Agrobacterium Isolates Research Articles

Diversity and functional traits of seed endophytes of Dysphania ambrosioides from heavy metal contaminated and non-contaminated areas.

Seed endophytes played a crucial role on host plants stress tolerance and heavy metal (HM) accumulation. Dysphania ambrosioides is a hyperaccumulator and showed strong tolerance and extraordinary accumulation capacities of multiple HMs. However, little is known about its seed endophytes response to field HM-contamination, and its role on host plants HM tolerance and accumulation. In this study, the seed endophytic community of D. ambrosioides from HM-contaminated area (H) and non-contaminated area (N) were investigated by both culture-dependent and independent methods. Moreover, Cd tolerance and the plant growth promoting (PGP) traits of dominant endophytes from site H and N were evaluated. The results showed that in both studies, HM-contamination reduced the diversity and richness of endophytic community and changed the most dominant endophyte, but increased resistant species abundance. By functional trait assessments, a great number of dominant endophytes displayed multiple PGP traits and Cd tolerance. Interestingly, soil HM-contamination significantly increased the percentage of Cd tolerance isolates of Agrobacterium and Epicoccum, but significantly decreased the ration of Agrobacterium with the siderophore production ability. However, the other PGP traits of isolates from site H and N showed no significant difference. Therefore, it was suggested that D. ambrosioides might improve its HM tolerance and accumulation through harboring more HM-resistant endophytes rather than PGP endophytes, but to prove this, more work need to be conducted in the future.

Potential uses of Lipopolysaccharide and Exopolysaccharide Isolated from Agrobacterium spp.

A gram-negative bacterium, Agrobacterium, synthesizes lipopolysaccharide and exopolysaccharide, both indispensable in establishing complex interactions with plant hosts. These polysaccharides have biocontrol applications. This study intended to determine: 1) LPS potential as insecticide; 2) EPS production capacity. LPSs obtained from Agrobacterium isolates were tested for insecticidal activity against test insects, damaging stored grains. Agrobacterial LPS elicits insect mortalities comparable to Permethrin (standard insecticide). Typical strains (At2, At3 At5, At6) incited 80% mortality of tested insects. EPS yield was quantified at regular intervals under varying physical parameters. Maximum EPS yield of 12.25 g/L was recorded for strain At1. Antibacterial activity of all isolates (before and after UV light exposure) was tested against Gram-positive and Gram-negative reference strains. Agrobacteria exposed to UV light exhibited antibacterial activity against Gram-positive reference strains. Therefore, agrobacterial LPS can be future candidate for biocontrol of insect pests in grain stocks. Moreover, agrobacteria can not only be prospective candidates for industrial scale EPS production but can also potentially help in treating infections by antibiotic resistant Gram-positive bacteria. Hence, same native isolates carry numerous benefits for the community.

Isolation and Pathogenecity Study of Agrobacterium From Rhizospheric Soil of Albizia Saman

Isolation and Pathogenecity Study of Agrobacterium From Rhizospheric Soil of Albizia Saman

Isolation and Characterization of Agrobacterium Strains from Soil: A Laboratory Capstone Experience.

In this investigation, the students’ goal was to isolate and characterize Agrobacterium strains from soil. Following selection and enrichment on 1A-t medium, putative Agrobacterium isolates were characterized by Gram stain reaction and biochemical tests. Isolates were further evaluated using polymerase chain reaction (PCR) with different primer sets designed to amplify specific regions of bacterial deoxyribonucleic acid (DNA). Primer sets included AGRH to identify isolates that were members of the Rhizobiaceae, BIOVAR1 primers to identify members of Agrobacterium biovar group I, and a third set, VIRG, to determine presence of virG (only present in pathogenic Agrobacterium strains). During the investigation, students applied previously learned techniques including serial dilution, use of selective/differential media, staining protocols, biochemical analysis, molecular analysis via PCR, and electrophoresis. Students also gained practical experience using photo documentation to record data for an eventual mock journal publication of the capstone laboratory experience. Pre- and post-evaluation of class content knowledge related to the techniques, protocols, and learning objectives of these laboratories revealed significant learning gains in the content areas of Agrobacterium–plant interactions (p ≤ 0.001) and molecular biology (p ≤ 0.01). The capstone journal assignment served as the assessment tool to evaluate mastery and application of laboratory technique, the ability to accurately collect and evaluate data, and critical thinking skills associated with experimental troubleshooting and extrapolation. Analysis of journal reports following the capstone experience showed significant improvement in assignment scores (p ≤ 0.0001) and attainment of capstone experience learning outcomes.

Comparative study of diagnostic methods used for monitoring of common grape vine (Vitis vinifera L.) crown gall (Agrobacterium vitis Ophel & Kerr) in Slovenia

Agrobacterium vitis causes common grape vine (Vitis vinifera L.) crown gall disease that destroyed a lot of Slovenian vineyards more than a decade ago. Eighty isolates of Agrobacterium spp. collected during monitoring in 2006 were identified as A. vitis and A. tumefacies by pehA and multiplex PCR method. Tumor-inducing capacity of these strains was assessed on test plants and with PCR methods for detection of the Ti plasmid responsible for tumor induction. With VCF3/VCR3 primer pair six false negatives and no false positives were detected. The high genetic diversity of pathogenic Agrobacterium spp. strains affects the performance of molecular methods, thus biological test should be performed where results from molecular methods are doubtful.

First Report of Agrobacterium vitis as Causal Agent of Crown Gall Disease of Grapevine in Tunisia.

Since October 2011, a serious outbreak of crown gall disease was observed on 1- and 2-year-old grapevines (Vitis vinifera L.) cv. Superior Seedless in several vineyards located in the region of Regueb in the center of Tunisia. Fifty isolates of Agrobacterium were isolated on a tartrate medium from galls of affected plants. To prepare template DNA, cell suspensions were lysed in 0.25% sodium-azide (NaN3) buffer prepared in 1% Triton X-100 by heating the samples at 95°C for 10 to 15 min (1). The strains were differentiated using a multiplex PCR assay with a combination of VIRFF1/VIRFR2 and VIRD2S4F716/VIRD2S4R1036 primers (2), which detect regions of virF and virD2 genes, respectively, in A. vitis strains carrying octopine or nopaline Ti plasmids and A. vitis vitopine strains. In order to differentiate A. vitis strains from A. tumefaciens strains, PGF/PGR (4), a polygalacturonase specific primer set, was added to the mixture in multiplex PCR. The isolates segregated into three main groups. The first group carries octopine type Ti plasmids, the second carries vitopine type Ti plasmids, and the third group carries both octopine and vitopine type Ti plasmids. The polygalacturonase gene sequence from 10 isolates showed 94 to 97% identity to the sequences of A. vitis previously deposited in the NCBI GenBank database (Accession No. CP000633.1gb). The biochemical test results corresponded to the results of genetic analysis. The ability to aerobically convert lactose to 3-ketolactose was tested by spotting bacteria onto medium containing lactose and flooding plates with a layer of Benedict's reagents after incubation at 28°C for 48 h. Acid production from glucose was tested by spotting bacterial strains onto potato dextrose agar (PDA) medium supplemented with CaCO3. Alkali production from L-tartrate was tested by streaking bacteria on AB minimal medium supplemented with L-tartrate and growth in salt medium was tested by streaking on nutrient broth supplemented with 2% NaCl. All isolates except one were negative in 3-ketolactose. They were negative in acid clearing on PDA-CaCO3, grew in 2% NaCl, and produced alkali from tartarate. Pathogenicity of all 50 strains was tested on 1-month-old tomato plants (Lycopersicum esculentum cv. Riograndi). Plants were inoculated on the stem by pricking one to three times through a drop of inoculum (108 CFU/ml) at three inoculation sites. Sterile distilled water was used as control treatment. Plants were grown for 4 weeks at 23 ± 3°C and symptoms were recorded. Typical tumors developed at the inoculation sites and no symptoms were observed on the control plants. In Tunisia, crown gall disease was observed only on stone fruit trees and only A. tumefaciens Biovar 1 have been reported and assigned to four genomic species G4, G6 G7, and G8 basically on the recA sequencing (3). To our knowledge, this is the first report of A. vitis determined as the causal agent of grapevine crown gall in Tunisia.

OCCURRENCE OF CROWN GALL OF THE GRAPEVINE IN TUNISIA AND CHARACTERIZATION OF TUNISIAN AGROBACTERIUM VITIS AND A. TUMEFACIENS STRAINS

The occurrence of crown gall disease in Tunisian vineyards was investigated. Crown gall symptoms were detected in several vineyards in the northern and central regions of the country but with low incidence (9.72%). Different Agrobacterium species were isolated and char- acterized. Morphological characterization, molecular as- says and pathogenicity tests were conducted to identify Agrobacterium isolates. A set of 380 isolates with mor- phologically Agrobacterium-like colonies on MG medi- um was obtained from different parts of diseased vines and rhizospheric soils. Eighty out of them were chosen for detailed study in comparison with Agrobacterium reference strains obtained from the French CFBP col- lection. Biovars 1, 2 and 3 were first distinguished on the three semi-selective media 1A, 2E and 3AN respec- tively. Further, the isolates were differentiated by BOX- PCR analysis. It revealed that 11 Tunisian isolates could be identified as A. tumefaciens and four as A. vitis. The assignment of six A. tumefaciens strains to genomic species was achieved using the housekeeping gene recA. Results confirmed those of the BOX-PCR analysis and showed 99% similarity of three strains with the A. tumefaciens G4 strain (CFBP7128) and a comparable similarity (99%) of two strains was recorded with the A. tumefaciens G4 strain (CFBP7126). A non pathogenic strain was identified as A. tumefaciens G7 with 98% similarity to CFBP 7129. Two strains were confirmed to belong to the A. vitis by analysis of their 16S genes. The virulence and tumourigenicity of these Tunisian isolates were analyzed by vir-PCR and pathogenicity tests on carrot disks and indicator plants. The study revealed a relatively low percentage of highly tumourigenic isolates in Tunisia. Only 16% of the 300 isolates tested on car- rot disks proved to be highly tumourigenic (++) and to form typical tumours around the cambium of carrot slices, whereas 36%, induced not clearly discernible tu- mours (+). The rest of the strains (48%) were negative (-). Of the 80 isolates tested on indicator plants, only two elicited tumours on tomato and tobacco plants. The vir-PCR test of 10 strains revealed the pathogenicity of five of them.

Protocol for Isolation and Identification of Agrobacterium Isolates from Stone Fruit Plants and Sensitivity of Native A. tumefaciens Isolates against Agrocin Produced by A. radiobacter Strain K84

Agrobacteria from rhizosphere soil and galls developed on stone fruit plants, were isolated on D1 medium. Agrobacterium isolates, belonging to biovar-1, developed glistening, transparent, convex colonies with cherry red centre at maturity on medium 1A and biovar-2 developed typical transparent to red coloured colonies on 2E. Single cell colonies of Agrobacterium took red colour when streaked on yeast extract mannitol agar supplemented with Congo red. Pathogenic Agrobacterium isolates (A. tumefaciens) developed galls whereas non-pathogenic isolates (A. radiobacter) failed to develop galls on stems of artificially inoculated 4-week old potted tomato plants. Agrocin production was detected in A. radiobacter isolates without and after exposure to chloroform. The results on number of flagella and their arrangement confirmed by scanning electron microscopy revealed that individual cells of all the native isolates possess single flagellum at their sub-polar ends. The isolate showed luxurious growth on Hofer’s alkaline, produced ketolactose and showed no acid production with bromothymol blue. All the native A. tumefaciens isolates were sensitive to agrocin produced by strain K84 which indicated their nopaline utilizing behaviour. All native Agrobacterium isolates showed 97–100 % sequence homology with A. radiobacter and A. tumefaciens.

Gamma-caprolactone stimulates growth of quorum-quenching Rhodococcus populations in a large-scale hydroponic system for culturing Solanum tuberosum

Bacteria degrading quorum sensing (QS) signals have been proposed as biocontrol agents able to quench QS-dependent expression of virulence symptoms caused by Pectobacterium on potato plants. We report here that gamma-caprolactone (GCL) treatment stimulated growth of the native QS-degrading bacterial community in an industrial plant hydroponic system for culturing Solanum tuberosum. Post-GCL treatment, QS-degrading bacteria were mainly identified as Rhodococcus isolates, while Agrobacterium isolates dominated under similar untreated conditions. Most of the assayed Rhodococcus isolates exhibited efficient biocontrol activity for protecting potato tubers. Analytical chemistry approach revealed the rapid degradation of GCL introduced in the plant cultures.

Evidence of pAgK84 transfer from Agrobacterium rhizogenes K84 to natural pathogenic Agrobacterium spp. in an Italian peach nursery

Nine Italian peach nurseries, which use Agrobacterium rhizogenes strain K84 to protect plants from crown gall, were monitored for three years with the aim of determining whether transconjugant populations may arise following plasmid exchanges between K84 and autochtonous soil agrobacteria. Six hundred and seventy‐eight Agrobacterium isolates were obtained from 120 tumours developed on apricot and peach rootstocks that had been treated in pre‐planting with the antagonist. Agrobacteria were characterized for pathogenicity, biovar, opine catabolism and agrocin 84 sensitivity. Colony hybridization was used for screening the isolates harbouring plasmids pTi and/or pAgK84. Analysis of plasmid content and Southern blotting were performed on putative transconjugant agrobacteria found in tumours collected from one nursery where a biological control breakdown was observed. The RFLP analysis of 16S + IGS regions showed that pAgK84 was transferred from the antagonist to virulent and avirulent soil agrobacteria belonging to different ribotypes. Pathogenic transconjugants, inoculated on GF677 rootstocks, were not controlled in vivo by K84 and stably maintained pTi and pAgK84 in the bacterial cells for at least one year. At the end of a biocontrol trial, new transconjugant tumorigenic agrobacteria originated by the transfer of pAgK84 to the pathogen. Virulent and avirulent transconjugants may represent a real threat for biological control by K84 strain since all of them produced agrocin and were insensitive to it. Survival in soil of these populations could make the future application of K84 ineffective.

Mode of Action of the Antibacterial Compound Ar26 Produced by Agrobacterium vitis Strain E26 with Activity against A. tumefaciens, A. rhizogenes and A. vitis

AbstractThe biological control bacterium Agrobacterium vitis strain E26 was previously shown to produce an antibacterial compound, Ar26. This compound was involved in the biocontrol process and inhibited grapevine crown gall‐causing A. vitis strains in vitro by an unknown mechanism. This work was undertaken to determine the antibacterial properties and mode of action of Ar26. In a well agar plate diffusion assay against 29 tumorigenic isolates of Agrobacterium spp., Ar26 displayed broad inhibitory activity against 27. All of the 10 A. vitis, 8 of 9 A. tumefaciens and 9 of 10 A. rhizogenes strains were sensitive to this compound. Agrobacterium vitis strains were more sensitive to Ar26 than A. tumefaciens or A. rhizogenes strains, with larger inhibition zones and lower minimal inhibitory concentration (MIC). Ar26 exhibited a bactericidal effect against A. vitis. This compound did not cause bacterial cell lysis, as determined by morphological observation with an electronic microscope. Also, no leakage of cytoplasmic materials from cells of A. vitis occurred after treatment with Ar26 at concentrations equivalent to the MIC. However, an inhibition of the incorporation of radiolabelled precursors into DNA, RNA and protein was observed after treatment with Ar26. Results obtained suggest that Ar26 inhibited DNA, RNA and protein syntheses in tumorigenic A. vitis.

포도나무 뿌리혹병 발생생태 및 포도 재배포장의 Agrobacterium 속의 밀도 조사

To investigate the severity of crown gall disease on grapevine, the ratio of healthy vs. galled grapevines and the presence of the pathogen of the disease in soil were measured in Korean vineyards. In field and greenhouse cultivations, the crown gall incidence of the cv. Kyoho grapevines was and and those of cv. Campbell Early was and , respectively. The higher populations of Agrobacterium spp. were isolated from soils of grapeveins with crown gall than from soils of noninfected vineyards. Based on the colony shapes and growth on plates, 480 isolates of Agrobacterium spp. from 21 soil samples were collected. Only 13 isolates out of 480 developed the gall on inoculated grapevines.

Phylogenetic and symbiotic characterization of rhizobial bacteria nodulating Argyrolobium uniflorum in Tunisian arid soils

Forty-two bacterial isolates from root nodules of Argyrolobium uniflorum growing in the arid areas of Tunisia were characterized by phenotypic features, RFLP, and sequencing of PCR-amplified 16S rRNA genes. The isolates were found to be phenotypically diverse. The majority of the isolates tolerated 3% NaCl and grew at temperatures up to 40 degrees C. Phylogenetically, the new isolates were grouped in the genera Sinorhizobium (27), Rhizobium (13), and Agrobacterium (2). Except for the 2 Agrobacterium isolates, all strains induced nodulation on Argyrolobium uniflorum, but the number of nodules and nitrogen fixation efficiency varied among them. Sinorhizobium sp. strains STM 4034, STM 4036, and STM 4039, forming the most effective symbiosis, are potential candidates for inoculants in revegetalisation programs.

Distribution of Agrobacterium tumefaciens Biovars in Jordan and Variation of Virulence

One hundred and nine Agrobacterium isolates were recovered from 68 samples(51 plant tumor and 17 soil) that were collected from different habitats in Northern Jordan. The isolated cultures were grouped into 3 biovars based on their biochemical characteristics and biovar I, II, and III comprised a total number of 46, 41, and 22 isolates, respectively. Isolates of biovar I were obtained primarily from the diseased peach, oak and rose plants, whereas isolates of biovar II and ill were obtained mostly from apple and grape plants, respectively. Twenty-nine isolates were found to be virulent to at least one of the tested hosts such as carrots, chickpeas, garden peas and tomato plants with a response of tumor formation or tumor with roots induction. Our result suggested that A. tumefaciens strains from tumor of various plants and soil of Jordan were diverse and they have a variation in their virulence.

Eucalyptus occidentalis plantlets are naturally infected by pathogenic Agrobacterium tumefaciens

In the Sidi M’djahed nursery (Algeria), over 60,000 eucalyptus (Eucalyptus occidentalis) plantlets exhibited tumour-like growths localized at the crown of the plants that resembled crown galls caused by Agrobacterium tumefaciens. Bacteria colonizing the galls were isolated and purified. Most (22 out of 24) of the isolates had cultural and biochemical characteristics similar to those of strains of the biovar 1 of A. tumefaciens. Twenty out of 22 Agrobacterium isolates induced tumour formation on various test plants. In PCR experiments, DNA extracted from these virulent strains yielded an amplification signal corresponding to a 247-bp fragment located within the virulence region of nopaline type Ti plasmid. Consistent with this, the opine nopaline was detected in the tumours induced on test plants – but not on eucalyptus plants. Nopaline was degraded by the 20 pathogenic isolates that were also sensitive to agrocin 84, indicating the presence of a nopaline-type pTi in these strains. The chromosomal region encoding the 16S rRNA was analyzed in a sub-population of the pathogenic agrobacterial isolates. The analyzed strains were found to belong to the ribogroup of the reference strain B6. Interestingly, Eucalyptus camaldulensis and Eucalyptus cladocalyx grown in the same nursery and in the same soil substrate developed no galls.

Genetic Characterization of Epilithic Cyanobacteria and Their Associated Bacteria

Epilithic phototrophic biofilms develop inside Roman Necropolis and Catacombs on rock surfaces exposed to artificial light sources and are composed by a microbial consortium dominated by cyanobacteria. In this work, six non-axenic cultures of Leptolyngbya sp. strains isolated from biofilms from different Roman hypogea and maintained in cultures from 11 to 20 years were analysed along with their associated bacteria isolated in culture. The employment of PCR-fingerprinting techniques, using HIP1 and ERIC derived primers, allowed the clustering in three groups of the six Leptolyngbya strains and the typing of their isolated bacteria. The bacterial fingerprinting patterns were in agreement with the 16S rRNA gene sequencing and showed the presence in Leptolyngbya isolates of Pseudomonas, Stenotrophomonas, Agrobacterium and Bacillus representatives that were detected also in biofilms sampled from catacombs.

Induction of root‐mat symptoms on cucumber plants by Rhizobium, but not by Ochrobactrum or Sinorhizobium, harbouring a cucumopine Ri plasmid

Ochrobactrum CSL 2573, Rhizobium CSL 2411 and Sinorhizobium CSL 2611 strains harbouring the Agrobacterium cucumopine Ri plasmid (pRi), previously were shown to induce root‐mat symptoms in an in vitro cucumber cotyledon assay. In whole‐plant, rockwool‐grown cucumber host tests Rhizobium CSL 2411 was shown to be as efficient an inducer of root‐mat symptoms as the virulent Agrobacterium radiobacter strain NCPPB 4042, which also harbours a cucumopine pRi. Conjugal transfer of pRi to ingressing, avirulent Agrobacterium isolates was observed within root tissues with symptoms. Ochrobactrum CSL 2573 and Sinorhizobium CSL 2611 were not able to induce root‐mat symptoms on plants. Rhizobium CSL 2411 and Ochrobactrum CSL 2573 were reisolated from inoculated plants, but Sinorhizobium CSL 2611 was not detected or isolated from inoculated plants 68 days after inoculation. It was postulated that the differences in pathogenicity observed between the in vitro and in situ host tests were caused by a lack of proper attachment to inoculated root tissues by pRi‐harbouring Ochrobactrum and Sinorhizobium in the whole‐plant host tests.

Genes for utilization of deoxyfructosyl glutamine (DFG), an amadori compound, are widely dispersed in the family Rhizobiaceae

Amadori compounds form spontaneously in decomposing plant material and can be found in the rhizosphere. As such, these compounds could influence microbial populations by serving as sources of carbon, nitrogen and energy to microorganisms expressing suitable catabolic pathways. Two distinct sets of genes for utilization of deoxyfructosyl glutamine (DFG), an Amadori compound, have been identified in isolates of Agrobacterium spp. One, the soc gene set, is encoded by pAtC58, a 543 kb plasmid in A. tumefaciens strain C58. The second, mocD dissimilates DFG formed in the pathway for catabolism of mannopine (MOP) a non-Amadori, imine-type member of the mannityl opine family characteristic of certain Ti and Ri plasmids. To assess the level of dispersal of these two Amadori-utilizing systems, isolates of Agrobacterium spp. and related bacteria in the family Rhizobiaceae were examined by Southern analysis for homologs of socD and mocD. Homologs of mocD were associated only with Ti plasmid-encoded pathways for catabolism of MOP. Homologs of socD were more widely distributed, being detectable in many but not all of the isolates of Agrobacterium, Sinorhizobium, and Rhizobium spp. tested. However, this gene was never associated with the virulence elements, such as the Ti and Ri plasmids, in these strains. Regardless of genus most of the isolates containing socD homologs could utilize DFG as sole source of carbon, nitrogen and energy. Correlation studies suggested that mocD has evolved uniquely as part of the mannityl opine catabolic pathway while socD has evolved for the general utilization of Amadori compounds. Certain isolates of Agrobacterium and Rhizobium that lacked detectable homologs of socD and mocD also could utilize DFG suggesting the existence of additional, unrelated pathways for the catabolism of this Amadori compound. These results suggest that Amadori compounds constitute a source of nutrition that is important to microflora in the rhizosphere.

Molecular Typing of Agrobacterium Species Isolates From Catheter-Related Bloodstream Infections

Agrobacterium isolates from intravenous catheters of three hospitalized patients were initially identified as A. tumefaciens, but inability to produce 3-ketolactose revealed that two of them were A. vitis. However, rDNA analysis correlated all of the isolates to A. tumefaciens. Pulsed-field gel electrophoresis analysis ascertained the nosocomial transmission of the infection.

Tumorigenic Agrobacterium sp. Isolated from Weeping Fig in Spain.

Agrobacterium-like colonies were recovered onto Roy-Sasser's medium from a young tumor (4 cm in diameter) on the stem of weeping fig (Ficus benjamina L.), 10 cm from the crown. The galled plant was collected in 1999 from a garden center in Valencia, Spain. After colony purification and tomato and weeping fig plant inoculations, one nonpathogenic and five Agrobacterium isolates that were tumorigenic in both plant species were characterized. On the basis of biovar classification tests, the nonpathogenic isolate was identified as belonging to biovar 1 of Agrobacterium (now called A. tumefaciens), whereas the tumorigenic isolates could not be assigned to any of the known Agrobacterium biovars. The isolates were positive for oxidase, growth in 2% NaCl, production of alkali from l-tartaric acid, and production of acid from mannitol-CaCO3 and negative for 3-ketolactose production, growth and pigmentation in ferric ammonium citrate, growth at 35°C, citrate utilization, acid production from sucrose and melezitose, and alkali production from malonic acid. Nopaline was the unique opine found in galls induced in weeping fig plants inoculated with the pathogenic isolates. Moreover, all isolates utilize the opine nopaline, but not octopine, manopine, agropine, chrysopine, cucumopine, or mikimopine. They were susceptible to agrocin 84 produced by strain K84. Heat-treated bacterial suspensions of these isolates yielded the expected amplification product using polymerase chain reaction (PCR) with the FGPtmr530/FGPtmr701' primers pair from the tmr gene (3). Aerial gall disease was first reported on F. benjamina in Florida (1), and the isolated agrobacteria belongs to a new species named A. larrymoorei (2). Later, tumorigenic agrobacteria from weeping fig galls were isolated in Italy and the Netherlands (4). Our data suggest that the tumorigenic strains isolated in Spain differ greatly from those first described in the United States (1) on the basis of alkali production from l-tartaric acid, chrysopine detection on tumors, susceptibility to agrocin 84, and tmr amplification, but they might be similar to some of the Italian isolates (4). To our knowledge, this is the first report of isolation of tumorigenic Agrobacterium sp. from F. benjamina L. in Spain.