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Bu çalışmada asma uru etmenine karşı biyolojik mücadele etmenlerinin topraktan izole edilmesi amacıyla Kahramanmaraş ili ve Kahramanmaraş'ın Aydınkavak, Türkoğlu, Narlı, Ekinözü ve Göksun ilçelerinde bulunan bağ alanlarından diğerlerine göre daha sağlıklı gözüken asmaların kök bölgelerinden 15 toprak ve kök örnekleri alınmıştır. Alınan bu toprak ve kök örneklerinden Kahramanmaraş Sütçü İmam Üniversitesi Bitki Koruma laboratuarrında bakteri izolasyonu yapılmıştır. İzolasyon sonucu elde edilen 468 izolatın 8 izolatı asma uru etmeni olan Agrobacterium vitis'in gelişimini engellediği belirlenmiştir. Engelleme zonlarının yarıçapları ölçmek suretiyle en etkin izolatın A1-24 olduğu belirlenmiştir.Engelleme zonu oluşturan 8 izolatın morfolojik, fizyolojik ve biyokimyasal özelliklerinden gram boyama, oksidaz, katalaz, KOH ile gram reaksiyonu ve floresan özellikleri belirlenmiştir. Bu izolatların tanılanması için kullanılan Biolog tanılama sistemine göre Bacillus gibsonii, Lactococcus lactis subsp. lactis, Brochotrix campestris, Stenotrophomonas rhizophila ve Pseudomonas aeruginosa türleri olduğu belirlenmiştir. In this study in order to isolate biological control agents against vine tumor agent, 15 soil and root samples were taken from healthier plants? root areas in vineyards in Kahramanmaraş and its towns, Aydınkavak, Türkoğlu, Narlı, Ekinözü and Göksun. Bacteria isolation stdies were conducted these samples in Plant Protection Laboratory of Sütçü İmam University in Kahramanmaraş. 468 isolates were obtained and 8 of them were demonstrated to form inhibition zone against the vine tumor Agrobacterium vitis. According to measurement of radius of inhibition zones, the most effective isolate was identified as A1-24.Gram staining, oxidase, catalase, gram reaction with KOH and flourescence tests were carried out to reveal morphological, physiological and biochemical characteristics of 8 isolates that formed inhibition zone. According to biological diagnosis system these isolates were identifical as Bacillus gibsonii, Lactococcus lactis subsp. lactis, Brochotrix campestris, Stenotrophomonas rhizophila and Pseudomonas aeruginosa. 60

The Ti plasmid of the Agrobacterium vitis nopaline-type strain AB4 was subcloned and mapped. Several regions of the 157 kb Ti plasmid are similar or identical to parts of the A. vitis octopine/cucumopine (o/c)-type Ti plasmids, and other regions are homologous to the nopaline-type Ti plasmid pTiC58. The T-DNA of pTiAB4 is a chimaeric structure of recent origin: the left part is 99.2% homologous to the left part of the TA-DNA of the o/c-type Ti plasmids, while the right part is 97.1 % homologous to the right part of an unusual nopaline T-DNA recently identified in strain 82.139, a biotype Il strain from wild cherry. The 3′ non-coding regions of the ipt genes from pTiAB4 and pTi82.139 are different from those of other ipt genes and contain a 62 by fragment derived from the coding sequence of an ipt gene of unknown origin. A comparison of different ipt gene sequences indicates that the corresponding 62 by sequence within the coding region of the AB4 ipt gene has been modified during the course of its evolution, apparently by sequence transfer from the 62 by sequence in the 3′ non-coding region. In pTi82.139 the original coding region of the ipt gene has remained largely unmodified. The pTiAB4 6b gene differs from its pTi82.139 counterpart by the lack of a 12 by repeat in the 3′ part of the coding sequence. This leads to the loss of four glutamic acid residues from a series of ten. In spite of these differences, the ipt and 6b genes of pTiAB4 are functional. Our results provide new insight into the evolution of Agrobacterium Ti plasmids and confirm the remarkable plasticity of these genetic elements. Possible implications for the study of bacterial phylogeny are discussed.

Summary The sugar and fatty acid composition of 9 strains belonging to the 3 biovars of Agrobacterium tumefaciens and to Agrobacterium rubi was investigated. All strains showed a complex composition of the sugar moieties which comprised mostly 6-deoxyhexoses, hexoses and acidic sugars, such as GalA, 3-deoxy-lyxo-heptulosaric acid and Kdo. More striking however, was the different pattern in DOC-PAGE indicating that biovar 3 strains (Agrobacterium vitis), as well as Agrobacterium rubi contained an R-type LPS (or an R-type with only a very truncated O-chain). In contrast, strains of biovars 1 and 2 showed very long O-chains comprising more than 20 O-repeating units. The fatty acid profiles of the lipid A moieties of the LPS of biovars 1, 2 and 3 were characterized by the predominance of hydroxylated fatty acids which comprised 2–5 different 3-OH-fatty acids, as well as (n-2) hydroxylated long chain fatty acids, such as 27-OH-28 : 0. Biovar 2 strains were characterized by a high content of the rare iso-branched 3-OH-15 : 0, which was lacking in the other biovars and may serve as a characteristic chemotaxonomic marker. The presence of the rare 3-OH-18 : 0 and 3-OH-18 : 1 in 7 out of 9, or 5 out of 9 strains, respectively, was conspicuous.

Summary In order to study evolutionary relationships of the crown gall pathogen on grapevine, Agrobacterium vitis , bacterial genomes were analyzed by genomic fingerprinting. Additionally, A. vitis specific IS elements were used as chromosomal markers to establish genetic relations obtained by restriction analysis. DNA fragments up to 1 Mb in size were produced by digestion of genomic DNA with the rare cutting restriction endonucleases Xba I, Sfi I, and Spe I and separated by pulsed field gel electrophoresis (PFGE). As the result of the macrorestriction analysis of 42 strains six genomic groups with homologous restriction patterns could be identified. Based on similarity coefficients of more than 0.8 A. vitis strains were allocated to one of the six genome types. For every genome type defined, a distinct IS element pattern was found. The synopsis of IS element patterns and genomic fingerprinting allowed the construction of a phylogenetic tree on the evolution of different genome types within A. vitis .

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

The octopine/cucumopine (o/c) strains of Agrobacterium vitis are divided into limited (LHR) and wide host range (WHR) strains. We present here the first map of a Ti plasmid from a WHR o/c strain, Tm4. pTiTm4 (252 kb) has homology with the octopine Ti plasmid pTiAch5, the nopaline plasmid pTiC58, and the Ri plasmid pRiHRI. Approximately 155 kb of pTiTm4 is homologous with the LHR o/c Ti plasmids pTiAB3 and pTiAg57, the remaining part is pTiTm4-specific. Our data suggest that the evolution of the o/c Ti plasmids was accompanied by large-scale changes in plasmid structure. The origin of replication of pTiTm4 was localized by a functional assay.

The analysis of housekeeping recA gene sequences from 138 strains from 13 species or genomic species of Agrobacterium, nine being biovar 1 genomospecies, and the others Agrobacterium larrymoorei, Agrobacterium rubi, Agrobacterium sp. NCPPB 1650, and Agrobacterium vitis and one “former” Agrobacterium species, Rhizobium rhizogenes, led to the identification of 50 different recA alleles and to a clear delineation of the 14 species or genomospecies entirely consistent with that obtained by amplified fragment length polymorphism (AFLP) analysis. The relevance of a recA sequencing approach for epidemiological analyses was next assessed on agrobacterial Tunisian isolates. All Tunisian isolates were found to belong to the Agrobacterium tumefaciens/biovar 1 species complex by both biochemical tests and rrs sequencing. recA sequence analysis further permitted their unambiguous assignment to A. tumefaciens genomospecies G4, G6, G7, and G8 in total agreement with the results of an AFLP-based analysis. At subspecific level, several Tunisian recA alleles were novel, indicating the power and accuracy of recA-based typing for studies of Agrobacterium spp.

The vitopine Ti plasmid pTiS4 of Agrobacterium vitis has an unusual T-DNA organization. The pTiS4 oncogenes, localized by screening selected pTiS4 clones for growth-inducing activity, are localized on three T-DNAs, whereas in all other characterized Ti plasmids one or two T-DNAs are found. The nucleotide sequences and predicted amino acid sequences of the pTiS4 oncogenes set them apart from the corresponding genes from other Ti or Ri plasmids. The oncogenes induce the same type of reaction on various test plants as the well-known pTiAch5 oncogenes but the pTiS4 ipt gene induces considerably more shoots than its Ach5 homologue. We have also identified the gene coding for vitopine synthase as well as a vitopine synthase pseudogene. Both sequences show homology to the octopine synthase gene. In terms of both nucleotide sequence and overall organization, the pTiS4 T-DNAs appear to be only distantly related to previously characterized T-DNAs.

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.

Rahnella aquatilisHX2, a biocontrol agent for grapevine crown gall caused byAgrobacterium vitis, produces an antibacterial substance that inhibits the growth ofA. vitisin vitro. In this study, we show that MH15 and MH16, two Tn5-induced mutants of HX2, have lost their abilities to inhibitA. vitisand have reduced biocontrol activities; they grow in logarithmic phase at a rate similar to that of the wild type and have single Tn5insertions. 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, designatedpqqAthroughpqqF. 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-framegdhdeletion mutant has the same phenotypes as the Tn5mutant of MH16. Complementation of both deletion and Tn5 gdhmutants restored the affected phenotypes to wild-type levels. Our results suggest that an antibacterial substance plays a role in biocontrol ofA. vitisby HX2.

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.

ELECTRONIC NOSE AS A RAPID AND INNOVATIVE TOOL FOR THE DIAGNOSIS OF GRAPEVINE CROWN GALL The electronic nose (E-nose) is an innovative device developed for microbiological, food/drink and environmental safety, and medical applications. E-nose comprises appropriate sensor arrays, able to detect and discriminate several flavours and odours from different sources. In the last decade, E-nose is being applied to recognize plant diseases caused from different quarantine bacterial pathogens such as Erwinia amylovora, Ralstonia solanacearum and Clavibacter michiganensis subsp. sepedonicus. In grapevines, tumourigenic strains of Agrobacterium vitis cause crown gall disease which may give serious problems in nurseries and vineyards. For the first time, a portable E-nose (PEN3) was used to discriminate between galled and healthy vines (controls), experimentally inoculated with two different strains of A. vitis and water, respectively. E-nose data were related to the increase of inoculation site diameters measured 9 months after inoculation with the pathogen. Preliminary gas chromatography-mass spectrometry (GC-MS) analysis were performed to identify volatile species from grapevine samples and to optimize experimental conditions for sensorial analysis. Solid phase microextraction (SPME) CAR/PDMS fibers were used to concentrate trace level vapour emissions. Spectra from tumoured vines and controls had almost identical volatile compositions, except the key compound styrene detected in infected samples. In plants, styrene formation is compatible with decarboxylation of cinnamic acid involved in secondary metabolism of plants. All data (E-nose sensors responses and diameter increases) from samples were processed with Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA). PCA application allowed a discrete separation of the samples into two groups, according to the presence or absence of disease symptoms. Moreover, the LDA model allowed the correct sample classification between healthy and galled vines (83.3%, cross validation). Tumours caused by the two strains of the pathogen were not discriminated according to the volatile compound composition identified by GC-MS. Although a larger number of grapevine samples should be analysed to create a more robust model, our results give interesting clues to go further with research on the diagnostic potential of this innovative system associated with multi-dimensional chemometric techniques. Keywords: E-nose; Agrobacterium vitis; GC-MS.

İç Anadolu bölgesinde, farklı çeşitlerle asma yetiştiriciliği giderek önem kazanmaktadır. 2007?2008 yıllarında, Konya iline bağlı 24 ilçe ve 14 adet farklı asma çeşidinde bakteriyel hastalıkların belirlenmesi amacıyla çalışmalar yürütülmüştür. Bakteriyel ur hastalığı, Agrobacterium vitis'in tanılanmasında, Roy ve Sasser, King B, PDA besi yerlerinde gelişim, 37 ºC' de gelişim, 3-ketolaktoz oluşumu, % 2' lik NaCl' de gelişim, L- tartarik asitten alkali oluşumu, sitrat kullanımı, pektolitik aktivite, oktopine ve napolin kullanımı, endoglukonaz aktivitesi, litmus milk testi, eritritol' den ve melezitoz'dan asit oluşumu, malonik asitten alkali oluşumu, demir amonyum sitrat, PDA+CaCO3'da asit temizleme testleri esas alınmıştır. Patojenisite testlerinde, Sultani Çekirdeksiz çeşidi asma fidanları, Datura sp. ve ayçiçeği bitkilerine yapılan 108 hücre/ml bakteriyel inokulasyon sonucu tipik ur oluşumu gözlenmiştir. Moleküler tanı, peh A ve virA genlerinin PCR amplifikasyonu ile yapılmıştır. Toplam 309 asma örneğinden 280' inde A. vitis belirlenirken, izolatların çoğunluğunun tümörojenik karakterde olduğu gözlemlenmiştir. Elde edilen bulgulara göre, il genelinde asmalardaki etmenle bulaşıklığın %90.61' lik oranla büyük önem taşıdığı, patojenin mevcut çeşitler içersinde en fazla Sultani Çekirdeksiz, Cardinal, Hafızali ve en az Ekşi Kara çeşitlerinde görüldüğü tespit edilmiştir., 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 on14 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 37ºC, 3-ketolactose production, growth on 2% NaCl, production of alkaline from L-tartaric acid, using of citrate, pectolytic activity, using of octopine and napolin, endogluconase activity, litmus milk test, acid from erytritol and melesitose, alkaline production from malonic acid, 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, Datura sp. and sunflower plants. It was made amplification of peh A 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 data, rate of incidence has been as 90.61% has importance and within all of the cultivars; pathogen was shown the most on cv. Sultani Seedless, cv. Cardinal, cv. Hafızali and at the least on cv. Sour Black in the province.

Tumor formation in Vitis species and hybrids, incited by Agrobacterium tumefaciens, was altered by chemical, physical, developmental, and genetic variables. Knowledge of the effect of these variables was used to develop a stringent in vitro assay system to select parents for a study of genetic factors that modulate tumor formation. Tumor formation was reduced by short day preconditioning of assay plants and by inoculation of the morphological apex of isolated stem segments. Pretreatment of plants with auxin or cytokinin altered specificity in various combinations of strains and host genotypes. All Vitis species and hybrids formed tumors in response to strains designated as limited host range, but some displayed a necrotic reaction (cell death at and below site of inoculation) or a null response (same as the response to inoculation with an avirulent strain) to strains designated as wide host range (VC Knauf, CG Panagopoulos, EW Nester [1982] Phytopathology 72: 1545-1549). Screens of F(1) progeny, derived from crosses of null, necrotic, and tumor-producing phenotypes, demonstrated that the null and the necrotic phenotypes were modulated by dominant and recessive host genes. The extent of cellular necrosis in the necrotic phenotype was modified by the morphological location of the inoculation site, by the presence of buds on the host stem, and by deletion of the tryptophane monooxygenase locus gene of the Ti-plasmid.

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.

Tn5-induced mutations in Agrobacterium vitis F2/5 resulted in both altered grape necrosis and tobacco leaf panel collapse phenotypes, suggesting that the underlying mechanisms of the reactions are related. The reaction on tobacco resembles the classical hypersensitive response (HR) caused by several plant pathogenic bacteria in that it is observable within 14 h, is inhibited by treatment of plants with metabolic inhibitors, and results in the inability to recover the pathogen from the necrotic zone. Strains of A. vitis differ with regard to their efficiency of causing the reaction on tobacco. An EcoRI fragment from one mutant, M6, which is necrosis-altered and HR-minus, was cloned and sequenced. Sequence analysis revealed that the Tn5 insertion occurred in a region that shares significant homology with genes involved in long chain fatty acid production by the marine bacteria Shewanella spp. and Moritella marina. Complementation of M6 with a cosmid clone from an F2/5 DNA library restored the tobacco HR and grape necrosis phenotypes.

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

TEZ6192 Tez (Doktora) -- Çukurova Üniversitesi, Adana, 2006. Kaynakça (s.82-94) var. xiv, 103 s. : rnk. ; 29 cm. In this study vineyards in Mersin, Adana, Osmaniye, Hatay, Gaziantep, Adyyaman and Kahramanmaraş provinces were surveyed and infected plant samples by crown gall agent were collected. Infected plants were recorded in vineyards of Mersin, Gaziantep, Hatay, Adyyaman and Kahramanmaraş provinces. Forty-seven tumorigenic bacterial isolates were isolated from galls of infected plants. All isolates were identified as Agrobacterium vitis by classical bacteriological techniques, PCR, fatty acid analysis and pathogenicity tests on grape, tomato and kalanchoe plants. All isolates caused crown gall on grape shoots. No gall formation observed on tomato (Lycopersicum esculentum Mill. cv. Heinz 2274) and kalanchoe (Bryophyllum daigremontianum (Raym.-Hamet & E.P. Perrier) Berger) plants by seven and eight isolates, respectively. Totally 464 Plant Growth Promoting Rhizobacteria (PGPR) were isolated from 39 soil samples collected from vineyards of Mersin, Adana, Osmaniye, Hatay, Gaziantep and Kahramanmaraş. Sixty-three candidates PGPR isolates solubilized phosphorus on solid NBRIP medium. Ten candidate PGPR isolates which have solubilized phosphorus with the highest ratio were used in pot experiments. PGPR isolates, Ga7/3-6 (%45), Ga10/2-5 (%97), Os1/3-1 (%80), OsD1/3-1 (%98) ve HaD6/3-1 (%75), significantly reduced galls weight on grape plants in pot experiments. When lipopolysaccharide (LPS) extracted from A. vitis applied to cutting grapevine, gal size was reduced by 91.5% rate. Bu çalışmada Mersin, Adana, Osmaniye, Hatay, Gaziantep, Adıyaman ve Kahramanmaraş illerinde bulunan bağ alanları incelenmiş ve hastalıklı bitkilerden örnek alınmıştır. Mersin, Gaziantep, Hatay, Adıyaman ve Kahramanmaraş illerinde hastalıklı asma bitkilerindeki urlu dokulardan toplam 47 adet ur oluşturan patojen bakteri izole edilmiştir. Klasik bakteriyolojik tanılama teknikleri, PCR, yağ asit analizi ve patojenite testlerine göre izole edilen tüm izolatlar Agrobacterium vitis olarak tanılanmıştır. Patojenite testlerinde, tüm izolatlar bağda ur oluştururken, 7 izolat domateste (Lycopersicum esculentum Mill. cv. Heinz 2274) ve 8 izolat kalonşe (Bryophyllum daigremontianum (Raym.-Hamet & E.P. Perrier) Berger) bitkilerinde ur oluşturmamıştır. Mersin, Adana, Osmaniye, Hatay, Gaziantep ve Kahramanmaraş illerinde bulunan bağ alanlarından asma bitkisi büyümesini teşvik eden rizobakterileri (PGPR) izole etmek için 39 toprak örneği alınmıştır. Toprak örneklerinden 464 adet bakteri izole edilmiştir. Katı NBRIP besi yeri kullanılarak bu bakteri izolatlarından 63'ünün fosforu çözdüğü belirlenmiştir. Nicel olarak izolatların fosforu çözme miktarı belirlenmiş ve en yüksek fosfor çözen 10 izolat seçilerek saksı çalışmalarında kullanılmıştır. Saksı çalışması ile Ga7/3-6 (%45), Ga10/2-5 (%97), Os1/3-1 (%80), OsD1/3-1 (%98) ve HaD6/3-1 (%75) izolatlarının asmada ur ağırlığını önemli düzeyde azalttığı belirlenmiştir. A. vitis izolatından elde edilmiş lipopolisakkarid (LPS)'lerin asma çubuklarına 10 g/l konsantrasyonda infiltre edildiğinde ur büyüklüğünü %91,5 oranında azalttığı belirlenmiştir. Bu çalışma Ç.Ü. Bilimsel Araştırma Projeleri Birimi Tarafından Desteklenmiştir. Proje No:ZF2005D1, ZF2005KAP10, 105O007

Crown gall on grapevine is mainly caused by Agrobacterium vitis, which metabolizes tartrate. Competition experiments between a tartrate-utilizing strain and its non-utilizing derivative showed that tartrate utilization confers a selective advantage on grapevine.

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 5x10(2) and 1x10(3)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.

On the basis of 23S rRNA gene sequences, 1 universal forward and 4 taxon (species/biovar)-specific reverse primers were designed for multiplex PCR to aid in identification and differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2. In reactions with DNA of 119 bacterial strains belonging to: Agrobacterium, Allorhizobium, Mesorhizobium, Rhizobium, Sinorhizobium and Phyllobacterium, as well as phytopathogenic bacteria representing various genera, the primers developed for identification of A. vitis, A. rubi or Agrobacterium biovar 1 amplified only DNA of strains belonging to these taxa, producing fragments of the expected sizes: 478, 1006 and 184bp, respectively. However, in the case of the primer developed for identification of Agrobacterium biovar 2, the characteristic 1066bp PCR product was obtained not only with DNA of this biovar, but also with DNA of 3 atypical biovar 1 strains and some rhizobial strains. Differentiation between Agrobacterium biovar 2 and the other strains was possible using the restriction analysis of this product with endonuclease Alw26I. The method developed is an excellent tool for rapid classification of these 4 taxa of Agrobacterium.

The sequence of the rrnA operon and its flanking regions was determined for the Agrobacterium vitis type strain NCPPB3554. Compared to the earlier obtained rrnA sequence of A. vitis strain S4, several important differences were noted: the sequences diverged at the 5'-flanking region, within the 16S-23S intergenic region, and within the 23S rRNA sequence. The B8 stem-loop structure at the 5'-end of the 23S rRNA of strain NCPPB3554 was 142 nt shorter than that of strain S4. These findings have important consequences for the use of ribosomal RNA gene sequences in phylogenetic comparisons.

The sucrose phosphorylase gene of an isolate, Leuconostoc mesenteroides No. 165, was amplified by PCR, cloned on pUC118, and expressed in E. coli. The nucleotide sequence of the gene showed 96.3% similarity to that of L. mesenteroides ATCC12291 and 67% to that of Streptococcus mutans, but low similarity to the Agrobacterium vitis gene. The cloned gene, which fusing with lacZalpha, was expressed inducibly with IPTG in E. coli to produce an active enzyme in large quantities that accounted for about 50% of the total cell protein.

Combined inoculation of grapevine roots with Meloidogyne hapla and Agrobacterium vitis at the level of 10 2−3 bacteria/g of soil resulted in root infestation by A. vitis. When inoculation of M. hapla and A. vitis was applied, most of the feeder roots were dead and at points of secondary root branching, dark lesions could be observed. Along the secondary living roots, typical nematode induced galls could be seen. In many cases the infection stopped growth of the main or secondary roots. Plants inoculated with A. vitis alone did not show any galls or swellings on roots and its infection did not stop root growth. However in the M. hapla only treatment, small swellings or elongated galls were readily apparent on secondary roots. These galls were always exempt of A. vitis. Xylem sap, collected in spring 2 years after inoculation of A. vitis + M. hapla yielded many A. vitis cells demonstrating that the bacteria became systemic in plant vessels

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.

Rhizobium, Agrobacterium and Allorhizobium are genera within the bacterial family Rhizobiaceae, together with Sinorhizobium. The species of Agrobacterium, Agrobacterium tumefaciens (syn. Agrobacterium radiobacter), Agrobacterium rhizogenes, Agrobacterium rubi and Agrobacterium vitis, together with Allorhizobium undicola, form a monophyletic group with all Rhizobium species, based on comparative 16S rDNA analyses. Agrobacterium is an artificial genus comprising plant-pathogenic species. The monophyletic nature of Agrobacterium, Allorhizobium and Rhizobium and their common phenotypic generic circumscription support their amalgamation into a single genus, Rhizobium. Agrobacterium tumefaciens was conserved as the type species of Agrobacterium, but the epithet radiobacter would take precedence as Rhizobium radiobacter in the revised genus. The proposed new combinations are Rhizobium radiobacter, Rhizobium rhizogenes, Rhizobium rubi, Rhizobium undicola and Rhizobium vitis.

ÖZET Doktora Tezi ORTA ANADOLU BAĞLARINDA TAÇ URU' NA NEDEN OLAN Agrobaaerium vitis'İN BÖLGESEL DAĞILIMI VE BAZI BİYOLOJİK ÖZELLİKLERİ ÜZERİNE ARAŞTIRMALAR Nihal ARGUN Ankara Üniversitesi Fen Bilimleri Enstitüsü Bitki Koruma Anabilim Dalı Danışman: Prof. Dr. Salih MADEN Orta Anadolu'da. Ankara, Kırıkkale, Konya, Karaman ve Nevşehir illerinde 1000 da üzerinde bağ alanı olan 55 köy Agrobaaerium vitis yönünden incelenmiş, bunlardan 6' sı Ankara, 7' si Nevşehir, 9' u Konya, 2' si Kırıkkale ve 3' ü Karaman'a ait olmak üzere toplam 27 köyün bağlarının adı geçen bakteri ile bulaşık olduğu tesbit edilmiştir. Bir yaşlı dallardan öz su ekstraksiyonu ve hasta bitki urlarının parçalanması yoluyla yapılan iki farklı teknik karşılaştırılmış ve taze olması koşulu ile yılın her döneminde alınan urlardan yapılan izolasyonlarda daha fazla izolat elde edilmiştir. Ancak taze urların, en yoğun şekilde bahar aylarında oluştuğu gözlenmiştir. İzolasyonlar sonucu; ikisi özsudan olmak üzere toplam 57 A. vitis izolaü elde edilmiş ve hepsinin asma dallarında ur, sürgün ve kökte nekroz ve ayrıca tütünde aşın duyarlılık reaksiyonu (HR) oluşturduğu belirlenmiştir. Daha önceden biyolojik kontrol ajanı olduğu bilinen F 2/5 (patojen olmayan A. vitis) sera koşullarında elde edilen tüm izolatlara karşı denenmiştir. Adı geçen biyolojik kontrol ajanı olan A. viıis patojen tüm izolatlann asmadaki ur oluşturma kapasitesini çok büyük oranda engellemiştir. Asmada ur oluşturma yeteneklerinin incelenmesi sırasında, oluşturduğu ur büyüklüğüne göre en virulent izolat olarak belirlenen Tr2, Orta Anadolu Bölgesi' nde yaygın üretimi yapılan üzüm çeşit ve anaçlarına inokule edilerek bunların reaksiyonları belirlenmiştir. Sonuçta; Hafızali ve Cardinal üzüm çeşitlerinde ur oluşmazken. Alicante Bouschet ve Kadın Parmağı en duyarlı çeşitler olarak bulunmuştur. Anaçlann çeşitlere göre daha dayanıklı olduğu, 44-53 M ve 1 103 P'nin ise denen diğer anaçlara göre daha dayanıklı olduğu tesbit edilmiştir. 2001, 84 sayfa ANAHTAR KELİMELER; Agrobaaerium vitis, asma, F2/5, çeşit ve anaç reaksiyonu ABSTRACT Ph.D. Thesis RESEARCHES ON REGIONAL DISSEMINATION AND SOME BIOLOGICAL CHARACTERISTICS OF Agrobaaerium ritis CAUSING CROWN GALL ON GRAPEVINES IN CENTRAL ANATOLIA Nihal ARGUN Ankara University Graduate School of Natural and Applied Sciences Department of Plant Protection Supervisor: Prof. Dr. Salih MADEN Crown gall on grapevines was searched at 55 villages of Ankara, Kınkkale, Konya, Karaman and Nevşehir provinces in Central Anatolia and 6, 2, 9, 3, 7 villages of those provinces were found infected respectively. Two different isolation methods, crushing of fresh galls in water and vacuum extraction of sap of the canes, were compared. More recovery of the bacterium was obtained from the fresh galls. As a result of these isolations, totally 57 Agrobaaerium viris isolates, 2 of which is from sap were obtained. All the isolates caused galls on grape canes, necrosis on the shoots and roots of grapevines also HR on tobacco plants. F2/5 (non-pathogen A. viris) known as a biological control agent was tested against all the obtained isolates in greenhouse. F2/5 inhibited tumorigenicity of almost all the isolates on grape. The reactions of common grape cultivars and rootstocks in Central Anatolia were determined by inoculating with the most virulent isolate as to size of galls on grape, Tr2. Consequently, no galls formed on cultivars of Hafizali and Cardinal. However Alicante Bouschet and Kadın Parmağı were found the most susceptible. Rootstocks were more resistant than cultivars, 44-53 M and 11 03 P were found more resistant than the other rootstocks in assay. 2001, 84 pages Key Words: Agrobaaerium vitis, grapevine, F2/5, cultivar and rootstock reaction 84

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

Tryptophan-2-monooxygenase genes occur in a number of bacteria and encode the conversion of tryptophan to the plant hormone precursor indole-3-acetamide. The role of these genes in the plant-bacteria interaction is often unclear. However, their function as a virulence determinant is established for Pseudomonas savastanoi and Agrobacterium tumefaciens. Some members of the Agrobacteria, such as Agrobacterium vitis have a limited host range. We have characterized the tryptophan-2-monooxygenase (iaaM) gene of A. vitis strain AG162 and show it is different from other A. vitis strains and related to iaaM of A. rhizogenes. The sequence of AG162 iaaM was deposited in the Genbank database under the accession number AF142716.

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.

Three tartrate utilization regions from Agrobacterium vitis strains involved in host specificity have been compared, to clearly define the borders of these regions and eventually identify specific sequences that could provide a mechanism of duplication of this region. A 10.8-kb conserved DNA fragment called the TAR element, found in different genetic contexts, was defined. A comparison of the two tartrate dehydrogenase genes (ttuC and ttuC') in each of the three TAR elements suggests that these genes co-evolve.

Agrobacterium vitis is the causal organism of crown gall in grapevine. Infection is particularly severe in areas that experience winter damage to the vine. Improving resistance to A. vitis will require a detailed knowledge about this organism. In this study, 18 grapevine isolates of A. vitis were obtained from different vineyards around Lubbock, Texas. Isolates were subjected to a phenotypic characterization using several biochemical tests. Isolates were also evaluated for host range, tumor morphology and opine utilization and the oncogenic regions of the T-DNA were compared at the molecular level by Southern hybridzation. Although all isolates were able to metabolize tartarte and grow on Roy-sasser media, there was high variability based on other biochemical tests. This variability was also observed in terms of tumor morphology and opine utilization. Southern hybridization analysis, however, showed that the oncogenic regions of A. vitis isolates have strong homology with previously characterized Agrobacterium T-DNA genes. This study suggests that the local isolates were probably of clonal origin. Tobacco was used as a model to test whether transgenic plants with TDNA auxin biosynthetic genes in an antisense orientation would block tumor morphology. The expression of iaaM antisense RNA resulted in an approximately 45% reduction in the number of tumors. The iaaM antisense also affected tumor morphology by reducing gall size. Plants expressing iaaH antisense had fewer tumors than the control, but more than 60% of samples formed roots when infected with Agrobacterium. The incomplete suppression of tumor formation may be due to the presence of other loci on the T-DNA that are also involved in auxin biosynthesis. Although a complete inhibition of tumorgenesis was not achieved, the fact that both tumor frequency and morphology changed, indicated that further manipulation of phytohormone genes using antisense technology may result in complete resistance to Agrobacterium tumefaciens species in transgenic plants.

The incompatibility properties of two tumor-inducing (Ti) and seven tartrate (Tr) plasmids, derived from various Agrobacterium vitis strains, were characterized using incRh1, incRh2, incRh3, and incRh4 clones which were established for the identification and classification of Agrobacterium plasmids. The tested A. vitis plasmids could be allocated into four groups on the basis of their incompatibility with incRh1, incRh2, and incRh4 clones. The two octopine tumor-inducing plasmids, pTiAT6 and pTiAB3, expressed incompatibility both to incRh1 and to incRh2 clones. Three pTrs could not be allocated either to incRh1-4 and incAg1 or to the wide-host-range incP1, incQ, and incW groups.

Octopine and nopaline strains of Agrobacterium tumefaciens differ in their ability to induce tumors on Nicotiana glauca. The presence of a virF locus on the octopine Ti plasmid makes N. glauca a host plant for these strains, indicating that the VirF protein is a host-range determinant. Here we show the presence of a virF locus not only on the Agrobacterium vitis octopine/cucumopine plasmids pTiAg57 and pTiTm4, but also on the nopaline Ti plas-mids pTiAT1, pTiAT66a, and pTiAT66b. On the octopine Ti plasmids from A. tumefaciens the virF gene is located between the virE locus and the left border of the T-region. In contrast, the virF gene on Ti plasmids of A. vitis is located at the very left end of the vir-region near the virA locus. The virF gene of pTiAg57 has been sequenced and codes for a protein of 202 amino acids with a molecular mass of 22,280 Da. Comparison showed that the virF gene from A. vitis strain Ag57 is almost identical to that from A. tumefaciens octopine strains. The transcription of the pTiAg57 virF is inducible by the plant phenolic compound acetosyringone through the presence of a vir-box consensus sequence in its promoter region. The VirF protein from pTiAg57 can complement octopine A. tumefaciens strains deleted for virF as shown by tumor formation on N. glauca.

Agrocin-minus mutants of nontumorigenic Agrobacterium vitis strain F2/5 controlled grape crown gall as well as the wild-type strain, indicating that agrocin is not a major factor in the mechanism of biological control. Relative levels of attachment to grape cells by tumorigenic and biocontrol strains were also measured. Attachment of tumorigenic strains (CG49 and K306) and biological control strains (F2/5 and agrocin-minus mutant 1077) was often reduced when mixtures of the strains were applied. However, high populations (103to 105CFU/ml) of all strains attached following mixed inoculations, suggesting that competition for attachment sites is also not a factor in the mechanism of biological control. Transfer of T-DNA to grape by CG49 was prevented or greatly inhibited in the presence of F2/5 or 1077 as measured by expression of the GUS reporter gene. The Ti plasmid virulence genes, however, were induced by exudates from grape shoots that had been inoculated with F2/5. Sonicated and autoclaved preparations of F2/5 and 1077 did not control crown gall or inhibit T-DNA transfer. Control by F2/5 is specific to grape, since gall formation on tomato, sunflower, and Kalanchoe daigremontiana were not inhibited.

The genetic diversity among 16 strains of Erwinia amylovora, chosen to represent different host plant origins and geographical regions, was investigated by RAPD analysis. One strain of Erwinia herbicola and one of Agrobacterium vitis were used as outgroups. Ninety-eight different RAPD fragments were produced by polymerase chain reaction amplification with six different 10-mer primers. RAPD banding profiles were found that enabled the Erw. amylovora strains to be distinguished from one another. Cluster analysis based on the number of RAPD fragments shared between strains showed that strains of Erw. amylovora isolated from subfamily Pomoideae formed a single group, whereas two strains from Rubus (subfamily Rosoideae) formed a second group. Two strains isolated from Asian pear on Hokkaido, Japan, formed a third group. Sets of RAPD fragments were identified that enabled each of the two host-range groups and one geographical region (Hokkaido) of Erw. amylovora strains to be unambiguously distinguished from one another and from the outgroups. This study shows that strains of Erw. amylovora exhibit genetic diversity detectable by RAPD analysis, and that molecular and statistical analysis of RAPD fragments can be used both to distinguish between strains and to determine relatedness between them.

DNA sequencing of the Agrobacterium vitis pehA gene revealed a predicted protein with an M(r) of 58,000 and significant similarity to the polygalacturonases of two other plant pathogens, Erwinia carotovora and Ralstonia (= Pseudomonas or Burkholderia) solanacearum. Sequencing of the N terminus of the PehA protein demonstrated cleavage of a 34-amino-acid signal peptide from pre-PehA. Mature PehA accumulated primarily in the periplasm of A. vitis and pehA+ Escherichia coli cells during exponential growth. A. vitis PehA released dimers, trimers, and monomers from polygalacturonic acid and caused less electrolyte leakage from potato tuber tissue than did the E. carotovora and R. solanacearum polygalacturonases.

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. References: (1) A. Abolmaaty et al. Microbios 101:181, 2000. (2) F. Bini et al. Vitis 47:181, 2008. (3) D. Costechareyre et al. Microb. Ecol. 60:862, 2010. (4) E. Szegedi and S. Bottka. Vitis 41:37, 2002.

A highly variable region of DNA was found between positions 115 and 388 (Escherichia coli numeration) of the large subunit (ls) rRNA genes of 55 rhizobial and agrobacterial strains. In each case this heterogeneity was accompanied by the presence of a new rRNA species approximately 130 bp long. This novel rRNA species corresponded to the 5'-end of the ls rRNA genes. An additional rRNA processing site was located in the central region of the remaining ls rRNA of many of the Rhizobium leguminosarum and Rh. etli strains, and in all of the agrobacteria studied, excepting the type strain of Agrobacterium vitis NCPPB 3554 and Agrobacterium sp. strain ChAg4.

The effect of nine factors on the outcome of classic in vitro screens testing the antagonistic action of endophytic bacterial isolates from grape vines against virulent Agrobacterium vitis has been examined. These factors were (i) the strain of A. vitis, (ii) the strain of endophyte, (iii) the growth medium of the pathogen, (iv) the growth medium of the endophyte, (v) the temperature of growth of the pathogen, (vi) the temperature of growth of the endophyte, (vii) the pH of growth of the pathogen, (viii) the pH of growth of the endophyte, and (ix) the medium of the assay plate. Analyses of variance of the full factorial design incorporating main effects and two- and three-way interactions accounted for 66% of the variance. All nine factors had a significant effect on the diameter of inhibition zones (p

Rhizobium galegae infects goat’s rue (Galega orientalis and G. officinalis) in a very host specific manner. Nodules with an apical meristem are formed. R. galegae strains only form effective (nitrogen-fixing) nodules on their original host. Strains from G. orientalis infect G. officinalis, and give rise to almost fully developed nodules which fail to fix nitrogen, and vice versa (Lindstrom, 1989). G. orientalis is a new, perennial forage legume for Northern conditions. It is very persistent, resistant to disease and suitable for making silage. The plant and its rhizobia originate in the Caucasus and the plant requires inoculation with compatible rhizobia to grow well. Our laboratory has focused on the ecology of nitrogen fixation, the taxonomy of R. galegae, molecular biology of the interaction between symbionts and the development and application of molecular tools for identification and classification of the rhizobia. R. galegae is the only Rhizobium species described so far that belongs to the same phylogenetic branch as Agrobacterium vitis, A. rubi and biovar 1 (A. tumefaciens and A. radiobacter, Fig. 1). We are interested in the basis for the host specificity in our system and in the evolution of nodulation functions. This report describes the phylogeny of common nodulation genes and nodD genes of R. galegae and the identification of another nodulation gene region.

Crown gall tumors, collected from branches of 1-year-old weeping fig (Ficus benjamina L.) trees, yielded both tumorigenic and nonpathogenic agrobacteria. On the basis of classical diagnostic tests, the nonpathogenic strains were identified as Agrobacterium tumefaciens, whereas the tumorigenic strains could not be assigned to any of the known terrestrial Agrobacterium spp. The tumorigenic strains also differed from other members of the genus by producing more acid from mannitol. According to cluster analysis of carbon substrate oxidation (GN Microplate; Biolog, Inc.) and fatty acid content, the tumorigenic fig strains were distinct from strains of A. tumefaciens, Agrobacterium rhizogenes, Agrobacterium vitis, and Agrobacterium rubi. Furthermore, they had unusual opine metabolism, inducing tumors that synthesized nopaline and three recently discovered opines: chrysopine (d-lactone of N-1-deoxy-D-fructosyl-L-glutamine, and N-1-deoxy-D-fructosyl-L-glutamine, and N-1-deoxy-D-fructosyl-5-oxo-L-proline. The nonpathogenic A. tumefaciens strains present in the same tumors were unable to degrade any of the opines tested. The phylogenetic position of the tumorigenic fig strain AF3.10 was inferred from comparing its rrs (i.e., 16S rRNA gene) sequence with those from the type strains of Agrobacterium and Rhizobium species. The analysis showed that strain AF3.10 clustered with A. tumefaciens and A. rubi but not with A. vitis and was far removed from A. rhizogenes. However, the sequence was significantly different from those of A. tumefaciens and A. rubi to suggest that the tumorigenic fig strain may be a new Agrobacterium species that is as different from A. tumefaciens and A. rubi as these two species are from one another.

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. References: (1) J. H. Haas et al. Appl. Environ. Microbiol. 61:2879, 1995. (2) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (3) J. Pulawska et al. Syst. Appl. Microbiol. 29:470, 2006. (4) K. Suzaki et al. J. Gen. Plant Pathol. 70:342, 2004.

The 16S rRNA sequences of seven representative Agrobacterium strains, eight representative Rhizobium strains, and the type strains of Azorhizobium caulinodans and Bradyrhizobium japonicum were determined. These strains included the type strains of Agrobacterium tumefaciens, Agrobacterium rhizogenes, Agrobacterium radiobacter, Agrobacterium vitis, Agrobacterium rubi, Rhizobium fredii, Rhizobium galegae, Rhizobium huakuii, Rhizobium leguminosarum, Rhizobium loti, Rhizobium meliloti, and Rhizobium tropici. A phylogenetic analysis showed that the 15 strains of Agrobacterium and Rhizobium species formed a compact phylogenetic cluster clearly separated from the other members of the alpha subclass of the Proteobacteria. However, Agrobacterium species and Rhizobium species are phylogenetically entwined with one another, and the two genera cannot be separated. In the Agrobacterium species, the strains of biovar 1, biovar 2, Agrobacterium rubi, and Agrobacterium vitis were clearly separated. The two biovars exhibited homogeneity in their phenotypic, chemotaxonomic, and phylogenetic characteristics, and two species should be established for the two biovars. We considered the nomenclature of the two biovars, and revised descriptions of Agrobacterium radiobacter (for the biovar 1 strains) and Agrobacterium rhizogenes (for the biovar 2 strains) are proposed. The name Agrobacterium tumefaciens is rejected because the type strain of this species was assigned to Agrobacterium radiobacter, and consequently the description of the genus Agrobacterium is revised.

The octopine/cucumopine (o/c) Ti plasmids of the grapevine-associated Agrobacterium vitis strains constitute a family of related DNA molecules. Restriction maps were established of two limited-host-range o/c Ti plasmids, pTiAg57 and pTiAB3, and of the wide-host-range o/c Ti plasmid pTiHml. Together with the previously obtained map of the wide-host-range o/c Ti plasmid pTiTm4, about 1000 kb were mapped with a resolution of 0.2 kb, allowing a detailed comparison of the various structures. One region of the o/c Ti plasmids is highly conserved and differs mainly by the presence or absence of relatively small DNA fragments (0.9–2.7 kb); the other region has been modified more extensively and carries large sequences specific for each Ti plasmid type. The sequence similarity within large conserved regions shows that these plasmids have diverged recently and that their evolution was driven by large-scale genetic events rather than single nucleotide changes. These results have important implications for studies on bacterial evolution.

Within the Agrobacterium vitis group the vitopine strains represent a special subclass. Vitopine bacteria carry Ti plasmids with little or no homology with the well-characterized T-DNAs of Agrobacterium tumefaciens or Agrobacterium rhizogenes. The 262-kb Ti plasmid of the vitopine strain S4 was cloned and mapped. Homology studies with the octopine Ti plasmid pTiAch5, the nopaline Ti plasmid pTiC58, and the agropine/mannopine Ri plasmid pRiHRI identified several regions of homology. The origin of replication was localized to within 2.5 kb.

Agrobacterium tumefaciens biovar 3 causes both crown gall and root decay of grapes. All biovar 3 strains, regardless of their tumorigenicity, produce in culture a single polygalacturonase with a pI around 4.5. A. tumefaciens biovar 3 strain CG49 was mutagenized with Tn5 by using pSUP2021 as a suicide vector. A mutant strain, CG50, lacking polygalacturonase activity was isolated. The mutation was due to a single Tn5 insertion in an 8.5-kb EcoRI fragment that also contained the polygalacturonase structural gene. The polygalacturonase-encoding pehA gene was cloned in Escherichia coli by using the plasmid pBluescript as a vector. Activity-stained isoelectric focusing gel analysis demonstrated that E. coli cells harboring the pehA+ recombinant plasmid pCPP2067 produced a polygalacturonase in culture with the same pI as the enzyme produced by CG49. The pehA gene was localized within a 2.5-kb HindIII-SalI fragment. This fragment was used as a probe in Southern hybridization analysis and showed that no closely related genes are present in A. tumefaciens biovars 1 or 2, Rhizobium leguminosarum, or Bradyrhizobium japonicum. The polygalacturonase mutant was unable to induce root decay in grapes (Vitis vinifera cv. Chardonnay) and was substantially less tumorigenic than the wild type in grape stems when low levels of inoculum were used, although both strains were equally tumorigenic in potato disc assays. The results indicate that polygalacturonase is a virulence factor in both the root decay and crown gall incited in grapes by A. tumefaciens biovar 3.