Grapevine Crown Gall Research Articles

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.

Identification of Agrobacterium vitis as a causal agent of grapevine crown gall in Serbia

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.

Electronic nose as an innovative tool for the diagnosis of grapevine crown gall

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.

Strain E26 of Agrobacterium vitis, a Biological Control Agent of Grapevine Crown Gall, Does Not Contain virA and virG Pathogenic Determinants

AbstractRisk assessment of biological control agents (BCAs) for the control of plant diseases in the field and/or laboratories has now become a necessary procedure before developing and producing novel BCAs. Agrobacterium vitis strain E26 is a promising potential biocontrol agent of grapevine crown gall disease. However, much less is understood about its safety or environmental risks. In this study, polymerase chain reaction (PCR) and Southern blot analyses were used to determine whether five essential virulence genes (virA, virG, iaaH, iaaM and ipt) were present in strain E26. Primers and probes were designed based on the conserved regions of each gene. The overall results obtained indicated that A. vitis strain E26 does not contain the virA and virG determinants, suggesting that this strain would be unlikely to elicit crown gall symptoms in either host or non‐host plants. It seems that the iaaH, iaaM, or ipt gene were not present in strain E26 either. An applicable new approach combining PCR and Southern blot analyses to examine the pathogenicity of potential BCAs, particularly BCAs from the genus of Agrobacterium spp. was described.

Studies on the diagnosis and biological control of grapevine crown gall and phylogenetic analysis of tumorigenic Rhizobium vitis

Crown gall of grapevine, caused by Rhizobium vitis (Ti) (=Agrobacterium vitis (Ti), A. tumefaciens biovar 3), is the most important bacterial disease of grapevine throughout the world, but there is neither an effective control method nor a rapid and simultaneous diagnostic method. I will describe the development of diagnosis method based on multiplex polymerase chain reaction (PCR), phylogenetic analysis of R. vitis strains and biological control of grapevine crown gall using R. vitis (nonpathogenic) strain VAR03-1.

Mutations that disrupt either the pqq or the gdh gene of Rahnella aquatilis abolish the production of an antibacterial substance and result in reduced biological control of grapevine crown gall.

Rahnella aquatilis HX2, a biocontrol agent for grapevine crown gall caused by Agrobacterium vitis, produces an antibacterial substance that inhibits the growth of A. vitis in vitro. In this study, we show that MH15 and MH16, two Tn5-induced mutants of HX2, have lost their abilities to inhibit A. vitis and have reduced biocontrol activities; they grow in logarithmic phase at a rate similar to that of the wild type and have single Tn5 insertions. They are also impaired in producing pyrroloquinoline quinone (PQQ) or glucose dehydrogenase (GDH). Complementation of MH15 and MH16 with cosmid clones of CP465 and CP104 from an HX2 DNA library restored the antibiosis, biocontrol, and PQQ or GDH production phenotypes. A 6.7-kb BamHI fragment from CP465 that fully restored the MH15-affected phenotypes was cloned and sequenced. Sequence analysis of the mutated DNA region resulted in the identification of seven open reading frames (ORFs), six of which share significant homology with PQQ-synthesizing genes in other bacteria, designated pqqA through pqqF. Meanwhile, A 5.5-kb PstI fragment from CP104 fully complemented the MH16 mutant and contained a single ORF highly similar to that of genes coding for GDHs. An in-frame gdh deletion mutant has the same phenotypes as the Tn5 mutant of MH16. Complementation of both deletion and Tn5 gdh mutants restored the affected phenotypes to wild-type levels. Our results suggest that an antibacterial substance plays a role in biocontrol of A. vitis by HX2.

Surface motility and associated surfactant production inAgrobacterium vitis

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. 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. 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. Agrobacterium vitis is able to produce a characteristic swarming phenotype that is regulated by a complex QS system. Swarming activity is unique to Ag. vitis among Agrobacterium sp. and may be associated with the ability of the pathogen to colonize grapevines.

Quorum-sensing signal production byAgrobacterium vitisstrains and their tumor-inducing and tartrate-catabolic plasmids

Agrobacterium vitis strains, their tumor-inducing (pTi) and tartrate utilization (pTr) plasmid transconjugants and grapevine tumors were analyzed for the presence of N-acyl-homoserine lactones (AHLs). All wild-type A. vitis strains produced long-chain signals. PCR analysis of the A. vitis long-chain AHL synthase gene, avsI, showed the predicted amplicon. Agrobacterium tumefaciens UBAPF2 harboring various A. vitis pTi plasmids produced N-(3-oxo-octanoyl)-l-homoserine lactone encoded also by pTis of A. tumefaciens. UBAPF2 transconjugants carrying pTrs except for pTrTm4 and pTrAB3, also produced an AHL. UBAPF2 transconjugants carrying pTrAT6, pTrAB4 and pTrRr4 or pTiNi1 produced two additional AHLs not observed in the corresponding wild-type strains. We also provide evidence for in situ production of AHLs in grapevine crown gall tumors of greenhouse and field origin.

Grapevine crown gall caused by Rhizobium radiobacter (Ti) in Japan

In 2005, characteristic symptoms of crown gall on grapevines (Vitis vinifera L. cv. Muscat of Alexandria, and cv. Seto Giants) were observed in a commercial greenhouse-orchard in Okayama Prefecture, Japan. Isolations from diseased tissues consistently yielded bacterial colonies that were white, glistening, and produced abundant polysaccharide on potato semi-synthetic agar (PSA) medium. Ten representative isolates were chosen for further characterization. A multiplex polymerase chain reaction (PCR) assay showed these strains were not Rhizobium vitis but did possess a Ti plasmid. The bacteriological characteristics of the isolates corresponded well with R. radiobacter. The almost complete 16S ribosomal DNA sequences of isolates AT06-1 and AT06-2, selected from 10 grapevine isolates, were determined and corresponded to sequences of R. radiobacter. The pathogenicity of the isolates was tested on young grapevine and tomato (Lycopersicon esculentum Mill.) plants. Gall symptoms developed on both plant species after inoculation, and bacteria with the same colony morphology as those inoculated were reisolated. Based on these results, the isolates were identified as R. radiobacter (Ti). This report is the first of the occurrence of R. radiobacter (Ti) on grapevine in Japan. Phylogenetic analyses using the partial nucleotide sequences of virC operon located on a Ti plasmid showed that the isolate of R. radiobacter (Ti) isolated from grapevine and some strains of R. vitis (Ti) belonged to the same monophyletic group, which differed from the groups of R. radiobacter (Ti) isolated from plants other than grapevine and of the majority of R. vitis (Ti) strains isolated from grapevine.

Biological control of grapevine crown gall: purification and partial characterisation of an antibacterial substance produced by Rahnella aquatilis strain HX2

Strain HX2 of Rahnella aquatilis has been previously reported as a potential biological control agent (BCA) of grapevine crown gall. The production of an antibacterial substance (ABS) was suggested to be an important factor in the biocontrol process. This study was undertaken to determine the antibacterial properties and mode of action of ABS. Isolation and purification of ABS from culture broth of strain HX2 was achieved by methanol exaction, chromatography on macroporous resin and silica gel, and high-performance liquid chromatography (HPLC). Physical and chemical characteristic analysis revealed that ABS may be a thermostable and alkali-sensitive substance containing sugar(s) and an unknown 285-nm absorbed substance. Antibacterial activity assays revealed that ABS displayed a broad activity spectrum against all the 13 test isolates of phytopathogenic bacteria including the genera of Agrobacterium, Clavibacter, Pectobacterium, Pseudomonas, and Xanthomonas. Agrobacterium spp. strains were more sensitive to ABS than other tested strains, with larger inhibition zones and lower minimal inhibitory concentration (MIC). ABS exhibited a bactericidal effect against A. vitis both in vitro and in vivo. This compound did not cause bacterial cell lysis, as determined by morphological observation with an electron microscope. Also, no leakage of cytoplasmic materials from cells of A. vitis occurred after treatment with ABS at concentrations equivalent to the MIC. However, an inhibition of the incorporation of radiolabelled precursors into RNA and protein was observed after treatment with ABS. These results suggest that ABS inhibits RNA and protein syntheses in tumorigenic A. vitis.

Identification of an rsh gene from a Novosphingobium sp. necessary for quorum-sensing signal accumulation.

The stringent response is a mechanism by which bacteria adapt to environmental stresses and nutritional deficiencies through the synthesis and hydrolysis of (p)ppGpp by RelA/SpoT enzymes. Alphaproteobacteria and plants contain a single Rsh enzyme (named for RelA/SpoT homolog) that is bifunctional. Here we report the identification of a new species of bacteria belonging to the genus Novosphingobium and characterization of an rsh mutation in this plant tumor-associated isolate. Isolate Rr 2-17, from a grapevine crown gall tumor, is a member of the Novosphingobium genus that produces the N-acyl-homoserine lactone (AHL) quorum-sensing (QS) signals. A Tn5 mutant, Hx 699, deficient in AHL production was found to have an insertion in an rsh gene. The Rsh protein showed significant percent sequence identity to Rsh proteins of alphaproteobacteria. The Novosphingobium sp. rsh gene (rsh(Nsp)) complemented the multiple amino acid requirements of the Escherichia coli relA spoT double mutant by restoring the growth on selection media. Besides QS signal production, the rsh mutation also affects soluble polysaccharide production and cell aggregation. Genetic complementation of the Hx 699 mutant with the rsh(Nsp) gene restored these phenotypes. This is the first discovery of a functional rsh gene in a member of the Novosphingobium genus.

Biological Control of Crown Gall of Grapevine, Rose, and Tomato by NonpathogenicAgrobacterium vitisStrain VAR03-1

A nonpathogenic strain of Agrobacterium vitis VAR03-1 was tested as a biological control agent for crown gall of grapevine (Vitis vinifera). When roots of grapevine, rose (Rose multiflora), and tomato (Lycopersicon esculentum) were soaked in a cell suspension of antagonists before planting in soil infested with tumorigenic A. vitis, A. rhizogenes, and A. tumefaciens, respectively, treatment with VAR03-1 significantly reduced the number of plants with tumors and disease severity in the three plant species. The inhibitory effects of treatment with VAR03-1 and the nonpathogenic A. rhizogenes strain K84 on crown gall of rose and tomato were almost identical, and the inhibitory effect of VAR03-1 on grapevine was superior to that of K84. Moreover, VAR03-1 greatly controlled crown gall of grapevine due to tumorigenic A. vitis in the field. VAR03-1 established populations averaging 10(6) colony forming units (CFU)/g of root in the rhizosphere of grapevine and persisted on roots for 2 years. VAR03-1 was bacteriocinogenic, producing a halo of inhibition against those three species of Agrobacterium. This is the first report that a nonpathogenic strain, VAR03-1, can effectively control crown gall caused by tumorigenic A. vitis, A. rhizogenes, and A. tumefaciens.

The quorum-sensing system AvsR-AvsI regulates both long-chain and short-chain acyl-homoserine lactones in Agrobacterium vitis E26

Agrobacterium vitis strain E26 is a nonpathogenic bacterium isolated from grape crown gall. In this study, the identification of a luxR-luxI type quorum-sensing system in strain E26 is reported. This system is involved in the induction of hypersensitive response on tobacco, but not its biocontrol activity. The deduced components AvsI(E26) and AvsR(E26) show the greatest similarity to AvsI(F2/5) and AvsR(F2/5), respectively from A. vitis strain F2/5. The mutant in AvsI(E26) abolished the production of both long-chain and short-chain acyl-homoserine lactones signals as well as the ability to cause hypersensitive response on tobacco. Complementaion of avsI (E26) and avsR (E26) genes restored the lost phenotypes to the level of wild type E26. In pot trial, no significant difference on biocontrol efficiency against grapevine crown gall was found between the wide type E26 and its quorum sensing negative mutants.

Biological control of grapevine crown gall by nonpathogenic Agrobacterium vitis strain VAR03-1

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.

Inhibition of crown gall formation by Agrobacterium radiobacter biovar 3 strains isolated from grapevine

Graft unions of nursery stock of grapevine (Vitis vinifera L.) collected in Japan yielded pathogenic and nonpathogenic strains of Agrobacterium. On the basis of classical diagnostic tests, a sequence analysis, and a multiplex polymerase chain reaction method previously reported, the pathogenic strain was identified as Agrobacterium tumefaciens biovar 3, whereas the nonpathogenic strains were assigned to Agrobacterium radiobacter biovar 3. Stems of tomato (Lycopersicon esculentum Mill.) seedlings were inoculated with both A. tumefaciens biovar 3 strain G-Ag-27 as a pathogen and one of the control strains isolated from grapevine or A. radiobacter biovar 2 strain K84 as competitors to assay the suppression of gall formation caused by the pathogen. In a test with a 1 : 1 pathogen/nonpathogen cell ratio, all A. radiobacter biovar 3 strains reduced gall incidence and size compared to that of the positive control inoculated only with the pathogen. Strain VAR03-1 was especially effective in reducing the incidence of gall formation on grapevine and reduced gall size by 84%–100% of those on the positive control. Many tested nonpathogenic biovar 3 strains were bacteriocinogenic, causing an inhibition zone against A. tumefaciens biovar 3 strains on YMA medium. Strain VAR03-1 was the most effective against indicator strains and appears to be a promising agent for controlling crown gall of grapevine.

Characterization of Agrobacterium tumefaciens strains isolated from grapevine

Grapevine crown gall is predominantly induced by Agrobacterium vitis , but rarely, Agrobacterium tumefaciens may also cause this disease. We have collected 18 grapevine isolates of A. tumefaciens from distinct geographical regions and various grapevine varieties. Characterization of these strains showed that 7 of the 18 strains carried A. vitis -type octopine/cucumopine Ti plasmids, utilized tartrate, were avirulent on pea and did not respond to the virC-specific PCR primers like A. vitis . Eleven of the 18 strains carried A. tumefaciens -type Ti plasmids, did not grow on tartrate, were virulent on pea and reacted positively to virC-specific PCR primers. Two of these strains induced octopine/agropine-, and two strains induced agropine producing tumors on grapevine and/or Kalanchoe tubiflora plants. The remaining 7 strains showed the characteristic properties of A. tumefaciens nopaline strains and were sensitive to agrocin 84.

Human Exposure Estimates for Phthalates

Human Exposure Estimates for Phthalates

Identification of grapevine crown gall bacteria isolated in Japan.

Recently, the incidence of crown gall disease has been gradually increasing in some grapevine varieties including ‘Kyoho’ in Japan. Twenty-five bacterial isolates, which were obtained from the galls of grapevines collected in Iwate, Yamanashi, Nagano and Shimane Prefectures, were identified as Agrobacterium tumefaciens (Smith et Townsend 1907) Conn 1942 based on their ability to induce galls on grapevine, tomato and sunflower using the needle prick inoculation method and also based on 90 cultural, physiological and biochemical characteristics. Although the isolates differed from the reference strains belonging to A. tumefaciens biovars 1 and 2 in 15 and 14 characteristics, respectively, they were similar to biovar 3 with respect to the features presented in Bergey's Manual (1984) except for the cleavage of ethanol and propionate. Based on these results it was suggested that the isolates belonged to A. tumefaciens biovar 3.

A Monoclonal Antibody Specific toAgrobacterium tumefaciensBiovar 3 and its Utilization for Indexing Grapevine Propagation Material

AbF21-1D3G7C8 does not react with A. tumefaciens biovars 1 or 2, or with other plant pathogens, symbionts, or saprophytes. AbF21-1D3G7C8 is useful for laboratory diagnosis of A. tumefaciens biovar 3, and thus for detection and diagnosis of grapevine crown gall, indexing of propagation wood, and quarantine testing. A. tumefaciens biovar 3 was identified in grapevine cultivars and rootstocks from New York and Washington states by means of the monoclonal antibody

Cucumopine—a new T-DNA-encoded opine in hairy root and crown gall

A new opine, cucumopine, has been identified by high voltage paper electrophoresis in hairy roots induced by Agrobacterium rhizogenes. Cucumopine gives a positive reaction with the Pauly imidazole reagent, the phenanthrene quinone reagent and alkaline silver nitrate. This substance was also found in spontaneous grapevine crown gall tumors. It is degraded by A. rhizogenes and by grapevine isolates of A. tumefaciens. Cucumopine was purified from carrot hairy root culture supernates and grapevine crown gall tumours.