pTrAB3 plasmid) and TAR-II (on the AB3 Ti plasmid). TAR-I and TAR-II were structurally and functionally analyzed and are similar to the TAR-III region from the tartrate utilization plasmid pTrAB4 of the nopaline-type A. vitis strain AB4 (Crouzet and Otten, J. Bacteriol. 1995, 177:6518-6526). The minimal tartrate utilization region of TAR-I contains four genes (ttuA-ttuD). The ttuC gene is homologous to the tartrate dehydrogenase gene from Pseudomonas putida. Outside the minimal region a second ttuC-like gene is found (ttuC') which is transcribed and complements a ttuC mutant. Most grapevine isolates carry one or two of the three characterized TAR regions and show a considerable degree of polymorphism around these regions.

Effect of Heat Treatments on Grape Bud Mortality and Survival ofAgrobacterium vitis in vitroand in Dormant Grape Cuttings

No differences were observed in the heat sensitivity of four strains of <i>Agrobacterium vitis</i>, the cause of grape crown gall. At 50°C, the calculated D values of cells that were grown to stationary growth phase in LB broth were about 2.5 (time in minutes required for population to be reduced by ninety percent). The effect of heat treatments on survival of <i>A. vitis</i> in dormant cuttings was also measured. <i>A. vitis</i>-infected dormant grape cuttings (containing 5.0 x 10² to 2.7 x 10⁴ cfu per unit of sampled tissue) were submersed in a water bath at 50°C for 30 or 60 minutes, 30°C for two hours followed by 50°C for 90 minutes, or at 55°C for 30 minutes. Treatments significantly reduced the numbers of cuttings with detectable <i>A. vitis</i>; however, none of the treatments totally eradicated the pathogen. Therefore, <i>A. vitis</i> cells surviving in cuttings appear to be more heat-tolerant than cells grown <i>in vitro</i>. Using heat treatment to reduce systemic <i>A. vitis</i> in cuttings may be a good strategy for managing crown gall in vineyards. The 50°C for 60 minutes treatment always resulted in improved bud germination as compared to untreated buds. The effect of treatments of 50°C for 90 minutes and 55°C for 30 minutes on bud germination was variable depending on cultivar.

Further Evidence that the Vitopine-type pTi's ofAgrobacterium vitisRepresent a Novel Group of Ti Plasmids

To study the incompatibility properties of the vitopine Ti plasmids of Agrobacterium vitis, pPM739 containing the cloned ori region of pTiS4 was introduced by triparental mating into seven Agrobacterium tumefaciens strains carrying incRh-1, incRh-2, and incAg-1 plasmids and into eight A. vitis strains. All strains containing pPM739 retained their original plasmids and virulence or the ability to grow on tartrate, except for the three vitopine strains S4, Sz1, and NW11. Furthermore, pTiS4 was stably maintained in S4 cells following introduction of the ori/inc clones of the incRh-1, incRh-2, and incRh-3 plasmids. These results show that vitopine Ti plasmids represent a novel incompatibility group for which we propose the name incRh-4.

Sequence and mutational analysis of a tartrate utilization operon from Agrobacterium vitis

The grapevine is the natural host of the tumorigenic bacterium Agrobacterium vitis. Most of the A. vitis isolates can use tartrate, an unusually abundant compound in grapevine. The nopaline strain, AB4, contains a 170-kb conjugative plasmid (pTrAB4) encoding tartrate utilization. A 5.65-kb pTrAB4 region which enables non-tartrate-utilizing Agrobacterium tumefaciens to grow on tartrate was sequenced and mutagenized with the transcriptional fusion transposon Tn5-uidA1. This DNA fragment contains four intact open reading frames (ORFs) (ttuABCD) required for tartrate-dependent growth. The mutant phenotypes of each ORF, their homologies to published sequences, and their induction patterns allowed us to propose a model for tartrate utilization in A. vitis. ttuA encodes a LysR-like transcriptional activator and is transcribed in the absence of tartrate. ttuB codes for a protein with homology to transporter proteins and is required for entry of tartrate into bacteria. ttuC codes for a tartrate dehydrogenase, while ttuD lacks homology to known sequences; the growth properties of ttuD mutants suggest that TtuD catalyzes the second step in tartrate degradation. A fifth incomplete ORF (ttuE) encodes a pyruvate kinase which is induced by tartrate and required for optimal growth. Although the ttuABCD fragment allows growth of A. tumefaciens on tartrate, it does not provide full tartrate utilization in the original A. vitis background.

A full factorial analysis of nine factors influencing in vitro antagonistic screens for potential biocontrol agents.

The effect of nine factors on the outcome of classic in vitro screens testing the antagonistic action of endophytic bacterial isolates from grape vines against virulent Agrobacterium vitis has been examined. These factors were (i) the strain of A. vitis, (ii) the strain of endophyte, (iii) the growth medium of the pathogen, (iv) the growth medium of the endophyte, (v) the temperature of growth of the pathogen, (vi) the temperature of growth of the endophyte, (vii) the pH of growth of the pathogen, (viii) the pH of growth of the endophyte, and (ix) the medium of the assay plate. Analyses of variance of the full factorial design incorporating main effects and two- and three-way interactions accounted for 66% of the variance. All nine factors had a significant effect on the diameter of inhibition zones (p < 0.001). An examination of the three-way interactions revealed that generalizations were difficult to draw; each target agrobacterium had a specific response to a given antagonistic isolate. It was possible to determine that the growth history of bacterial strains, before they were administered to an assay plate to test for antagonism (especially the composition of the growth medium and the temperature of growth), had a profound effect on the outcome of the test. Generally the more chemically defined media produced less inhibition whereas the lower growth temperature of 15 degrees C produced more inhibition. These findings could be relevant to in situ inhibitory activity. The method used to conduct the inhibitory screen (order of strain application and the medium of the assay plate) had a profound influence on the results. These influences add to the caution necessary in the use of in vitro antagonistic screens for finding successful biocontrol agents.

A new Agrobacterium strain isolated from aerial tumors on Ficus benjamina L

Crown gall tumors, collected from branches of 1-year-old weeping fig (Ficus benjamina L.) trees, yielded both tumorigenic and nonpathogenic agrobacteria. On the basis of classical diagnostic tests, the nonpathogenic strains were identified as Agrobacterium tumefaciens, whereas the tumorigenic strains could not be assigned to any of the known terrestrial Agrobacterium spp. The tumorigenic strains also differed from other members of the genus by producing more acid from mannitol. According to cluster analysis of carbon substrate oxidation (GN Microplate; Biolog, Inc.) and fatty acid content, the tumorigenic fig strains were distinct from strains of A. tumefaciens, Agrobacterium rhizogenes, Agrobacterium vitis, and Agrobacterium rubi. Furthermore, they had unusual opine metabolism, inducing tumors that synthesized nopaline and three recently discovered opines: chrysopine (d-lactone of N-1-deoxy-D-fructosyl-L-glutamine, and N-1-deoxy-D-fructosyl-L-glutamine, and N-1-deoxy-D-fructosyl-5-oxo-L-proline. The nonpathogenic A. tumefaciens strains present in the same tumors were unable to degrade any of the opines tested. The phylogenetic position of the tumorigenic fig strain AF3.10 was inferred from comparing its rrs (i.e., 16S rRNA gene) sequence with those from the type strains of Agrobacterium and Rhizobium species. The analysis showed that strain AF3.10 clustered with A. tumefaciens and A. rubi but not with A. vitis and was far removed from A. rhizogenes. However, the sequence was significantly different from those of A. tumefaciens and A. rubi to suggest that the tumorigenic fig strain may be a new Agrobacterium species that is as different from A. tumefaciens and A. rubi as these two species are from one another.

Survival and Tumorigenicity ofAgrobacterium vitisin Living and Decaying Grape Roots and Canes in Soil

Agrobacterium vitis was recovered from living and decaying grape roots and canes 23 months after grapes were artificially inoculated with a mixture of six strains of the bacterium. Each strain contained a unique plasmid profile. Following inoculation, some plants were treated with the herbicide Roundup to speed up plant death and tissue decay. Roots and canes were assayed over time, and by comparing plasmid profiles of recovered strains it was determined that certain A. vitis strains used in the inoculum mixture were recovered more frequently than others. Profiles identical to those identified for each strain used in the inoculum mixture were observed at least twice in strains recovered during the experiment. Of 133 plasmid profiles that were observed, only 18 did not resemble any of the strains used in the inoculum mixture. Of 333 strains recovered from roots and canes, 321 were tumorigenic, indicating that this trait was stable throughout the experiment. A group of six strains having plasmid profiles identical to strain CG49 that were recovered over an 16-month period were further characterized using restriction fragment length polymorphic analysis of plasmid DNA, random amplified polymorphic DNA analysis of total genomic DNA, and ribofingerprinting of a chromosomal region including 1,479 bp (99.5%) of the 16S rDNA, the intergenic spacer between 16S and 23S rDNA genes, and 132 bp of the 23S rDNA gene. All six strains were shown to be identical to CG49.

A Rapid and Sensitive Method to DetectAgrobacterium vitisin Grapevine Cuttings Using the Polymerase Chain Reaction

Oligonucleotide primers were designed for amplification of the virA, pehA, and 6a genes of Agrobacterium species and evaluated for their potential use in the identification of Agrobacterium vitis. In conjunction with the polymerase chain reaction, the pehA-specific primer pair readily identified all strains of A. vitis that were tested. Four different extraction/purification methods were tested for the recovery of A. vitis DNA from grapevine cuttings. A protocol which relied on exposure to microwaves for cell lysis, followed by purification of DNA by ion exchange chromatography was the most effective in removing, from grapevine samples, inhibitors of the polymerase chain reaction. The reported method was both sensitive and rapid, making it amenable to routine diagnostic testing. It was demonstrated that the lysis in planta of A. vitis and analysis of the recovered DNA by polymerase chain reaction provided a more reliable indication of the occurrence of the bacteria in actively growing grapevines than did recovery of viable bacteria and growth on selective media.

Natural instability of Agrobacterium vitis Ti plasmid due to unusual duplication of a 2.3-kb DNA fragment.

The octopine/cucumopine (o/c) Ti plasmids of Agrobacterium vitis carry two T regions, TA and TB. The TA region resembles the octopine TL region. The TB region contains the auxin synthesis genes TB-iaaM and TB-iaaH and the cucumopine synthesis gene cus. Within the group of o/c isolates, strains 2608 and 2641 are closely related. However, 2641 lacks the TB region. The restriction maps of pTi2608 and pTi2641 were established and showed that the TB deletion resulted from intramolecular recombination between two directly repeated sequences separated by 66 kb in pTi2608. The 2,294-bp repeated sequence lacks inverted repeats and does not duplicate its target site, indicating that it is not a classical bacterial insertion sequence (IS element). It was therefore called an RSAv element (repeated sequence of A. vitis). The RSAv element carries two open reading frames: ORF234 is homologous to the traR gene of the A. tumefaciens nopaline Ti plasmid pTiC58; ORF488 is homologous to the sucrose phosphorylase gene of Leuconostoc mesenteroides and the glucosyl transferase A gene of Streptococcus mutans. The RSAv repeat starts precisely at the start codon of ORF488 and ends two base pairs 3' of the stop codon of ORF234. The structural organization of the RSAv element suggests that the amplification event did not result from a random amplification process. A study of the distribution of the two RSAv copies (RSAv-1 and RSAv-2) in pTi2608 and other o/c isolates indicates that the ancestor o/c Ti plasmid contained only RSAv-1 and that this sequence was duplicated at one point during the divergent evolution of the o/c Ti plasmids.(ABSTRACT TRUNCATED AT 250 WORDS)

Crown Gall Resistance ofVitisspp. and Grapevine Rootstocks

Vitis riparia 'Gloire de Montpellier' (Gloire) was the most resistant Vitis species and clone tested following inoculations with strains of Agrobacterium vitis and A. tumefaciens. Although tumors failed to appear after infection, the pathogen A. vitis multiplied at the same rate in Gloire as in the crown gall-susceptible V. vinifera 'Chasselas'. Root decay was induced by strains of A. vitis on all the investigated Vitis genotypes. Similarly, roots and shoots of all Vitis genotypes produced vir gene-inducing compounds. This fact ruled out the possibility that crown gall resistance is related to the lack of vir gene induction. However, T-DNA from A. vitis strain CG49 transferred to V. riparia cells at a lower rate than to V. vinifera cells, as determined by evaluation of uidA gene (GUS) expression

Proposal for Rejection of Agrobacterium tumefaciens and Revised Descriptions for the Genus Agrobacterium and for Agrobacterium radiobacter and Agrobacterium rhizogenes

The 16S rRNA sequences of seven representative Agrobacterium strains, eight representative Rhizobium strains, and the type strains of Azorhizobium caulinodans and Bradyrhizobium japonicum were determined. These strains included the type strains of Agrobacterium tumefaciens, Agrobacterium rhizogenes, Agrobacterium radiobacter, Agrobacterium vitis, Agrobacterium rubi, Rhizobium fredii, Rhizobium galegae, Rhizobium huakuii, Rhizobium leguminosarum, Rhizobium loti, Rhizobium meliloti, and Rhizobium tropici. A phylogenetic analysis showed that the 15 strains of Agrobacterium and Rhizobium species formed a compact phylogenetic cluster clearly separated from the other members of the alpha subclass of the Proteobacteria. However, Agrobacterium species and Rhizobium species are phylogenetically entwined with one another, and the two genera cannot be separated. In the Agrobacterium species, the strains of biovar 1, biovar 2, Agrobacterium rubi, and Agrobacterium vitis were clearly separated. The two biovars exhibited homogeneity in their phenotypic, chemotaxonomic, and phylogenetic characteristics, and two species should be established for the two biovars. We considered the nomenclature of the two biovars, and revised descriptions of Agrobacterium radiobacter (for the biovar 1 strains) and Agrobacterium rhizogenes (for the biovar 2 strains) are proposed. The name Agrobacterium tumefaciens is rejected because the type strain of this species was assigned to Agrobacterium radiobacter, and consequently the description of the genus Agrobacterium is revised.

Studies on the Evolution of Agrobacterium vitis as Based on Genomic Fingerprinting and IS Element Analysis

Summary In order to study evolutionary relationships of the crown gall pathogen on grapevine, Agrobacterium vitis , bacterial genomes were analyzed by genomic fingerprinting. Additionally, A. vitis specific IS elements were used as chromosomal markers to establish genetic relations obtained by restriction analysis. DNA fragments up to 1 Mb in size were produced by digestion of genomic DNA with the rare cutting restriction endonucleases Xba I, Sfi I, and Spe I and separated by pulsed field gel electrophoresis (PFGE). As the result of the macrorestriction analysis of 42 strains six genomic groups with homologous restriction patterns could be identified. Based on similarity coefficients of more than 0.8 A. vitis strains were allocated to one of the six genome types. For every genome type defined, a distinct IS element pattern was found. The synopsis of IS element patterns and genomic fingerprinting allowed the construction of a phylogenetic tree on the evolution of different genome types within A. vitis .

Taxonomic Significance of the Lipopolysaccharide Composition of the Three Biovars of Agrobacterium tumefaciens

Summary The sugar and fatty acid composition of 9 strains belonging to the 3 biovars of Agrobacterium tumefaciens and to Agrobacterium rubi was investigated. All strains showed a complex composition of the sugar moieties which comprised mostly 6-deoxyhexoses, hexoses and acidic sugars, such as GalA, 3-deoxy-lyxo-heptulosaric acid and Kdo. More striking however, was the different pattern in DOC-PAGE indicating that biovar 3 strains (Agrobacterium vitis), as well as Agrobacterium rubi contained an R-type LPS (or an R-type with only a very truncated O-chain). In contrast, strains of biovars 1 and 2 showed very long O-chains comprising more than 20 O-repeating units. The fatty acid profiles of the lipid A moieties of the LPS of biovars 1, 2 and 3 were characterized by the predominance of hydroxylated fatty acids which comprised 2–5 different 3-OH-fatty acids, as well as (n-2) hydroxylated long chain fatty acids, such as 27-OH-28 : 0. Biovar 2 strains were characterized by a high content of the rare iso-branched 3-OH-15 : 0, which was lacking in the other biovars and may serve as a characteristic chemotaxonomic marker. The presence of the rare 3-OH-18 : 0 and 3-OH-18 : 1 in 7 out of 9, or 5 out of 9 strains, respectively, was conspicuous.

Rapid divergence of Agrobacterium vitis octopine-cucumopine Ti plasmids from a recent common ancestor.

The octopine/cucumopine (o/c) Ti plasmids of the grapevine-associated Agrobacterium vitis strains constitute a family of related DNA molecules. Restriction maps were established of two limited-host-range o/c Ti plasmids, pTiAg57 and pTiAB3, and of the wide-host-range o/c Ti plasmid pTiHm1. Together with the previously obtained map of the wide-host-range o/c Ti plasmid pTiTm4, about 1000 kb were mapped with a resolution of 0.2 kb, allowing a detailed comparison of the various structures. One region of the o/c Ti plasmids is highly conserved and differs mainly by the presence or absence of relatively small DNA fragments (0.9-2.7 kb); the other region has been modified more extensively and carries large sequences specific for each Ti plasmid type. The sequence similarity within large conserved regions shows that these plasmids have diverged recently and that their evolution was driven by large-scale genetic events rather than single nucleotide changes. These results have important implications for studies on bacterial evolution.

EFFECT OF TISSUE AND EXTRACTS FROM CROWN GALL-RESISTANT AND-SUSCEPTIBLE VITIS GENOTYPES ON VIR INDUCTION IN AGROBACTERIUM VITIS

A GUS marker was used in earlier experiments to show that crown gall resistant Vitis genotypes show significantly lower rates of transformation by A. vitis than the crown gall susceptible Vitis vinifera. In recent experiments a reporter gene (lacZ) fused with the virB promoter was used to determine whether the lower frequency of transformation resulted from reduced induction of Agrobacterium vitis virulence (vir) genes. With some A. vitis strains, addition of medium conditioned with grape tissue enhanced vir induction by the known inducer acetosyringone and significant vir induction also resulted without addition of acetosyringone when grape tissue was included in the induction medium. Enhancement of vir induction by grape extracts was not correlated with genotypic susceptibility to crown gall.