virD2 Genes Research Articles

The Type IV Secretion System (T4SS) Mediates Symbiosis between Bradyrhizobium sp. SUTN9-2 and Legumes.

There has been little study of the type IV secretion system (T4SS) of bradyrhizobia and its role in legume symbiosis. Therefore, broad host range Bradyrhizobium sp. SUTN9-2 was selected for study. The chromosome of Bradyrhizobium sp. SUTN9-2 contains two copies of the T4SS gene, homologous with the tra/trb operons. A phylogenetic tree of the T4SS gene traG was constructed, which exemplified its horizontal transfer among Bradyrhizobium and Mesorhizobium genera. They also showed similar gene arrangements for the tra/trb operons. However, the virD2 gene was not observed in Mesorhizobium, except M. oppotunistum WSM2075. Interestingly, the orientation of copG, traG, and virD2 cluster was unique to the Bradyrhizobium genus. The phylogenetic tree of copG, traG, and virD2 demonstrated that copies 1 and 2 of these genes were grouped in different clades. In addition, the derived mutant and complementation strains of T4SS were investigated in representative legumes Genistoids, Dalbergioids, and Millettiods. When T4SS copy 1 (T4SS1) was deleted, the nodule number and nitrogenase activity decreased. This supports a positive effect of T4SS1 on symbiosis. In addition, delayed nodulation was observed 7 dpi, which was restored by the complementation of T4SS1. Therefore, T4SS plays an important role in the symbiotic interaction between Bradyrhizobium sp. SUTN9-2 and its leguminous hosts. IMPORTANCE SUTN9-2 is a broad host range strain capable of symbiosis with several legumes. Two copies of T4SS clusters belonging to the tra/trb operon are observed on chromosomes with different gene arrangements. We use phylogenetic tree and gene annotation analysis to predict the evolution of the tra/trb operon of rhizobia. Our finding suggests that the gene encoding the T4SS gene among Bradyrhizobium and Mesorhizobium may have coevolution. In addition, Bradyrhizobium has a uniquely arranged copG, traG, and virD2 gene cluster. The results of T4SS1 gene deletion and complementation revealed its positive effect on nodulation. Therefore, T4SS seems to be another determinant for symbiosis. This is the first report on the role of T4SS in Bradyrhizobium symbiosis.

NATIVE BACTERIA IN RASPBERRY CROWN GALL REDUCE THE SEVERITY OF Agrobacterium tumefaciens

Native bacterial populations in crown galls caused by Agrobacterium tumefaciens may harbour bacteria of interest for biocontrol of this pathogen. In this study, we explored the density of native bacterial populations in crown galls of raspberry (Rubus ideaus) and evaluated their in vitro and in vivo antagonism against A. tumefaciens. Bacteria morphologically similar to A. tumefaciens were isolated from six gall samples and identified by virD2 gene sequencing. Bacterial population density was calculated by direct plate count on nutrient agar and R2A media. The in vitro antagonism efficiency index against A. tumefaciens of the most frequent bacteria was evaluated by dual confrontation on nutrient agar medium, and in vivo by inoculation of 1.5 x 108 UFC mL-1 in the root of tomato (Solanum lycopersiucm) plants under greenhouse conditions. By direct sequencing and biovar characterization, it was identified as A. tumefaciens biovar 1 in raspberry galls. Native bacterial populations in galls have variable density and their diversity is limited. By partial amplification of the 16S rRNA gene, 13 strains were identified with the highest frequency in the genera Pseudomonas (61.5 %), Bacillus (15.3 %), Alcaligenes (15.3 %) and Delftia (7.6 %). Among these, Alcaligenes faecalis showed the highest in vitro antagonism index (p ≤ 0.05) against A. tumefaciens, followed by Delftia sp. and Pseudomonas citronellolis. In vivo inoculation of tomato plants with these antagonists against Agrobacterium tumefaciens did not prevent infection; however, Alcaligenes faecalis significantly reduced (p ≤ 0.05) the severity of plant stem tumours. A. faecalis is the most efficient antagonist in vitro and in vivo against A. tumefaciens.

Establishment of Gypsophila paniculata root culture for biomass, saponin, and flavonoid production

Baby’s breath (Gypsophila paniculata L.) roots are valuable as pharmaceuticals due to the content of triterpenoid saponins and other bioactive phytochemicals. However, the long root harvest period and fluctuation of these constituent’s content are among the constraints to traditional agricultural production. The present study shows an efficient and reliable liquid root culture of G. paniculata cv. ‘Perfecta’ established in a shake flask system using different auxins, media, and sugars. Hairy root (HR) induction in G. paniculata through Rhizobium rhizogenes-mediated transformation was also investigated. α-Naphthaleneacetic acid (NAA) was optimized for adventitious roots (AR) biomass, saponin, and flavonoid production at 1 or 2 mg/L compared to IBA. Full strength Gamborg’s medium (B5) recorded higher saponin content; however, the highest yield of total saponin and total flavonoids was achieved by full strength Murashige and Skoog's (MS) medium. Sucrose was more essential for root growth and accumulated total saponins and flavonoids rather than fructose and glucose. Polymerase chain reaction (PCR) analysis showed that G. paniculata HR carried rolC gene of R. rhizogenes A4 strain in its genome but not virD2 gene. Compared to non-transformed root, saponin content of leaf and stem-derived HR was 2.7 and 2.3-fold, while total flavonoid content was 2.1 and 2.0-fold, respectively. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity was also higher in HR extracts than in non-transgenic roots. This study established an efficient protocol for G. paniculata root cultures for sustainable production of important natural saponins and flavonoids.

Unique TLR9 Activation by Helicobacter pylori Depends on the cag T4SS, But Not on VirD2 Relaxases or VirD4 Coupling Proteins

The genomes of the gastric bacterial pathogen Helicobacter pylori harbor multiple type-IV secretion systems (T4SSs). Here we analyzed components of three T4SSs, the cytotoxin-associated genes (cag) T4SS, TFS3 and TFS4. The cag T4SS delivers the effector protein CagA and the LPS-metabolite ADP-heptose into gastric epithelial cells, which plays a pivotal role in chronic infection and development of gastric disease. In addition, the cag T4SS was reported to facilitate conjugative transport of chromosomal bacterial DNA into the host cell cytoplasm, where injected DNA activates intracellular toll-like receptor 9 (TLR9) and triggers anti-inflammatory signaling. Canonical DNA-delivering T4SSs in a variety of bacteria are composed of 11 VirB proteins (VirB1-11) which assemble and engage VirD2 relaxase and VirD4 coupling proteins that mediate DNA processing and guiding of the covalently bound DNA through the T4SS channel. Nevertheless, the role of the latter components in H. pylori is unclear. Here, we utilized isogenic knockout mutants of various virB (virB9 and virB10, corresponding to cagX and cagY), virD2 (rlx1 and rlx2), virD4 (cag5, traG1/2) and xerD recombinase genes in H. pylori laboratory strain P12 and studied their role in TLR9 activation by reporter assays. While inactivation of the structural cag T4SS genes cagX and cagY abolished TLR9 activation, the deletion of rlx1, rlx2, cag5, traG or xerD genes had no effect. The latter mutants activated TLR9 similar to wild-type bacteria, suggesting the presence of a unique non-canonical T4SS-dependent mechanism of TLR9 stimulation by H. pylori that is not mediated by VirD2, VirD4 and XerD proteins. These findings were confirmed by the analysis of TLR9 activation by H. pylori strains of worldwide origin that possess different sets of T4SS genes. The exact mechanism of TLR9 activation should be explored in future studies.

Identification of the causal agents of crazy root disease on hydroponically cultivated cucumber plants in Poland

In April 2019, hydroponically cultivated cucumber plants with characteristic symptoms of crazy root disease were found in two different commercial production cucumber greenhouses in Poland. Due to excessive and inappropriate root growth, this disease led to a reduction in yield and deterioration of the general conditions of infected plants. Bacteria isolated from the roots were subjected to a morphological evaluation, as well as molecular, biochemical and pathogenicity tests. To identify the bacteria causing the disease, Agrobacterium-like colonies were subjected to PCR with primers complementary to the pathogenicity-related genes located on the crazy root-inducing plasmid (Ri-plasmid): the virD2A + virD2E primers complementary to the virD2 gene and the rolBF + rolBR primers complementary to the rolB gene. The pathogenicity of the isolated strains was studied in sunflowers and cucumbers. Twelve strains positive for the Ri plasmid, as determined by PCR, and pathogenic to sunflowers were identified based on sequence analysis of the 16S rRNA and recA genes. One strain was classified as belonging to the genus Pararhizobium, three to Rhizobium, and eight to Agrobacterium biovar 1, with the highest similarity to genomospecies G3. The results of the analyses suggest that these strains may belong to a new, thus far, undescribed species. To confirm this hypothesis, further phylogenetic studies are required.

Development of a real-time PCR assay using locked nucleic acid probe for the detection of octopine, nopaline, and vitopine strains of tumorigenic Allorhizobium vitis in grapevines

Crown gall disease caused by Allorhizobium vitis is one of the most destructive diseases in grapevine cultivation from an economical perspective. The bacterial pathogen exists systemically in grapevines and spreads through cuttings used for propagation material obtained from grapevines, living vines, and mother blocks, which poses a high risk for certified grapevine nursery material cultivation, nursery industry, and growers. Therefore, in this study, real-time PCR-based methods were developed for effectively and accurately determining the presence and the density of the bacterial pathogen in grapevine nursery material and the data used for indexing programs. Octopine-, nopaline-, and vitopine-catabolizing A. vitis strain-specific primer and locked nucleic acid (LNA) probe sets were determined depending on the ocs, nos, vis, vitopine iaaM, and vitopine virD2 genes. Primer-probe sets were then used to amplify the octopine synthase gene (62 bp) from octopine strains, the nopaline synthase gene (78 bp) from nopaline strains, and the vitopine synthase gene (60 bp) from vitopine strains, as well as the vitopine iaaM gene (60 bp) and the vitopine virD2 gene (66 bp) of A. vitis. The detection of A. vitis strains from bacterial suspension or extracted plant sap could be achieved within a short time (i.e., 20–25 min) with real-time PCR-based assays using the designed primer and probe sets. For the real-time PCR-based methods, the bacterial cell sensitivity threshold was defined as sensitivity threshold for one bacterial cell and at the DNA level, which was 1.4–1.9 fg. In real-time PCR-based methods, the corresponding PCR efficiencies for octopine and nopaline strains were 97.12% and 97.87%, respectively and for vitopine strains were 94.39%, 96.76%, and 99.01%. In this study, a new real-time PCR-based method was developed that uses the LNA probe, and different opine strains of A. vitis were detected and quantified from vegetative materials of infected grapevine.

First report of root mat disease in a hydroponic tomato production system caused by rhizogenic Agrobacterium biovar 1 in South Korea.

In June 2019, root mat disease was observed in hydroponically cultivated tomatoes in Jinju, South Korea, which occurred in at least 30% of the plants in the greenhouse. To isolate the causal bacteria, 10 g of infested tomato root mat sample was ground with 50 mL of sterile water. A 100-µL aliquot of the homogenate was serially diluted and spread on Mannitol-Glutamate (MG) medium amended with 0.1% yeast extract (MGY) and incubated at 28°C for 48 hours. Fifteen dominant colonies that formed on the MGY medium were purified and subjected to diagnostic polymerase chain reaction (PCR) based on the virD2-ipt gene loci. Because Ti-plasmid harbors both virD2 and ipt genes, Ri-plasmid-borne Agrobacterium species with only virD2 can be differentiated using virD2-ipt PCR. To amplify virD2, the primers 5'-ATG CCC GAT CGA GCT CAA GT-3' and 5'-TCG TCT GGC TGA CTT TCG TCA TAA-3' were used; for ipt amplification, the primers 5'-GAT CG(G/C) GTC CAA TG(C/T) TGT-3' and 5'-GAT ATC CAT CGA TC(T/C) CTT-3' were used. Amplification involved an initial 94°C for 1 min and then 40 cycles at 94°C, 50°C, and 72°C for 1 min at each temperature, with a final 5-min extension at 72°C. For three strains (GNIY2, J10, and J11), virD2 PCR products, but no ipt PCR products, were identified, indicating that three strains are Ri-plasmid-borne Agrobacterium species. A pathogenicity test was performed on 2-week-old tomato plants. Bacteria isolates (GNIY2, J10, and J11) cultured overnight in LB were made into a bacterial suspension (107 cfu/mL) in 50 mM phosphate buffer. Five tomato seedling roots were cut with sterilized scissors and soaked in each bacterial suspension for 1 hour. Phosphate buffer was used as a negative control. The inoculated tomato seedlings were transplanted in new pots and placed in a greenhouse with 25°C day and 20°C night temperature set points in natural light. After 9 weeks, all inoculated tomato plants produced overgrown roots, while the negative control plants had no symptoms. To satisfy Koch's postulates, re-isolation was performed from the diseased tomato and was the re-isolated bacteria were subject to partial 16S rDNA sequencing. Biovar tests performed as previously described revealed that all three isolates were biovar 1. A representative strain (GNIY2) was deposited in the Korean Agricultural Culture Collection (KACC 21759). To confirm the identity, four housekeeping genes of KACC 21759 were sequenced (16S rRNA, trpE, rpoB, and recA) and deposited in GenBank (accession nos. MT071560, MT444428, MT444429, and MT444430). Multilocus sequence analysis performed as previously described showed that the KACC 21759 strain was grouped in Agrobacterium genomospecies 4. This is the first report on mat root disease caused by Agrobacterium biovar 1 in hydroponic tomatoes in South Korea.

Effects of Agrobacterium tumefaciens strain types on the Agrobacterium-mediated transformation efficiency of filamentous fungus Mortierella alpina.

Four Agrobacterium tumefaciens strains AGL-1, C58C1, EHA105 and LBA4404 were tested for the effects of strain types on the transformation efficiency in Mortierella alpina. Results showed that AGL-1, C58C1 and EHA105 transformed M. alpina successfully. Among them, A. tumefaciens EHA105 was first proven successful transformation of M. alpina. AGL-1 and EHA105 had the highest transformation efficiency among the four strains, while LBA4404 failed to transform M. alpina. The reason leading tothe transformation efficiency difference among the four strains was explored bydetermining transcription levels of the virulence (vir) gene in the induction medium. Results showed that the expressions of virD1, virD2, virD4 and virE1 genes were obviously induced by acetosyringone in all the strains, and their transcriptional levels as well as virA's of AGL-1, C58C1 and EHA105 were higher than that of LBA4404, suggesting high transcriptional levels of vir genes were important for successful transformation. The study selected A. tumefaciens with high transformation efficiency of M. alpina, and would accelerate the genetic management of M. alpina. SIGNIFICANCE AND IMPACT OF THE STUDY: Oleaginous filamentous fungus Mortierella alpina is a commercial strain for the production of arachidonic acid. Genetic manipulation of M. alpina requires highly efficient transformation method. In this study, we explore the effect of Agrobacterium tumefaciens strain types on the transformation efficiency of M. alpina and select A. tumefaciens with the highest transformation efficiency, which accelerates the genetic manipulation of M. alpina. Besides, high transcriptional levels of virulence genes in A. tumefaciens were proven to play an important role for successful transformation.

Study creating and cloning hairy root lines in Talinum paniculatum plants

Talinum paniculatum Gaertn. contains flavonoid and saponins that have strong antioxidant properties, which are used to treat a number of conditions such as inflammation, allergies, stomach ulcers, ... However, the amount of flavonoid synthesized naturally in Talinum paniculatum plants is very low (about 0.897 mg /g fresh leaves). Therefore a method has been proposed for enhancing flavonoid content in Talinum paniculatum plants by applying tissue culture technique to produce the hairy roots to enhance biomass. This study present the results of optimization of the gene transfer process to create the hairy root lines through Agrobacterium rhizogenes (A. rhizogenes) in Talinum paniculatum plants. Of the three types of materials that infect by A. rhizogenes (cotyledon, stem, leaf tissue), leaf tissue is a suitable material for transforming and inducing hairy roots. Density of bacteria corresponding to OD600 value=0.6; concentration AS 100 μmol/l; Infection time of 10 minutes; 2 days of co-culture; cefotaxime concentrations of 500 mg/l are suitable conditions for inducing hairy roots from leaf tissue. In state of the liquid MS medium without growth regulator, shaking culture conditions are suitable for hairy roots growth. The test result of presence of rolC gene by PCR and the absence of the virD2 gene asserted that 5 lines of hairy roots were produced from Talinum paniculatum plants.
 

The complete nucleotide sequence of an IncP-2 megaplasmid unveils a mosaic architecture comprising a putative novel blaVIM-2-harbouring transposon in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa , bla VIM-2 has been mostly associated with a chromosomal location and rarely with a plasmid backbone. Until now, only three complete bla VIM-2 -carrying plasmid sequences have been described in this species. Here we explore the modular structure of pJB37, the first bla VIM-2 -carrying megaplasmid described in P. aeruginosa . The complete nucleotide sequence of plasmid pJB37 was determined with an Illumina HiSeq, with de novo assembly by SPAdes, annotation by RAST and searching for antimicrobial resistance genes and virulence factors. Conjugation assays were conducted. Megaplasmid pJB37 (464 804 bp long and GC content of 57.2%) comprised: an IncP-2 repA-oriV-parAB region; a conjugative transfer region ( traF , traG , virD2 and trbBCDEJLFGI genes); Tn 6356 , a new putative bla VIM-2 -carrying transposon; heavy metal (mercury and tellurite) resistance operons; and an arsenal of virulence genes. Plasmid pJB37 was transferable by conjugation to a spontaneous rifampicin-resistant mutant of P. aeruginosa PAO1. Here, a bla VIM-2 -harbouring In58 integron was associated with a new complex transposable structure, herein named Tn 6356 , suggesting that In58 was most likely acquired by insertion of this element. The mosaic arrangement exhibited by the pJB37 IncP-2 megaplasmid, which highlights the vast assembly potential of distinct genetic elements in a Pseudomonas widespread plasmid platform, gives new insights into bacterial adaptation and evolution.

Biochemical and genetic characterisation of Agrobacterium tumefaciens the causal agent of walnut crown gall disease in Iran

In 2009–2010, crown tumours were collected from walnut (Juglans regia L.) trees in northern Iran. Gram-negative, rod shaped and aerobic bacteria with circular, convex and white-coloured colonies on potato dextrose agar plus CaCO3 medium were isolated from galls. In pathogenicity tests, tomato seedlings were inoculated with all strains and tumours started to appear three weeks after inoculation. Strains yielded a 224 bp amplicon from the virD2 gene in PCR. When the 16S rRNA gene sequence of strains was compared by BLASTn with nucleotide sequences from GenBank, it showed 99.6% identity with the 16S rRNA sequence of Agrobacterium tumefaciens ATCC 33970. Based on phenotypic and genotypic properties, the bacterium that causes crown gall of walnut trees was identified as A. tumefaciens.

Identification and characterization of Agrobacterium spp. isolated from apricot in Serbia

Crown gall, caused by tumorigenic strains of Agrobacterium spp., is considered one of the most important diseases in stone fruit nurseries throughout the world. Since the crown gall disease has not been studied extensively in Serbia for more than 30 years, the objective of this study was to isolate, identify and characterize the bacterium associated with crown gall symptoms on one-year-old apricot trees. Samples were collected from a nursery in central Serbia and subjected to laboratory analysis. Bacteria were isolated from tumour tissue on yeast mannitol agar (YMA) and six gram-negative isolates were selected for further study. PCR assay using primers specific for virD2, virC, ipt and tms2 pathogenicity-associated plasmid genes revealed that isolates harbour Ti plasmids. All studied strains carrying Ti plasmids were classified as nopaline-type based on further molecular analysis. Using a multiplex PCR assay, targeting 23S rRNA gene sequences, and physiological and biochemical tests, five strains were assigned as A. rhizogenes/biovar 2 and the remaining one as A. tumefaciens/biovar 1. Identity of the strains was confirmed by sequence analysis of the 16S rRNA gene. In pathogenicity assay, all six strains caused tumour formation on inoculated carrot root discs, young tomato and sunflower plants.

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.

Development of a Magnetic Capture Hybridization Real-Time PCR Assay for Detection of TumorigenicAgrobacterium vitisin Grapevines

Agrobacterium vitis, the causal agent of grape crown gall, can have severe economic effects on grape production. The bacterium survives systemically in vines and, therefore, is disseminated in propagation material. We developed an assay for use in indexing programs that is efficient and sensitive for detecting A. vitis in grape tissue. Initially, real-time polymerase chain reaction (PCR) primers specific for diverse tumorigenic strains of A. vitis were developed using the virD2 gene sequence. To overcome the effects of PCR inhibitors present in plant tissue, DNA extraction methods that included magnetic capture hybridization (MCH), immunomagnetic separation (IMS), and extraction with the Mo Bio Powerfood kit were compared. The assays incorporating MCH or IMS followed by real-time PCR were 10,000-fold more sensitive than direct real-time PCR when tested using boiled bacterial cell suspensions, with detection thresholds of 10(1) CFU/ml compared with 10(5) CFU/ml. DNA extraction with the Powerfood DNA extraction kit was 10-fold more sensitive than direct real-time PCR, with a detection threshold of 10(4) CFU/ml. All three assays were able to detect A. vitis in healthy-appearing grapevine cuttings taken from infected vines.

Genetic diversity of Iranian Agrobacterium strains from grapevine

Crown gall of grapevine is an economically important disease in all areas where grapes are grown, but it is particularly severe in regions with cold climates. The purpose of this study was twofold: (1) to compare available Agrobacterium primer sets and identify a more reliable PCR analysis that can detect tumorigenic and non-pathogenic A. vitis and A. tumefaciens strains involved in the crown gall disease of grapevines in Iran and (2) to detect the genetic diversity of these strains by insertion sequence 50 (IS50) PCR as the genetic marker. Based on the results of morphological, physiological, biochemical, and pathogenicity tests and amplification of the virD2 gene fragment and subsequent comparison of IS50-PCR fingerprints of the strains, we identified 27 of all 40 strains tested as tumorigenic A. vitis, three strains as non-tumorigenic A. vitis, and ten strains as tumorigenic A. tumefaciens. The similarity of all pairwise combinations was determined using Jaccard’s coefficient, and cluster analysis was performed using the unweighted pair group method with arithmetic average (UPGMA) clustering. On the basis of the fingerprint patterns generated by IS50-PCR,, the isolated strains were differentiated into four clusters (A–D) at the 60% similarity level. These four clusters could be further divided into several subgroups at the 80% similarity level. These results demonstrate that there is a wide variation in the sensitivity patterns of different Agrobacterium strains to different antibiotics.

First Report of Agrobacterium vitis as the Causal Agent of Grapevine Crown Gall in Serbia.

In November 2010, a serious outbreak of crown gall disease was observed on 3-year-old grapevine (Vitis vinifera L.) cv. Cabernet Sauvignon grafted onto Kober 5BB rootstock in two commercial vineyards located in the South Banat District in Serbia. Large, aerial tumors were visible above the grafting point on grapevine trunks, and in most cases, the tumors completely girdled the trunk. From the gall tissues, white, circular, and glistening bacterial colonies were isolated on yeast mannitol agar medium. Eight, nonfluorescent, gram-negative, and oxidase-positive strains were isolated from seven tumor samples and selected for further identification. PCR assays with A/C' (1) and VCF3/VCR3 (4) primers corresponding to the virD2 and virC genes yielded 224- and 414-bp fragments, respectively, confirming that the strains harbored the plasmid responsible for pathogenicity. The strains were differentiated to the species/biovar level with a multiplex PCR assay targeting 23S rRNA gene sequences (3) and were identified as Agrobacterium vitis. The 16S rDNA gene sequence from one isolate (GenBank Accession No. JN185718) showed 99% identity to the sequences of A. vitis previously deposited in NCBI GenBank database. The physiological and biochemical test results corresponded to the results of genetic analysis (2). The strains grew at 35°C and in nutrient broth supplemented with 2% NaCl. They were negative in 3-ketolactose, acid clearing on PDA supplemented with CaCO3, and ferric ammonium citrate tests; nonmotile at pH 7.0; pectolytic at pH 4.5; utilized citrate; produced acid from sucrose and alkali from tartarate. Pathogenicity was confirmed by inoculation of three plants per bacterial strain on grapevine cv. Cabernet Franc and on a local cultivar of tomato (Lycopersicon esculentum L.). The 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 a negative control. Inoculated plants were maintained in a greenhouse at 24 ± 3°C. Typical tumors developed at the inoculation sites on tomatoes 3 weeks after inoculation and on grapevine 6 weeks after inoculation. No symptoms were observed on the control plants. Bacteria were reisolated from tumorigenic tissues and identified as pathogenic A. vitis by PCR. Crown gall disease was sporadically observed in vineyards in Serbia in previous years, but did not cause significant damage. Therefore, the causal agent was not studied in detail. To our knowledge, this is the first report of A. vitis determined as the causal agent of grapevine crown gall in Serbia.

Differentiation of Phytopathogenic agrobacterium spp.

Due to the difficulties in differentiation of phytopathogenic Agrobacterium spp. and lack of a standardized protocol, we carried out selection and evaluation of suitable methods based on the bacterial physiological, genetic and pathogenic properties. Strains of Agrobacterium tumefaciens, A. rhizogenes and A. vitis were differentiated using standard bacteriological and molecular methods. The biochemical and physiological tests confirmed authenticity of the strains. Two duplex PCR methods were conducted with four different primer pairs. In all strains, presence of plasmid virD2 and virC pathogenicity genes was detected. Chromosomal pehA gene was determined in A. vitis strain. Pathogenicity was confirmed on carrot slices and young plants of tomato and sunflower. Strains of A. tumefaciens and A. vitis were pathogenic on all test plants, while strain of A. rhizogenes induced characteristic symptoms only on carrot slices. The tests used in this study provided reliable discrimination between the three species and confirmed their identity as tumorigenic (Ti) Agrobacterium tumefaciens and A. vitis, and rhizogenic (Ri) A. rhizogenes.

Detection of Agrobacterium vitis by PCR using novel virD2 gene-specific primers that discriminate two subgroups

Tumour tissue samples were collected from vines grown in various regions of Italy and other parts of Europe and extracted for detection of Agrobacterium vitis. Fifty strains were isolated on agar plates and screened by PCR with consensus primers from the virD2 gene. They were confirmed as A. vitis with a species-specific monoclonal antibody. The isolates were further analyzed by PCR for their opine synthase genes and ordered into octopine, nopaline and vitopine strains. Primers designed on the octopine synthase gene did not detect octopine strains of Agrobacterium tumefaciens. For quantitative PCR, virD2 fragments were sequenced: two classes of virD2 genes were found and two primer sets designed, which detected octopine and nopaline strains or only vitopine strains. For simultaneous identification of all opine-type strains, multiplex real-time PCR with either primer pair and SYBR Green was performed: the combined sets of primers gave signals with DNA from any A. vitis strain. Specificity of the new primers for real-time PCR was evaluated using several unidentified bacterial isolates from grapevines and other plant species. An elevated level of non-specific background was observed when the combined primer sets were used in multiplex PCR assays. The real-time PCR protocol was also used to detect A. vitis cells directly from grapevine tumours; avoiding direct isolation procedures a sensitivity in the range of one to ten cells per assay was found. Inhibition of the PCR reaction by plant material was overcome by treating tumour extracts with a DNA purification kit as a step for the isolation of nucleic acids.

Transgenic Arabidopsis plants expressing Agrobacterium tumefaciens VirD2 protein are less susceptible to Agrobacterium transformation

SUMMARY Agrobacterium tumefaciens causes crown gall disease on many plant species and can result in considerable economic losses. Here we report a new strategy to control crown gall disease by over-expressing Agrobacterium tumefaciens VirD2 protein in plants. Transgenic Arabidopsis plants over-expressing virD2 from constitutive or wound-inducible promoters are less susceptible to Agrobacterium-mediated transformation. Additionally, the transient introduction of an A. tumefaciens virD2 gene in tobacco BY-2 cells reduces subsequent Agrobacterium-mediated transformation.

Novel constructions to enable the integration of genes into the Agrobacterium tumefaciens C58 chromosome.

We constructed several versatile sets of vectors that can be used to introduce any gene into the pgl/picA locus of the Agrobacterium tumefaciens C58 chromosome without affecting T-DNA transfer. One set contains a fragment containing the lacIq and lacZ genes and a multiple cloning site from pBluescriptII SK(+) inserted into a PstI site between the pgl and picA genes on an incPalpha plasmid. The resulting plasmid contains eight unique restriction endonuclease sites and the ability to use blue-white screening for the presence of an insert. A second plasmid also contains a beta-lactamase gene within this locus and provides a convenient ampicillin-carbenicillin resistance marker for the selection of genes integrated into the chromosome following double homologous recombination (homogenotization). A third plasmid contains, in addition to the lacZ, lacIq, and beta-lactamase genes within the pgl/picA locus, a sacRB gene cassette within the vector to counterselect against the presence of the vector within A. tumefaciens. To test this system, we introduced a wild-type virD2 gene into the A. tumefaciens chromosome at the pgl/picA locus. When a Ti plasmid harboring a deletion of virD2 was in this strain, the integrated virD2 gene complemented the virD2 deletion and the resulting transformation phenotype was identical to that resulting from A. tumefaciens strains harboring a wild-type virD2 gene located on a replicating plasmid.