Agrobacterium Isolates Research Articles

Occurrence and distribution of crown gall disease in Jordan

Field inspection indicated that crown gall disease occurs in various fruit-tree-growing areas in Jordan, including Almafraq, Alsalt, Alshobak, Altafila, Alyadoda, Amman, Irbid, Jerash, Jordan Valley and Madaba. Twohundred tumorigenic Agrobacterium isolates were obtained from stone fruit rootstocks (bitter almond, GF677, GF305, Myrobalan, Nemaguard and Mahaleb), pome fruit rootstocks (apple seedling, quince, and clonal rootstock MM106), grapevine, olive, pomegranate, carob, rose and Cichorium pumilum. The pathogenicity of these isolates was proved through artificial inoculation on tomato seedlings and Kalanchoe plants, and through tmr gene detection with PCR. Biochemical and physiological testing of the isolates indicated the occurrence of biovar 1 (60.5%), biovar 2 (23.5%), and biovar 3 (1%), in addition to an intermediate biovar (15%). Seventy-seven percent of isolates were found to be sensitive to agrocin 84; of these, most (66.9%) belonged to biovar 1.

Characterization of agrobacteria from weeping fig (Ficus benjamina)

Ficus benjamina plants, galled both at epi‐ and hypogeous parts, were observed in Italy and in The Netherlands, and these were the first records of the appearance of weeping fig crown gall in Europe. A total of 241 Agrobacterium isolates was obtained from 41 tumours and studied for their morphological, physiological and phytopathological characters. Two main groups of agrobacteria were distinguished by their colony morphology and through classical biovarietal tests that allowed allocation of 86 isolates into biovar 1 and 155 into an intermediate biovar rather different from any of the three biovars defined for agrobacteria. Most of the isolates were unable to utilize mannopine, nopaline or octopine as C and N sources; only 62 strains utilized nopaline. However, when nonopine‐utilizing strains were inoculated into F. benjamina, only nopaline was detected in the developing tumours. BIOLOG ML 1™ system analysis applied to 50 representative strains allowed identification of the biovar 1 isolates as Agrobacterium tumefaciens and most of the intermediate biovar isolates as the newly proposed species Agrobacterium fici. Analysis of sensitivity to a set of 14 antibiotics confirmed the allocation of the 50 strains into two well defined main clusters matching the BIOLOG identification. Out of 141 tumorigenic isolates, 66 were sensitive in vitro to agrocine 84, but four of these strains showed scarce or no sensitivity to the antagonist A. radiobacter K84 when tested in fig plants. The two types of agrobacteria could usually be isolated from the same tumours. Tumorigenic strains were able to induce tumours in six herbaceous plant species, in eight to 10 out of 12 woody plants and in six to eight out of nine Ficus species, indicating a wide host‐range Ti plasmid. Agrobacteria were able to survive and move in the vascular system of galled ficus plants and to induce tumour growth in stem‐cutting propagated plants. Moreover, agrobacteria were detected in many healthy F. benjamina plants as part of the endophytic microflora. These findings suggest potential for spread of the disease through latently infected plant propagation material produced as cuttings or by tissue culture.

Rapid Identification of Agrobacterium Species By Staircase Electrophoresis of Low Molecular Weight RNA Profiles

Several plant species are infected by different species of the genus Agrobacterium. One problem is that no rapid and sensitive method is available for the identification of isolates of Agrobacterium at the species level. The usefulness of LMW RNA profiles for the identification of Agrobacterium species was examined. The profiles of strains belonging to the proposed species were identical to those of the type strain of each species except in two cases. In A. radiobacter, two groups of strains with different tRNA profiles were detected and in A. rhizogenes two groups with different 5S rRNA zones were found. Nevertheless, with the LMW RNA profiles it was possible to assign any isolate to one of each group within these species. The results obtained showed that all isolates studied here can be assigned to a species of Agrobacterium and hence that LMW RNA profiles offer a suitable method for the identification of tumor-inducing bacteria.

Characterization of plasmid-borne and chromosome-encoded traits of Agrobacterium biovar 1, 2, and 3 strains from France.

We collected 111 Agrobacterium isolates from galls of various origins (most of them from France) and analyzed both their plasmid-borne and chromosome-encoded traits. Phenotypic analysis of these strains allowed their classification in three phena which exactly matched the delineation of biovars 1, 2, and 3. A fourth phenon was identified which comprises three atypical strains. The phenotypic analysis has also allowed us to identify 12 additional characteristics which could be used to identify the three biovars of Agrobacterium. Our results also suggest that biovar 1 and 2 represent distinct species. Analysis of plasmid-borne traits confirmed that tartrate utilization is a common feature of biovar 3 strains (now named Agrobacterium vitis) and of Agrobacterium grapevine strains in general. Among pathogenic strains of Agrobacterium, several exhibited unusual opine synthesis and degradation patterns, and one strain of biovar 3 induced tumors containing vitopine and a novel opine-like molecule derived from putrescine. We have named this compound ridéopine.

Evidence of migration and endophytic presence of Agrobacterium tumefaciens in rose plants

Agrobacterium tumefaciens was isolated from stem tumors of several rose cultivars showing that the bacterium is the causal agent of aerial galls in rose plants. No differences were observed in the characteristics of the Agrobacterium isolates from crown or aerial galls. Stem inoculation of ten rose cultivars showed that all of them were susceptible to A. tumefaciens but differences in the size of the resulting tumors were observed. The movement of A. tumefaciens in rose plants was demonstrated using two wild type strains and two antibiotic resistant mutants. Three months after inoculation, the inoculated strains were recovered in the roots, crown and below and above the inoculation site but low numbers of pathogenic Agrobacterium cells were isolated. New tumors appeared in 5% of the noninoculated wounds. A. tumefaciens was isolated from the stem at different distances from the tumor in naturally infected plants. In symptomless commercial plants, the isolation from the roots, crown and at different stem levels demonstrated the existence of systemic and latent infections in rose. Direct isolation using a nonselective and selective media with or without a previous enrichment step were efficient methods for isolating tumorigenic Agrobacterium from the different parts of rose plants.

Isolation of Agrobacterium sp. strain KNK712 that produces N-carbamyl-D-amino acid amidohydrolase, cloning of the gene for this enzyme, and properties of the enzyme.

Agrobacterium sp. strain KNK712, which produced N-carbamyl-D-amino acid amidohydrolase (DCase) was isolated from soil. The bacterium had D-specific hydantoinase activity also. Both enzymes are suitable for use in the production of D-amino acids. The DCase gene from Agrobacterium sp. strain KNK712 was cloned into Escherichia coli. The cloned DNA fragment contained one open reading frame, predicted to encode a peptide of 304 amino acids, with a calculated molecular weight of 34,285. The DCase gene was overexpressed under the control of the lac promoter, and DCase accounted for 50% of the soluble protein in the cells. The enzyme was purified and some properties were investigated. Both the optimum pH and the pH that gave greatest stability were about pH 7.0. The optimum temperature was 65 degrees C, and the enzyme was stable at 55 degrees C. The enzyme had strict specificity toward N-carbamyl-D-amino acids, and was inhibited by thiol reagents, Cu2+, Hg2+, Ag+, and ammonia.

Ti plasmid-encoded genes responsible for catabolism of the crown gall opine mannopine by Agrobacterium tumefaciens are homologs of the T-region genes responsible for synthesis of this opine by the plant tumor

Agrobacterium tumefaciens NT1 harboring pSaB4, which contains the 14-kb BamHI fragment 4 from the octopine/mannityl opine-type Ti plasmid pTi15955, grew well with agropine (AGR) but slowly with mannopine (MOP) as the sole carbon source. When a second plasmid encoding a dedicated transport system for MOP was introduced, these cells grew well with both AGR and MOP. Transposon insertion mutagenesis and subcloning identified a 5.7-kb region of BamHI fragment 4 that encodes functions required for the degradation of MOP. DNA sequence analysis revealed seven putative genes in this region: mocD (moc for mannityl opine catabolism) and mocE, oriented from right to left, and mocRCBAS, oriented from left to right. Significant identities exist at the nucleotide and derived amino acid sequence levels between these moc genes and the mas genes that are responsible for opine biosynthesis in crown gall tumors. MocD is a homolog of Mas2, the anabolic conjugase encoded by mas2'. MocE and MocC are related to the amino half and the carboxyl half, respectively, of Mas1 (MOP reductase), the second enzyme for MOP biosynthesis. These results indicate that the moc and mas genes evolved from a common origin. MocR and MocS are related to each other and to a putative repressor for the AGR degradation system encoded by the rhizogenic plasmid pRiA4. MocB and MocA are homologs of 6-phosphogluconate dehydratase and glucose-6-phosphate dehydrogenase, respectively. Mutations in mocD and mocE, but not mocC, are suppressed by functions encoded by the chromosome or the 450-kb megaplasmid present in many Agrobacterium isolates. We propose that moc genes derived from genes located elsewhere in the bacterial genome and that the tumor-expressed mas genes evolved from the bacterial moc genes.

Agrocin 434, a new plasmid encoded agrocin from the biocontrol Agrobacterium strains K84 and K1026, which inhibits biovar 2 agrobacteria

Agrocin 434 is a previously unrecorded inhibitor produced by Agrobacterium strains K84 and K1026, which also secrete agrocin 84. These strains are used for the biological control of crown gall disease of plants and agrocin 434 may contribute to the biocontrol processes. When strain K84 was cured of the plasmids which encode agrocin 84 production (pAgK84) and nopaline catabolism (pAtK84b), the derivative strains retained both agrocin 434 production and the remaining 300-400 kb plasmid. This plasmid was mobilized into a plasmidless recipient and the resulting transconjugants also produced agrocin 434. Processes essential for biosynthesis of agrocin 434 are thus encoded on the large plasmid, for which the designation pAgK434 is recommended. Bioassays of the inhibitory effects of agrocin 434 were done with 68 Agrobacterium isolates from Australia, Europe and North America. All the biovar 2 ( A. rhizogenes) isolates tested were inhibited, with the exception of the strains which produced agrocin 434 themselves. None of the biovar 1 ( A. tumefaciens), biovar 3 ( A. vitis), or A. rubi strains tested was sensitive to agrocin 434. Preliminary chemical analyses indicated that the compound is a di-substituted cytidine nucleoside.

Agrobacterium Infections in Humans: Experience at One Hospital and Review

Agrobacteria are noted primarily for their phytopathogenicity and when isolated from human clinical specimens are often considered contaminants or organisms of low pathogenicity. We report six cases at one hospital over a 6 1/2-year period in which infection was accompanied by a compatible clinical syndrome and review 19 cases reported in the literature. Fourteen of the 25 combined cases involved central venous catheter-associated infections. Six cases involved peritonitis, five of which occurred in patients undergoing continuous ambulatory peritoneal dialysis. Additional infections included two non-catheter-associated bacteremias, one prosthetic valve endocarditis, and two urinary tract infections. Most infections were community acquired, and restriction enzyme analysis of Agrobacterium isolates from eight patients at one hospital revealed unique patterns in each case without evidence for clonal dissemination of these strains. Agrobacterium isolates may be resistant to multiple antibiotics, and optimal therapy has not yet been determined. Agrobacteria should be recognized as opportunistic pathogens in the immunocompromised host, particularly in those with indwelling plastic catheters.

Plasmid Heterogeneity in Spanish Isolates of Agrobacterium tumefaciens from Thirteen Different Hosts

Plasmid DNA was isolated from 80 Spanish isolates of Agrobacterium tumefaciens from 13 hosts of several geographical and temporal origins. One to five plasmids occurred in all of the isolates studied. Plasmid sizes varied between 5 and greater than 1,000 MDa. Generally, there was no correlation between plasmid number or size and geographical origin, host, biovar, sensitivity to agrocin 84, or opine-catabolizing ability of the different isolates.

Characterization of an Unusual New Agrobacterium tumefaciens Strain from Chrysanthemum morifolium Ram

We characterized five isolates of Agrobacterium tumefaciens from naturally occurring galls on Chrysanthemum morifolium. The isolates are similar, possibly identical, members of a single strain of A. tumefaciens that we designate Chry5. The strain is a biotype I, as indicated by its response to both newly described and traditional biotype tests. Chry5 produces tumors on at least 10 plant species. It is unusual in its ability to form efficiently large tumors on soybean (Glycine max), a species normally refractory to transformation. Chry5 is unable to utilize octopine or mannopine as a carbon source. Although Chry5 can catabolize a single isomer each of nopaline and succinamopine, it differs from other known nopaline and succinamopine strains in its insensitivity to agrocin 84. This pattern of opine catabolism is unique among Agrobacterium strains examined to date. All five isolates of Chry5 contain at least two plasmids, one of which shares homology with pTiB6.

Agrobacterium vitis sp. nov. for Strains of Agrobacterium biovar 3 from Grapevines

Agrobacterium isolates from grapevines that were previously characterized as biovar 3 strains were compared with the type strains of the four previously described species of the genus Agrobacterium, Agrobacterium tumefaciens, Agrobacterium radiobacter, Agrobacterium rhizogenes, and Agrobacterium rubi. Ten grapevine isolates were distinguished from other Agrobacterium species phenotypically by a number of biochemical tests and by their reaction with a monoclonal antibody raised to biovar 3. Levels of DNA binding between the type strains of previously described species and three grapevine isolates were estimated by optical measurement of initial DNA renaturation rates. The levels of DNA binding between grapevine isolates were 78 to 92%, and the levels of DNA binding with other type strains ranged from 7 to 47%. Our results indicate that the grapevine isolates are distinct from the type strains of the four previously described Agrobacterium species. A new species, Agrobacterium vitis, is proposed; the type strain is strain NCPPB 3554.

Isolation of Agrobacterium sp., strain from the Azolla leaf cavity

We have isolated a bacterial strain from surface sterilized fronds of the aquatic fern Azolla filiculoides and infer that it occurs in leaf cavities, along with the nitrogen-fixing cyanobacterium Anabaena azollae. This strain grew slowly on rich media, appeared to be Gram-negative, and was identified using a bacteriological multitest system as Agrobacterium sp. This strain, designated AFSR-1, could grow in the presence of 400 ppm of ammonium sulphate. DNA/DNA hydrodization analysis showed that a 16 S ribosomal RNA gene is located in the AFSR-1 strain on an identical DNA restriction fragment as in a strain of Agrobacterium tumefaciens. In addition, it was found that strain AFSR-1 contained DNA regions homologous to the modulation genes nodABC and nodL of Rhizobium trifolii, AFSR-1 contains a cryptic plasmid, and the genetic transfer of a broad-host-range plasmid pLAFR3 to strain AFSR-1 was achieved.

Characteristics of bacteria isolated from soil and roots of fruit trees

A total of 142 strains of aerobic bacteria were isolated from soil (43 strains) and roots of peach (51 strains) and apple (48 strains) trees using YA agar medium (egg-albumin agar medium containing yeast extract). On the basis of the morphological, physiological, and chemotaxonomic characteristics, these strains were divided into 6 groups. The strains of group 1 were assigned to the genus Agrobacterium on the basis of the presence of flagella, ubiquinone system and cellular fatty acid composition. Of 70 strains of this group, 34 and 29 strains were isolated from the roots of peach and apple trees, respectively. Forty percent of the isolates of Agrobacterium exhibited antagonism (in vitro) against the fungus Rosellinia necatrix. Of the remaining groups, the strains of groups 2 and 3 were assigned to the genus Pseudomonas and Xanthomonas, respectively. Strains of group 2 were isolated from only the roots and those of group 3 from only soil. Gram-positive (group 5) and spore-forming (group 6) bacteria were i...

Opines in crown gall tumours induced by biotype 3 isolates of Agrobacterium tumefaciens

Pathogenic properties and opine markers of biotype 3 (grapevine) isolates of Agrobacterium tumefaciens (Smith and Townsend) Conn were compared with those of biotypes 1 and 2. By contrast with biotypes 1 and 2, grapevine isolates induced only small tumours on Kalanchoe daigremontiana Hamet and Perrier stems but produced large organogenic galls on decapitated shoot tips of this plant species. Grapevine agrobacteria were classified according to their opine catabolic properties and the type of opine they induced in Kalanchoe tubiflora Hamet and grapevine ( Vitis vinifera L.) tumours. Crown gall tissues obtained after infection with octopine-utilizing grapevine isolates contained octopine in very large amounts compared to biotype 1 tumours. A similar quantitative difference was not observed when nopaline production of tumours incited by biotypes 1 and 2 and by grapevine isolates was examined. Tumours formed by a third class of grapevine isolates synthesized a novel opine, for which we propose the trivial name vitopine. Octopine, nopaline, agropine and vitopine were also detected in healthy tissues of plants infected with the appropriate Agrobacterium isolates, indicating that these opines can selectively promote the colonization of the whole plant by agrobacteria. Biotype 3 octopine isolates induced octopine synthesis at the infection sites of a crown gall-resistant grapevine hybrid, showing that pathogenic agrobacteria can transform plants without tumour formation.

Long-Term Storage of Plant-Pathogenic Bacteria in Sterile Distilled Water

This study was made to determine the effectiveness of the preservation of plant-pathogenic bacteria in sterile distilled water. After 20 or 24 years of storage in distilled water, a very high percentage (90 to 92%) of the isolates of Agrobacterium tumefaciens and Pseudomonas spp. were still alive. Moreover, 12 of 13 viable (after 24 years) isolates of P. syringae subsp. syringae maintained their ability to produce syringomycin and were pathogenic to bean seedlings. The water-stored cells of two isolates of P. syringae subsp. syringae, when observed by electron microscopy, were smaller than cells of 24-h-old subcultures of bacterial cells grown in nutrient broth; the water-stored cells appeared plasmolysed with an electron-dense cytoplasm and thickened cell wall.

Susceptibility of Agrobacterium tumefaciens Strains to Two Agrocin-Producing Agrobacterium Strains

Sixty-five strains and isolates of Agrobacterium tumefaciens representing each of the known biotypes, were tested for in vitro and in vivo susceptibility to the agrocin-producing strains Agrobacterium radiobacter 84 and A. tumefaciens D286. No biotype 3 strain was susceptible to the effects of either of the agrocinogenic strains in vitro. On datura and tobacco, the best inhibition of tumor formation was obtained when the agrocinogenic strains were applied to wounds 24 h before the pathogens and by the concomitant application of agrocin producer and pathogen at cell ratios of 10:1 or 3:1; inhibition of infection tended to decrease progressively as the cell ratio decreased from 10:1 to 3:1 to 1:1. Generally, strain 84 was superior to D286 in inhibiting tumor formation. A combined cell suspension of 84 and D286 was as effective as 84 alone. The overall pattern of inhibition of tumor formation by biotype 1 and 2 pathogens resistant to the agrocinogenic strains in vitro was similar to that obtained with strains that were susceptible in vitro.

Isolation and characterisation ofAgrobacteriumspecies

Abstract Two hundred and sixty-two isolates of Agrobacterium. (Smith & Towns.) Conn. from nursery soil were characterised by biochemical tests. Biotypes 1 and 2 of Keane et al. (1970; Australian Journal of Biological Science 23: 585-95) were represented. In addition a third group comprising 37 isolates was obtained. Isolates from this group shared characteristics of both biotype 1 and biotype 2 Agrobacterium and were designated biotype 1-2. Both biotype 1 and biotype 2 Agrobacterium were abundant in the rhizosphere.

Erratum: Characterization of Agrobacterium isolates from stone fruits in Ontario

Erratum: Characterization of <i>Agrobacterium</i> isolates from stone fruits in Ontario

Characterization of Agrobacterium isolates from stone fruits in Ontario

Isolations made from 68 naturally infected peach, plum, and sweet cherry nursery stock from the Niagara Peninsula and southwestern Ontario yielded 30 pathogenic isolates of Agrobacterium. These isolates were separated into two groups by 17 physiological and biochemical tests. The predominant group, consisting of 27 isolates, was 3-ketolactose negative and corresponded to biotype 2, and the other, consisting of only three isolates, corresponded to biotype 1 of Agrobacterium radiobacter var. tumefaciens sensu Keane, Kerr, and New (1970). Among the pathogenic isolates, 25 were sensitive and 5 were resistant to the bacteriocin produced by strain 84 of A. radiobacter var. radiobacter sensu Keane. Kerr, and New (1970).