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Crown gall caused by Agrobacterium vitis (A. vitis) is one of the crucial issues restricting the to grape industry. In this study, Agrobacterium tumefaciens (Ag-8) was separated from the soil that could prevent the occurrence of grape crown gall. By the mutagenesis of Ag-8 transposon, the siaD gene deletion strain (Δ siaD) showed significantly lower efficacy in grape and tomato plants for controlling grape crown gall, but the relevant mechanism was not clear. The biofilm formation and motility of Δ siaD were significantly decreased, and the colonization ability of Δ siaD in tomato roots was significantly reduced. RNA-seq analysis showed that the expression of nemR significantly reduced in the Δ siaD and that the expression of nemR showed a high correlation with biofilm and motility. Further studies showed that the nemR gene deletion strain of Ag-8 (Δ nemR) showed significantly reduced motility, biofilm formation and control of grape crown gall compared to Ag-8, and the nemR gene complementary strain of Ag-8 (Δ nemR -comp) recovered to Ag-8 wild-type levels. The inoculation experiments of preventive, curative or simultaneous treatment further showed that the preferential inoculation with Ag-8 reduced the incidence of grape crown gall on tomato plants, and studies showed that the mutation of siaD affected the site competition between Ag-8 and A. vitis , and that the mutation of nemR was consistent with the previous results. This study provides a new strategy for the prevention and control of grape crown gall, which is of great significance to the grape industry to increase production and income. • Agrobacterium tumefaciens (Ag-8) could prevent the occurrence of grape crown gall. • SiaD or nemR deletion of Ag-8 was critical for controlling grapevine crown galls. • Biofilm formation and colonization ability of Δ siaD were significantly decreased. • Expression of nemR showed a high correlation with biofilm and motility. [ABSTRACT FROM AUTHOR]

Using Nanopore Q20+ sequencing, we report the complete genome of Allorhizobium ( Agrobacterium ) vitis strain CG957=AA25, isolated nearly 40 years ago from a grapevine crown gall in Afghanistan. The assembled genome size is 6 Mb, comprising a circular chromosome, a linear chromid, a Ti plasmid, and two non-Ti plasmids., Competing Interests: The authors declare no conflict of interest.

The experiment was conducted during 2016–2017 on Vitis vinifera cultivars (cv.) 'Superior Seedless', 'Razakı', 'Crimson Seedless', 'Sultana', 'Royal', and American rootstocks (41B, 1103P, 1616C, Ramsey, 5BB) at Department of Horticulture, Agriculture Faculty, Ege University, Turkey. This study was designed to investigate the effects of HWT on the quality of hard wood cuttings following the treatment. The hard wood cuttings that were hot water treated at 50 °C for 30 min which is the most common protocol against Agrobacterium vitis used worldwide. After HWT, cuttings (ungrafted) were planted in black polythene bags for rooting in greenhouse conditions. In this experiment, the effects of HWT on cuttings and root distribution (0–4 scala), root number, root length (cm), shoot length (cm), sprouting rate (%), rooting rate (%), fresh and dry root weight (g) were investigated. HWT was found to have variable effects on different characteristics of cultivar and rootstock cuttings. 41B saplings were negative affected from HWT for root distribution (0–4 scala), root number, shoot length (cm), sprouting rate (%), fresh and dry root weight (g). The HWT for 41B rootstock cuttings should be done carefully. 'Superior Seedless', 'Razakı' and 'Royal' were affected positively by HWT compared to non-HWT group cuttings for some criterians. [ABSTRACT FROM AUTHOR]

Aim. Detection of representatives of saprophytic (Bacillus thuringiensis, Alcaligenes faecalis, Lactobacillus plantarum) and pathogenic (Agrobacterium spp.) microbiota in flowers and berries of grape. Materials and Methods. Flowers, non-damaged green and ripe berries of grape were selected from a vineyard of Vitis vinifera L. cv Pinot noir located in Odessa region in May, July and September 2018, accordingly. Flowers or berries were homogenized and left for autofermentation process for 7 days. Real-Time polymerase chain reaction to detect species B. thuringiensis, L. plantarum, A. faecalis and Agrobacterium tumefaciens, Agrobacterium vitis was carried out with DNA isolated from the resulted fermented homogenate. Results. B. thuringiensis was prevalent among the tested species: these bacteria were identified in 50% of samples of grape flowers, in 90% of green berries and 20% of samples of ripe berries. L. plantarum was not detected in flowers, but these bacteria were identified in green and ripe berries (10% of tested samples). Results confirm literature data about the presence of B. thuringiensis and L. plantarum on grape. For the first time, we detected A. faecalis and Agrobacterium spp. in flowers and berries of grape. A. faecalis was found in 20% of flower samples, 60% of green berries and 20% of ripe berries. Agrobacterium spp. were detected in flowers and green berries (10% of samples), and in ripe berries these microorganisms were not found. Amount of detected species increased in green berries compared with the flowers, and decreased in ripe berries. Conclusions. For the first time, we detected A. faecalis and Agrobacterium spp. in flowers and berries of grape. Coexistence of phytopathogenic agrobacteria and their potential antagonists -- Bacillus thuringiensis, Alcaligenes faecalis and Lactobacillus plantarum in one ecological niche (flowers and green grape berries) was revealed. [ABSTRACT FROM AUTHOR]

Agrobacterium vitis and some strains of A. tumefaciens and A. rhizogenes cause a dangerous chronic disease on grape plants—crown gall. Molecular-genetic methods were used in the work to identify and study the diversity of pathogenic agrobacteria that cause crown gall in the vineyards of Krasnodar krai. The research material was 76 samples of young and lignified tumor-like outgrowths of crown gall of grapes collected from plants in 14 vineyards of Krasnodar krai with different origins of planting stock (Italy, France, Austria, Serbia, Bulgaria, and Russia). In all studied samples, the presence of A. vitis was identified using the PGF/PGR test-system for DNA analysis. When studying the diversity of agrobacteria by the type of Ti plasmids contained therein, four groups were identified: containing octopine-type Ti plasmids (59.5%), octopine/vitopine (13.5%), vitopine (13.5%), and nopaline type (13.5%). The obtained data indicate that crown gall of grapes in the vineyards of Krasnodar krai is caused by agrobacteria A. vitis, mainly of the octopine type. [ABSTRACT FROM AUTHOR]

Current detection methodologies for Agrobacterium vitis, causing crown gall of grapevines, are time intensive and lack the ability to quantify pathogen abundance in nursery stock and soil. Information on pathogen abundance is a key component to develop management strategies. The aim of this study was to develop a rapid and sensitive quantification assay for grapevine nursery stock and vineyard soil via droplet digital polymerase chain reaction targeting the virA gene. DNA isolated from roots of donnant grapevines originating from nurseries in Germany, California, and Ontario were tested for virA abundance. Bacterial numbers varied with grapevine origin; plants from California had the highest numbers. In addition, rhizosphere soil from two vineyards in the Okanagan valley in British Columbia was tested over a growing season. Sampling time during the season did not affect virA gene abundance. The older vineyard had higher soil A. vitis populations than the younger vineyard. The assay developed here has potential for use in national clean plant programs to prevent import of infected grapevine nursery stock and to test vineyard soil for abundance of the pathogen before planting. [ABSTRACT FROM AUTHOR]

Agrobacterium vitis-induced crown gall disease (tumor) is the greatest problem in vineyards. Much proceed has not been achieved in fight against crown gall disease. Producers either totally stub out the vines from already established vineyards or keep viticulture activities with palliative measures. This study was conducted for two years to investigate the effects of shoot regeneration on yield, quality and development of A. vitis-infected grapevines. Initially the grapevines with morphologic A. vitis symptoms were determined in experimental vineyard. Then, deep pruning (cutting) was performed 10-15 cm below the tumorous sections of the stems in 2010, 2011 and 2012 and shoot regeneration was stimulated. Data were gathered about yield, quality (number of clusters, cluster weight, berry weight, must TSS content, TA, maturation index) and vine growth parameters (number of buds, number of shoots, burst ratio, pruning weight). About 15% less development was observed in grapevines with shoot regeneration treatments in 2012, 25% in grapevines with shoot regeneration treatments in 2011 and 37% in grapevines with shoot regeneration treatments in 2010. With shoot regenerations in 2012, 2011 and 2010, yields decreased respectively by 49, 58 and 66%; cluster weights decreased respectively by 32, 37 and 47% and berry weights decreased respectively by 14, 19 and 21%. [ABSTRACT FROM AUTHOR]

The current strategies for the control of Agrobacterium vitis crown gall in grape are generally unsuccessful once the pathogen has established in vineyards. Experimental trials were conducted to evaluate the effectiveness of treatments based on non-pathogenic endophytes isolated from asymptomatic grapevines growing in vineyards with high incidence of crown gall and on microorganisms isolated from commercial products. Two-year in planta trials conducted on rootstocks treated with endophytic isolates showed the effectiveness of two bacterial endophytes, both in the genus Curtobacterium, and one fungal isolate in the genus Acremonium in reducing crown gall development. For the commercial biological control agents, Bacillus subtilis SR63 and Trichoderma asperellum T1 were the most effective strains against A. vitis, indicating commercial products could be reserves to draw upon to identify useful biocontrol agents. Based on the combination of data in this work, microorganisms, both endophytes and those formulated in commercial products, were identified that can potentially be exploited for the control of grapevine crown gall disease. [ABSTRACT FROM AUTHOR]

It was shown that crown gall is a chronic disease of the grapevine caused by agrobacteria. The disease causes significant damage to the viticulture industry in Krasnodar Territory. The causative agent of crown gall in the vineyards of this region was identified and studied using the polymerase chain reaction method. Young tumor formations found on grape plants in different areas of Krasnodar Territory were analyzed. The presence of agrobacteria of species Agrobacterium vitis differing in the type of Ti-plasmid (oktopine, nopaline, or vitopine) were detected by using test-systems for Agrobacterium-mediated genes ( pehA, virF, and virD2). [ABSTRACT FROM AUTHOR]

Complex strategies to control crown gall are reviewed: indexing of planting material, hot water treatment, cultural practices, treatments with chemical substances and plant extracts. Special attention is paid to the biological control. The short descriptions of the most well studied antagonistic strains are listed. The main problems of grape crown gall prevention are elucidated.

In 2010, a serious outbreak of crown gall disease was observed on grapevines (Vitis vinifera L. cv. Cabernet Sauvignon) in several commercial vineyards located in the Vojvodina province, Serbia. Bacteria were isolated from the young tumor tissue on nonselective YMA medium and five representative strains were selected for further identification. Tumorigenic (Ti) plasmid was detected in all strains by PCR using primers designed to amplify the virC pathogenicity gene, producing a 414-bp PCR product. The strains were identified as Agrobacterium vitis using differential physiological and biochemical tests, and a multiplex PCR assay targeting 23S rRNA gene sequences. In the pathogenicity assay, all strains induced characteristic symptoms on inoculated tomato and grapevine plants. They were less virulent on tomato plants in comparison to the reference strains of A. tumefaciens and A. vitis. [Ministarstva nauke Republike Srbije, br. III46008: Development of integrated management of harmful organisms in plant production in order to overcome resistance and to improve food quality and safety]

The uptake of exogenous solutes by prokaryotes is mediated by transport systems embedded in the plasma membrane. In many cases, a solute-binding protein (SBP) is utilized to bind ligands with high affinity and deliver them to the membrane-bound components responsible for translocation into the cytoplasm. In the present study, Avi_5305, an Agrobacterium vitis SBP belonging to Pfam13407, was screened by differential scanning fluorimetry (DSF) and found to be stabilized by d-glucosamine and d-galactosamine. Avi_5305 is the first protein from Pfam13407 shown to be specific for amino sugars, and co-crystallization resulted in structures of Avi_5305 bound to d-glucosamine and d-galactosamine. Typical of Pfam13407, Avi_5305 consists of two α/β domains linked through a hinge region, with the ligand-binding site located in a cleft between the two domains. Comparisons with Escherichia coli ribose-binding protein suggest that a cation-π interaction with Tyr168 provides the specificity for d-glucosamine/ d-galactosamine over d-glucose/ d-galactose. [ABSTRACT FROM AUTHOR]

A nonpathogenic strain of Agrobacterium (= Rhizobium) vitis, ARK-1, limits the development of crown gall of grapevine ( Vitis vinifera L.). Co-inoculation of grapevine shoots with ARK-1 and the tumorigenic (Ti) strain VAT03-9 at a 1:1 cell ratio resulted in significantly lower expression of the virulence genes virD2 and virE2 of VAT03-9 1 day after inoculation (dai) compared with expression levels when shoots were inoculated only with VAT03-9. In contrast, nonpathogenic A. vitis strain VAR06-30, which does not limit the development of crown gall of grapevine, co-inoculated with VAT03-9 did not affect expression levels of virD2 and virE2 under the same conditions. ARK-1 began to suppress the VAT03-9 population by seven dai, but no such effect was observed with VAR06-30 during the nine dai study period. Thus, the biological control activity of ARK-1 is likely based on the suppression of essential virulence genes. [ABSTRACT FROM AUTHOR]

Aim To molecularly and phenotypically characterize a selection of Agrobacterium-like isolates from grapevine canes, crowns, soil and tumours in plants grown under cold conditions. Methods and Results Most of the strains were biovar 3 ( Agrobacterium vitis), and the remaining were atypical biovar 1 ( Agrobacterium tumefaciens). All of them were tumourigenic on grapevine plants but differences in other hosts were observed. Chromosomal and plasmid-borne traits were analysed by gene amplification with four primer sets. Detection of the pectin enzyme hydrolase gene clearly distinguished A. vitis from the atypical A. tumefaciens. Regarding the virulence sensor gene, limited host range tumour-inducing plasmids were found in the atypical isolates. About opine utilization, most A. vitis and some A. tumefaciens contained octopine/cucumopine plasmids, but the nopaline-type was only detected in one A. tumefaciens. Conclusions The A. vitis strains were molecularly and phenotypically more homogeneous than those of A. tumefaciens, the latter displaying some typical A. vitis characteristics, suggesting an adaptation to life in grapevine. Significance and Impact of the Study The findings of this work will help to improve detection procedures of the pathogen, and demonstrate the pathogen diversity in cold vineyards, laying the groundwork for epidemiological studies and development of control strategies of the crown and cane gall disease. [ABSTRACT FROM AUTHOR]

A new banana leaf blight was found in Nanning city, China, during a 7-year survey (2003-2009) of the bacterial diseases on banana plants. Eight bacterial strains were isolated from affected banana leaves, and identified as an intraspecific taxon of Agrobacterium vitis based on their 16S rDNA sequence similarities with those of 37 randomly selected bacterial strains registered in GenBank database. The representative strain Ag-1 was virulent on banana leaves and shared similar growth and biochemical reactions with the reference strain IAM14140 of A. vitis. The strains causing banana leaf blight were denominated as A. vitis pv. musae. The traditional A. vitis strains virulent to grapevines were proposed to be revised as A. vitis pv. vitis. This is the first record of a new type of A. vitis causing banana leaf blight in China. [ABSTRACT FROM AUTHOR]

Rahnella aquatilis strain HX2 has the ability to promote maize growth and suppress sunflower crown gall disease caused by Agrobacterium vitis, A. tumefaciens, and A. rhizogenes. Pyrroloquinoline quinone (PQQ), a cofactor of aldose and alcohol dehydrogenases, is required for the synthesis of an antibacterial substance, gluconic acid, by HX2. Mutants of HX2 unable to produce PQQ were obtained by in-frame deletion of either the pqqA or pqqB gene. In this study, we report the independent functions of pqqA and pqqB genes in relation to PQQ synthesis. Interestingly, both the pqqA and pqqB mutants of R. aquatilis eliminated the ability of strain HX2 to produce antibacterial substance, which in turn, reduced the effectiveness of the strain for biological control of sunflower crown gall disease. The mutation also resulted in decreased mineral phosphate solubilization by HX2, which reduced the efficacy of this strain as a biological fertilizer. These functions were restored by complementation with the wild-type pqq gene cluster. Additionally, the phenotypes of HX2 derivatives, including colony morphology, growth dynamic, and pH change of culture medium were impacted to different extents. Our findings suggested that pqqA and pqqB genes individually play important functions in PQQ biosynthesis and are required for antibacterial activity and phosphorous solubilization. These traits are essential for R. aquatilis efficacy as a biological control and plant growth promoting strain. This study enhances our fundamental understanding of the biosynthesis of an environmentally significant cofactor produced by a promising biocontrol and biological fertilizer strain. [ABSTRACT FROM AUTHOR]

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Grapevines are affected by three major bacterial diseases worldwide, such as bacterial blight (Xylophilus ampelinus), Pierce’s disease (Xylella fastidiosa) and crown gall (Agrobacterium vitis). These bacteria grow in the vascular system of their host, thus they invade and colonize the whole plant, independently on symptom development. Latently infected propagating material is a major factor in their spreading. Therefore the use of bacteria-free planting stock has a basic importance in viticulture. Today several innovative diagnostic methods, mostly based on polymerase chain reaction, are available to detect and identify bacterial pathogens of grapevines. For production of bacteria-free plants, the use hot water treatment followed by establishment of in vitro shoot tip cultures is proposed.

Key message: Grapevine rootstock transformed with an Agrobacterium oncogene-silencing transgene was resistant to certain Agrobacterium strains but sensitive to others. Thus, genetic diversity of Agrobacterium oncogenes may limit engineering crown gall resistance. Abstract: Crown gall disease of grapevine induced by Agrobacterium vitis or Agrobacterium tumefaciens causes serious economic losses in viticulture. To establish crown gall-resistant lines, somatic proembryos of Vitis berlandieri × V. rupestris cv. 'Richter 110' rootstock were transformed with an oncogene-silencing transgene based on iaaM and ipt oncogene sequences from octopine-type, tumor-inducing (Ti) plasmid pTiA6. Twenty-one transgenic lines were selected, and their transgenic nature was confirmed by polymerase chain reaction (PCR). These lines were inoculated with two A. tumefaciens and three A. vitis strains. Eight lines showed resistance to octopine-type A. tumefaciens A348. Resistance correlated with the expression of the silencing genes. However, oncogene silencing was mostly sequence specific because these lines did not abolish tumorigenesis by A. vitis strains or nopaline-type A. tumefaciens C58. [ABSTRACT FROM AUTHOR]

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 10¹ CFU/ml compared with 105 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 104 CFU/ml. All three assays were able to detect A. vitis in healthy-appearing grapevine cuttings taken from infected vines. [ABSTRACT FROM AUTHOR]

Nontumorigenic Agrobacterium vitis strain F2/5 is able to prevent crown gall caused by tumorigenic A. vitis on grape but not on other plant species such as tobacco. Mutations in a quorum-sensing transcription factor, aviR, and in caseinolytic protease (clp) component genes clpA and clpPl resulted in reduced or loss of biological control. AH mutants were complemented; however, restoration of biological control by complemented clp A and clpPl mutants was dependent on the copy number of vector that was used as well as timing of application of the complemented mutants to grape wounds in relation to inoculation with pathogen. Mutations in other quorum-sensing and clp genes and in a gene associated with polyketide synthesis did not affect biological control. It was determined that, although F2/5 inhibits transformation by tumorigenic A. vitis strains on grape, it does not affect growth of the pathogen in wounded grape tissue over time [ABSTRACT FROM AUTHOR]

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 characterized. Morphological characterization, molecular assays and pathogenicity tests were conducted to identify Agrobacterium isolates. A set of 380 isolates with morphologically Agrobacterium-like colonies on MG medium 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 collection. Biovars 1, 2 and 3 were first distinguished on the three semi-selective media 1A, 2E and 3AN respectively. 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 carrot disks proved to be highly tumourigenic (++) and to form typical tumours around the cambium of carrot slices, whereas 36%, induced not clearly discernible tumours (+). 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. [ABSTRACT FROM AUTHOR]

Graft unions of nursery stock of grapevine ( Vitis vinifera L.) collected in Japan yielded non-pathogenic strains of Agrobacterium. On the basis of classic diagnostic tests, a sequence analysis and a previously reported multiplex PCR method, the non-pathogenic strains ARK-1, ARK-2 and ARK-3 were identified as Agrobacterium vitis. Stems of grapevine seedlings were inoculated with both a cell suspension of seven mixed strains of A. vitis (Ti) as a pathogen and one of a new strain or A. vitis strain VAR03-1, one of the biological control agents against crown gall previously reported, as competitors to assay the suppression of tumour formation caused by the pathogen. In a test with a 1:1 cell ratio of pathogen/nonpathogen, strains ARK-1, ARK-2 and ARK-3 reduced the tumour incidence.. In particular, strain ARK-1 was strongest at inhibiting tumour formation in this study. Strain ARK-1 established populations on roots of grapevine tree rootstock and persisted on roots for a year. ARK-1, ARK-2 and ARK-3 did not produce a halo of inhibition against A. vitis (Ti) strain on YMA medium. Moreover, strain ARK-1 did not reduce tumour incidence on the stems of grapevine when ARK-1 was dead or only culture filtrate was used. This result indicates the possibility that these new strains inhibit grapevine crown gall in planta by a different mechanism other than VAR03-1. In particular, one of the new strains, named ARK-1, was most effective in inhibiting tumour formation on grapevine and appears to be a promising new agent to control grapevine crown gall. [ABSTRACT FROM AUTHOR]

Agrobacterium vitis strain E26 is a promising biocontrol agent of grapevine crown gall, an economically important disease of grape worldwide. In this report, we developed a Plating-PCR method that allows specific detection and quantification of E26 by combining classical microbiological techniques with molecular tools. Random amplified polymorphic DNA fingerprints were used to differentiate E26 from other A. vitis strains. A differentially amplified fragment from E26 was sequenced and characterized as a sequence characterized amplified region (SCAR) marker. Two primer pairs were then designed and evaluated for their specificity against E26. One of the two SCAR primer pairs, 740F/R, was further selected for specific detection of strain E26. A plating assay coupled to PCR with the SCAR primers 740F/R allowed the assessment of population dynamics of E26 in non-sterile grape rhizosphere soil under controlled conditions. [ABSTRACT FROM AUTHOR]

SUMMARY The characterization of Tn5 transposon insertional mutants of Agrobacterium vitis strain F2/5 revealed a gene encoding a predicted LysR-type transcriptional regulator, lhnR (for 'LysR-type regulator associated with HR and necrosis'), and an immediate upstream operon consisting of three open reading frames ( lhnABC) required for swarming motility, surfactant production and the induction of a hypersensitive response (HR) on tobacco and necrosis on grape. The operon lhnABC is unique to A. vitis among the sequenced members in Rhizobiaceae. Mutagenesis of lhnR and lhnABC by gene disruption and complementation of Δ lhnR and Δ lhnABC confirmed their roles in the expression of these phenotypes. Mutation of lhnR resulted in complete loss of HR, swarming motility, surfactant production and reduced necrosis, whereas mutation of lhnABC resulted in loss of swarming motility, delayed and reduced HR development and reduced surfactant production and necrosis. The data from promoter- green fluorescent protein ( gfp) fusions showed that lhnR suppresses the expression of lhnABC and negatively autoregulates its own expression. It was also shown that lhnABC negatively affects its own expression and positively affects the transcription of lhnR. lhnR and lhnABC constitute a regulatory circuit that coordinates the transcription level of lhnR, resulting in the expression of swarming, surfactant, HR and necrosis phenotypes. [ABSTRACT FROM AUTHOR]

Aims: Natural and synthetic antimicrobial peptides (AMPs) are of increasing interest as potential resistance conferring elements in plants against pathogen infection. The efficacy of AMPs against pathogens is prescreened by in vitro assays, and promising AMP candidates are introduced as transgenes into plants. As in vitro and in planta environments differ, a prescreening procedure of the AMP efficacy in the plant environment is desired. Here, we report the efficacy of the purified synthetic peptide D4E1 against the grapevine-infecting bacterial pathogens Agrobacterium vitis and Xylophilus ampelinus in vitro and describe for the first time an in planta prescreening procedure based on transiently expressed D4E1. Methods and Results: The antimicrobial effect of D4E1 against Ag. vitis and X. ampelinus was shown by a reduction in colony-forming units in vitro in a traditional plate-based assay and by a reduction in bacterial titres in planta as measured by quantitative real-time PCR (qPCR) in grapevine leaves transiently expressing D4E1. A statistically significant reduction in titre was shown for X. ampelinus, but for Ag. vitis, a significant reduction in titre was only observed in a subset of plants. Conclusions: The titres of both grapevine-infecting bacterial pathogens were reduced in an in vitro assay and for X. ampelinus in an in planta assay by D4E1 application. This widens the applicability of D4E1 as a potential resistance-enhancing element to additional pathogens and in a novel plant species. Significance and Impact of the Study: D4E1 is a promising candidate to confer enhanced resistance against the two tested grapevine bacterial pathogens, and the applied transient expression system proved to be a valuable tool for prescreening of D4E1 efficacy in an in planta environment. The described prescreening procedure can be used for other AMPs and might be adapted to other plant species and pathogens before the expensive and tedious development of stably transgenic lines is started. [ABSTRACT FROM AUTHOR]

Background: The flagellotropic phage 7-7-1 infects motile cells of Agrobacterium sp H13-3 by attaching to and traveling along the rotating flagellar filament to the secondary receptor at the base, where it injects its DNA into the host cell. Here we describe the complete genomic sequence of 69,391 base pairs of this unusual bacteriophage. Methods: The sequence of the 7-7-1 genome was determined by pyro(454)sequencing to a coverage of 378-fold. It was annotated using MyRAST and a variety of internet resources. The structural proteome was analyzed by SDS-PAGE coupled electrospray ionization-tandem mass spectrometry (MS/MS). Results: Sequence annotation and a structural proteome analysis revealed 127 open reading frames, 84 of which are unique. In six cases 7-7-1 proteins showed sequence similarity to proteins from the virulent Burkholderia myovirus BcepB1A. Unique features of the 7-7-1 genome are the physical separation of the genes encoding the small (orf100) and large (orf112) subunits of the DNA packaging complex and the apparent lack of a holin-lysin cassette. Proteomic analysis revealed the presence of 24 structural proteins, five of which were identified as baseplate (orf7), putative tail fibre (orf102), portal (orf113), major capsid (orf115) and tail sheath (orf126) proteins. In the latter case, the N-terminus was removed during capsid maturation, probably by a putative prohead protease (orf114). [ABSTRACT FROM AUTHOR]

Abstract: The incidence of crown gall disease recently has been gradually increased in Egyptian vineyards and grapevine nurseries. Twelve isolates of tumorigenic bacteria were isolated from grapevines galls which are collected from different areas in Egypt. The isolates were identified as Agrobacterium vitis based on their reaction with a monoclonal antibody raised to A. vitis, their ability to induce galls and necrosis on grapevine, their reactions to standard biochemical and physiological tests and also by polymerase chain reaction amplification of specific Ti plasmid using polygalacturonase specific primers. All isolates were able to induce galls on grapevine. The isolates differed between itself in host range. All of these isolates except two were able to induce galls on kalanchoe, sunflower, datura, tobacco and chenopodium. Only five isolates produced galls on tomato. All isolates caused necrosis on grapevine explants. The genetic diversity was evaluated for these isolates by comparing DNA samples using multiplex PCR with three specific primer pairs. The isolates were segregated into three main groups, the first group that is isolated carry octopine type Ti plasmids; the second group that is isolated carry vitopine Ti plasmids and the third group that is isolated carry both octopine and vitopine type Ti plasmids. To our knowledge, this is the first report on the identification of A. vitis in Egypt. [Copyright &y& Elsevier]

Grapevine has an importance gradually increasing with various cultivars of grapevine in Central Anatolia Region. With the aim of determining of bacterial diseases, the researches were carried on 14 various cultivars of grapevine and in 24 district of Konya Province in 2007-2008. In the identification of bacterial gall disease, Agrobacterium vitis, growth on Roy and Sasser, King's B, PDA mediums, growth at 37 °C, 3-ketolactose production, growth on 2% NaCl, production of alkaline from L- tartaric acid and malonic acid, using of citrate, at pH=4,5 pectolytic activity, at pH=7 motility, litmus milk test, acid from erythrytol and melesitose, ferric ammonium citrate, acid cleaning on PDA+CaCO3 tests were based. In the pathogenicity tests, it was observed formation of gall typically in the result of bacterial inoculation with 108 cfu/ml concentrations on seedlings of grapevine cultivar Sultani Seedless, tomato and sunflower plants. It was made amplification of pehA and virA genes by PCR for molecular identification. While A. vitis was determined on 280 plants from totally 309 plant samples, the most of isolates had been observed tumorogenic character. Obtaining the results, rate of incidence has been as 90.61% has big importance and within all of the cultivars, pathogen was shown the most on cv. Sultani Seedless, cv. Cardinal, cv. Hafizali and at the least on cv. Sour Black in the province. [ABSTRACT FROM AUTHOR]

To examine the biocontrol activity of broad-range antagonists Serratia plymuthica IC1270, Pseudomonas fluorescens Q8r1-96 and P. fluorescens B-4117 against tumourigenic strains of Agrobacterium tumefaciens and A. vitis. Under greenhouse conditions, the antagonists, applied via root soak prior to injecting Agrobacterium strains into the wounded stems, significantly suppressed tumour development on tomato seedlings. A derivative of P. fluorescens Q8r1-96 tagged with a gfp reporter, as well as P. fluorescens B-4117 and S. plymuthica IC1270 marked with rifampicin resistance, stably persisted in tomato tissues for at least 1 month. Mutants of P. fluorescens Q8r1-96 and S. plymuthica IC1270 deficient in 2,4-diacetylphloroglucinol or pyrrolnitrin production, respectively, also proficiently suppressed the tumour development, indicating that these antibiotics are not responsible for the observed biocontrol effect on crown gall disease. The volatile organic compounds (VOCs) produced by the tested P. fluorescens and S. plymuthica strains inhibited the growth of A. tumefaciens and A. vitis strains in vitro. Solid-phase microextraction-gas chromatography-mass spectrometry analysis revealed dimethyl disulfide (DMDS) as the major headspace volatile produced by S. plymuthica IC1270; it strongly suppressed Agrobacterium growth in vitro and was emitted by tomato plants treated with S. plymuthica IC1270. 1-Undecene was the main volatile emitted by the examined P. fluorescens strains, with other volatiles, including DMDS, being detected in only relatively low quantities. S. plymuthica IC1270, P. fluorescens B-4117 and P. fluorescens Q8r1-96 can be used as novel biocontrol agents of pathogenic Agrobacterium. VOCs, and specifically DMDS, might be involved in the suppression of oncogenicity in tomato plants. However, the role of specific volatiles in the biocontrol activity remains to be elucidated. The advantage of applying these antagonists lies in their multiple activities against a number of plant pathogens, including Agrobacterium. [ABSTRACT FROM AUTHOR]

truncated form of the Ti-plasmid virE2 gene from Agrobacterium tumefaciens strains C58 and A6, and A. vitis strain CG450 was transferred and expressed in somatic embryos of grapevine rootstocks 110 Richter ( Vitis rupestris × V. berlandieri), 3309 Couderc ( V. rupestris × V. riparia) and Teleki 5C ( V. berlandieri × V. riparia) via Agrobacterium-mediated transformation to confer resistance to crown gall disease. Transformation was confirmed in 98% of the 322 lines by enzyme-linked immunosorbent assay for the neomycin phosphotransferase II protein and 97% of 295 lines by polymerase chain reaction for the truncated virE2 transgene. Southern blot analysis revealed the insertion of truncated virE2 at one to three loci in a subset of seven transgenic 110 Richter lines. In vitro resistance screening assays based on inoculations of shoot internode sections showed reduced tumorigenicity and very small galls in 23 of 154 transgenic lines. Non-transformed controls had a 100% tumorigenicity rate with very large galls. Disease resistance assay at the whole plant level in the greenhouse revealed seven transgenic lines (3 lines of 110 Richter, 2 lines of 3309 Couderc and 2 lines of Teleki 5C) were resistant to A. tumefaciens strain C58 and A. vitis strains TM4 and CG450 with a substantially reduced percentage of inoculation sites showing gall as compared to controls. No association was found between the level of resistance to crown gall disease and the source Agrobacterium strain of virE2. Taken together, our data showed that resistance to crown gall disease can be achieved by expressing a truncated form of virE2 in grapevines. [ABSTRACT FROM AUTHOR]

Abstract: For the first time, a portable electronic nose was used to discriminate between healthy and galled grapevines, experimentally inoculated with two tumourigenic strains of Agrobacterium vitis. The volatile profile of target cutting samples was analysed by headspace solid phase microextraction coupled with gas chromatography–mass spectrometry. Spectra from tumoured samples revealed the presence of styrene which is compatible with decarboxylation of cinnamic acid involved in secondary metabolism of plants. Principal Component Analysis confirmed the difference in volatile profiles of infected vines and their healthy controls. Linear Discriminant Analysis allowed the correct discrimination between healthy and galled grapevines (83.3%, cross-validation). Although a larger number of samples should be analysed to create a more robust model, our results give novel interesting clues to go further with research on the diagnostic potential of this innovative system associated with multi-dimensional chemometric techniques. [Copyright &y& Elsevier]

Bioagents and chemicals were applied to one-year old grafted vines (Ancelotta/420A) in glasshouse and field experiments set up at the Vivai Cooperativi Rauscedo (VCR, Pordenone, Italy). In the glasshouse, holes were drilled in vines on the rootstock and the holes were charged with suspensions of different strains of Pseudomonas spp., and with the biofungicides BS-F4 and Serenade, both based on Bacillus subtilis, before inoculation with a vitopine Agrobacterium vitis strain. The growth retardant Regalis and the resistance inducer Bion were applied to the vines two weeks before inoculation with the pathogen. Six months after inoculation, disease incidence was lowest when BS-F4 had been applied. In the field trial, the vines were wounded by making a cut in the crown, after which they were dipped into the antagonist suspensions just before inoculation with the pathogen. In the two weeks before inoculation, the root systems of the vines were dipped into Regalis and Bion solutions at 7 day intervals. Only these resistance inducers and BS-F4 significantly reduced disease severity. The results indicate that a potential for defence against A. vitis may exist even in susceptible grapevine cultivars, and that this potential can be activated by diverse elicitors. [ABSTRACT FROM AUTHOR]

Aims: Agrobacterium vitis is the causal agent of crown gall of grapevine. Surface motility (swarming), an important mechanism for bacterial colonization of new environments and a previously unknown behaviour of Ag. vitis, was demonstrated. Methods: Surface motility assays were performed on half-strength potato dextrose agar (Difco) containing 0·75% agar. To test for surfactant production, a drop-collapse test was used. Quorum-sensing (QS) negative and complemented mutants were tested for swarming activity. Results: Ninety-one Agrobacterium strains representing – Agrobacterium tumefaciens (17 strains), Agrobacterium rhizogenes (14 strains) and Ag. vitis (60 strains) were tested for swarming and production of surfactant. All Ag. vitis strains expressed a surface-related motility. In contrast, none of 17 strains of Ag. tumefaciens or 14 strains of Ag. rhizogenes exhibited this behaviour. Surface motility in Ag. vitis was associated with surfactant secretion; both of which are regulated by a QS system previously associated with induction of a hypersensitive response on tobacco and necrosis on grape. An aviR (belongs to luxR family) mutant was surface motility negative and did not produce surfactant. An avsI mutant (autoinducer synthase) was also surface motility negative and was complemented with an Ag. tumefaciens clone expressing avsI. Conclusions: Agrobacterium vitis is able to produce a characteristic swarming phenotype that is regulated by a complex QS system. Significance and Impact of the Study: Swarming activity is unique to Ag. vitis among Agrobacterium sp. and may be associated with the ability of the pathogen to colonize grapevines. [ABSTRACT FROM AUTHOR]

Summary: An Agrobacterium vitis-specific DNA fragment (pAVS3) was generated from PCR polymorphic bands amplified by primer URP 2R. A. vitis specificity of this fragment was confirmed by Southern hybridization with genomic DNA from different Agrobacterium species. Sequence-characterized amplified region (SCAR) markers were developed for A. vitis specific detection, using 24-mer oligonucleotide primers designed from the flanking ends of the 670bp insert in pAVS3. The SCAR primers amplified target sequences only from A. vitis strains and not from other Agrobacterium species or other bacterial genera. First round PCR detected bacterial cells between 5×102 and 1×103 cfu/ml and the detection sensitivity was increased to as few as 2cfu/ml by nested PCR. This PCR protocol can be used to confirm the potential presence of infectious A. vitis strains in soil and furthermore, can identify A. vitis strains from naturally infected crown galls. [Copyright &y& Elsevier]

Crown gall in grapevine, caused by tumorigenic Agrobacterium vitis strains, can cause severe losses in most viticulture regions worldwide. The only effective means of control is through cultivation practices. One non-tumorigenic A. vitis strain, E26, can prevent crown gall infection when applied to wounds on grapevine prior to or simultaneous with inoculation of tumorigenic strains. ME19, a Tn5 mutant of strain E26, was impaired in terms of its ability to be chemo-attracted by grapevine root tissue extracts and its attachment to grapevine roots, and had reduced biological control activity; it did not significantly differ from the wild-type strain of E26 in phenotypes of agrocin production, growth in minimum medium or swarming activity. Complementation of ME19 with the cosmid clone of CP1543 from an E26 DNA library restored the chemotaxis, attachment, and biocontrol phenotypes. A 7·3-kb KpnI fragment from CP1543 was cloned and sequenced, and sequence analysis revealed that the Tn 5 insertion occurred in a region that shares a significant homology with genes coding for methyl-accepting chemotaxis proteins (MCPs) in many bacteria. Complementation of the mcp gene mutants restored the affected phenotypes to the level of wild-type E26. An in-frame deletion mutant of the mcp gene was generated and was determined to have the same phenotypes as the original Tn5 mutant. [ABSTRACT FROM AUTHOR]

The 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. [ABSTRACT FROM AUTHOR]

Here, we identify a cluster of eight genes on chromosome 2 of Agrobacterium vitis that is associated with the ability of the bacterium to cause a hypersensitive response on tobacco and a necrosis of grape shoot explants. Three of these genes share a high level of structural and sequence similarity to clusters of genes in other bacteria that encode the enzymes for biosynthesis of polyketides and long-chain polyunsaturated fatty acids. No similar gene clusters were discovered in sequenced genomes of other members of Rhizobiales. [ABSTRACT FROM AUTHOR]

A nonpathogenic strain of Agrobacterium vitis VAR03-1 was tested as a biological control agent against crown gall of grapevine ( Vitis vinifera L.). A mixture of the nonpathogenic strain VAR03-1 and a tumorigenic strain G-Ag-27 of A. vitis at cell ratios of 1 : 1, 3 : 1, 9 : 1, and 99 : 1 significantly inhibited gall formation and size on stems of tomato ( Lycopersicon esculentum Mill.). Strain VAR03-1 also inhibited gall formation on stems of both tomato and grapevine at a 1 : 1 cell ratio with several tumorigenic A. vitis strains isolated from different fields of grapevine in Japan. In biological control tests, when roots of grapevine and tomato seedlings were soaked in a cell suspension of strain VAR03-1 for 24 h before a 1-h soaking in a cell suspension of the pathogen and subsequent planting in pots of infested soil, strain VAR03-1 significantly reduced the incidence of gall formation on both plants. [ABSTRACT FROM AUTHOR]