The Ecology of Agrobacterium vitis and Management of Crown Gall Disease in Vineyards.

Agrobacterium vitis is the primary causal agent of grapevine crown gall worldwide. Symptoms of grapevine crown gall disease include tumor formation on the aerial plant parts, whereas both tumorigenic and nontumorigenic strains of A. vitis cause root necrosis. Genetic and genomic analyses indicated that A. vitis is distinguishable from the members of the Agrobacterium genus and its transfer to the genus Allorhizobium was suggested. A. vitis is genetically diverse, with respect to both chromosomal and plasmid DNA. Its pathogenicity is mainly determined by a large conjugal tumor-inducing (Ti) plasmid characterized by a mosaic structure with conserved and variable regions. Traditionally, A. vitis Ti plasmids and host strains were differentiated into octopine/cucumopine, nopaline, and vitopine groups, based on opine markers. However, tumorigenic and nontumorigenic strains of A. vitis may carry other ecologically important plasmids, such as tartrate- and opine-catabolic plasmids. A. vitis colonizes vines endophytically. It is also able to survive epiphytically on grapevine plants and is detected in soil exclusively in association with grapevine plants. Because A. vitis persists systemically in symptomless grapevine plants, it can be efficiently disseminated to distant geographical areas via international trade of propagation material. The use of healthy planting material in areas with no history of the crown gall represents the crucial measure of disease management. Moreover, biological control and production of resistant grape varieties are encouraging as future control measures.

The Impacts of Tumorigenic and Nontumorigenic Agrobacterium vitis Strains on Graft Strength and Growth of Grapevines.

The effects of tumorigenic and nontumorigenic strains of Agrobacterium vitis on graft strength and growth of grapevines was studied. A procedure was developed for inoculating graft interface surfaces with A. vitis and for measuring the force required to break grafts at different time points. Cuttings were soaked in an aqueous suspension of bacteria, about 106 CFU/ml, and bacteria were spread onto the graft interface during the grafting procedure. Tumorigenic strain CG49 caused reduced bud germination and increased callus (crown gall) at the graft union and at the base of cuttings at 30 days postinoculation (dpi) and significantly reduced shoot growth by 60 dpi whereas, at the same time points, nontumorigenic strain F2/5 inhibited callus formation but did not affect bud germination or shoot growth. Graft strength was enhanced at 30 dpi with CG49, presumably because the crown gall callus served to secure the union; graft strength was weakened by F2/5 over the same period. Between 30 and 60 dpi, the greatest increase in graft strength was observed in the water control. Following graft union inoculations, the A. vitis population increased more than 1,000-fold within 5 days.

Inhibition of Grape Crown Gall by Agrobacterium vitis F2/5 Requires Two Nonribosomal Peptide Synthetases and One Polyketide Synthase.

Agrobacterium vitis nontumorigenic strain F2/5 is able to inhibit crown gall disease on grapevines. The mechanism of grape tumor inhibition (GTI) by F2/5 has not been fully determined. In this study, we demonstrate that two nonribosomal peptide synthetase (NRPS) genes (F-avi3342 and F-avi5730) and one polyketide synthase gene (F-avi4330) are required for GTI. Knockout of any one of them resulted in F/25 losing GTI capacity. We previously reported that F-avi3342 and F-avi4330 but not F-avi5730 are required for induction of grape tissue necrosis and tobacco hypersensitive response. F-avi5730 is predicted to encode a single modular NRPS. It is located in a cluster that is homologous to the siderophore vicibactin biosynthesis locus in Rhizobium species. Individual disruption of F-avi5730 and two immediate downstream genes, F-avi5731 and F-avi5732, all resulted in reduced siderophore production; however, only F-avi5730 was found to be required for GTI. Complemented F-avi5730 mutant (ΔF-avi5730(+)) restored a wild-type level of GTI activity. It was determined that, over time, populations of ΔF-avi4330, ΔF-avi3342, and ΔF-avi5730 at inoculated wound sites on grapevine did not differ from those of ΔF-avi5730(+) indicating that loss of GTI was not due to reduced colonization of wound sites by mutants.

Distribution of Agrobacterium vitis in Grapevines and Its Relevance to Pathogen Elimination.

Agrobacterium vitis, the cause of crown gall disease on grapevine, survives internally in vines and can be spread in cuttings for propagation. The possibility of generating pathogen-free vines through tissue culture makes it essential to understand the distribution of the pathogen in grapevines. A highly sensitive magnetic capture hybridization procedure along with real-time polymerase chain reaction were used to measure the distribution of tumorigenic A. vitis in dormant canes and green shoots of grapevines. Tumorigenic A. vitis was distributed from the basal to apical nodal and internodal tissues of canes as well as in nonlignified green shoots. In experiments conducted in 2013, A. vitis was detected in up to 17% of shoot tips and 52% of meristems of greenhouse-grown plants initiated from known A. vitis-contaminated cuttings. A lower frequency of detection was observed from surface-disinfected shoot tips (7%) as compared with nondisinfected tips (37%), suggesting epiphytic survival on green tissues. In 2014, vines propagated from cuttings collected from crown gall-infected vines from a different vineyard yielded lower incidences of A. vitis from shoot tips, and the bacterium was not detected in meristems. Tumorigenic A. vitis was also detected in cuttings of wild grapevines (Vitis riparia) that were collected both adjacent to and far removed from commercial vineyards.

An Sfp-type PPTase and associated polyketide and nonribosomal peptide synthases in Agrobacterium vitis are essential for induction of tobacco hypersensitive response and grape necrosis.

An Sfp-type phosphopantetheinyl transferase (PPTase) encoding gene F-avi5813 in Agrobacterium vitis F2/5 was found to be required for the induction of a tobacco hypersensitive response (HR) and grape necrosis. Sfp-type PPTases are post-translation modification enzymes that activate acyl-carry protein (ACP) domains in polyketide synthases (PKS) and peptidyl-carrier protein (PCP) domains of nonribosomal peptide synthases (NRPS). Mutagenesis of PKS and NRPS genes in A. vitis led to the identification of a PKS gene (F-avi4330) and NRPS gene (F-avi3342) that are both required for HR and necrosis. The gene immediately downstream of F-avi4330 (F-avi4329) encoding a predicted aminotransferase was also found to be required for HR and necrosis. Regulation of F-avi4330 and F-avi3342 by quorum-sensing genes avhR, aviR, and avsR and by a lysR-type regulator, lhnR, was investigated. It was determined that F-avi4330 expression is positively regulated by avhR, aviR, and lhnR and negatively regulated by avsR. F-avi3342 was found to be positively regulated by avhR, aviR, and avsR and negatively regulated by lhnR. Our results suggest that a putative hybrid peptide-polyketide metabolite synthesized by F-avi4330 and F-avi3342 is associated with induction of tobacco HR and grape necrosis. This is the first report that demonstrates that NRPS and PKS play essential roles in conferring the unique ability of A. vitis to elicit a non-host-specific HR and host-specific necrosis.

Reconciliation of sequence data and updated annotation of the genome of Agrobacterium tumefaciens C58, and distribution of a linear chromosome in the genus Agrobacterium.

Two groups independently sequenced the Agrobacterium tumefaciens C58 genome in 2001. We report here consolidation of these sequences, updated annotation, and additional analysis of the evolutionary history of the linear chromosome, which is apparently limited to the biovar I group of Agrobacterium.

Development of a magnetic capture hybridization real-time PCR assay for detection of tumorigenic Agrobacterium vitis in grapevines.

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

A host-specific biological control of grape crown gall by Agrobacterium vitis strain F2/5: its regulation and population dynamics.

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 clpP1 resulted in reduced or loss of biological control. All mutants were complemented; however, restoration of biological control by complemented clpA and clpP1 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.

Identification of an operon, Pil-Chp, that controls twitching motility and virulence in Xylella fastidiosa.

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce's disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.

LasR receptor for detection of long-chain quorum-sensing signals: identification of N-acyl-homoserine lactones encoded by the avsI locus of Agrobacterium vitis.

Bacterial biosensor strains have greatly facilitated the rapid discovery, isolation, and study of quorum-sensing systems. In this study, we determined the relative sensitivity of a LasR-based E. coli bacterial bioluminescence biosensor JM109 (pSB1075) for 13 diverse long-chain N-acyl-homoserine lactones (AHLs) including oxygen-substituted and -unsubstituted AHLs containing 14, 16, and 18 carbons and with and without double bonds. Furthermore, we show by bioassay, HPLC, and GC/MS that four long-chain AHLs of the C16-HSL family are encoded by the avsI gene of Agrobacterium vitis strain F2/5, a non-tumorigenic strain that inhibits pathogenic strains of A. vitis from causing crown gall on grape. The four C16-HSLs include: C16-HSL, N-hexadecanoyl homoserine lactone; 3-oxo-C16-HSL, N-(3-oxohexadecanoyl)homoserine lactone; C16:1-HSL, N-(cis-9-octadecenoyl)homoserine lactone; and 3-oxo-C16:1-HSL, N-(3-oxo-cis-11-hexadecenoyl)homoserine lactone. Thus, the LasR-based bioluminescent biosensor tested in this study should serve as a useful tool for the detection of various long-chain AHLs with and without double bonds as well as those oxylated at the third carbon from uninvestigated species.

Resistance to crown gall disease in transgenic grapevine rootstocks containing truncated virE2 of Agrobacterium.

A 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.

Genome sequences of three agrobacterium biovars help elucidate the evolution of multichromosome genomes in bacteria.

The family Rhizobiaceae contains plant-associated bacteria with critical roles in ecology and agriculture. Within this family, many Rhizobium and Sinorhizobium strains are nitrogen-fixing plant mutualists, while many strains designated as Agrobacterium are plant pathogens. These contrasting lifestyles are primarily dependent on the transmissible plasmids each strain harbors. Members of the Rhizobiaceae also have diverse genome architectures that include single chromosomes, multiple chromosomes, and plasmids of various sizes. Agrobacterium strains have been divided into three biovars, based on physiological and biochemical properties. The genome of a biovar I strain, A. tumefaciens C58, has been previously sequenced. In this study, the genomes of the biovar II strain A. radiobacter K84, a commercially available biological control strain that inhibits certain pathogenic agrobacteria, and the biovar III strain A. vitis S4, a narrow-host-range strain that infects grapes and invokes a hypersensitive response on nonhost plants, were fully sequenced and annotated. Comparison with other sequenced members of the Alphaproteobacteria provides new data on the evolution of multipartite bacterial genomes. Primary chromosomes show extensive conservation of both gene content and order. In contrast, secondary chromosomes share smaller percentages of genes, and conserved gene order is restricted to short blocks. We propose that secondary chromosomes originated from an ancestral plasmid to which genes have been transferred from a progenitor primary chromosome. Similar patterns are observed in select Beta- and Gammaproteobacteria species. Together, these results define the evolution of chromosome architecture and gene content among the Rhizobiaceae and support a generalized mechanism for second-chromosome formation among bacteria.

Xanthomonas axonopodis pv. glycines soybean cultivar virulence specificity is determined by avrBs3 homolog avrXg1.

Three races of Xanthomonas axonopodis pv. glycines were identified on pustule disease resistant and susceptible soybean cultivars based on virulence phenotype. For race 3, an avrBs3 homolog, avrXg1 was identified that conferred resistance expressed as a hypersensitive response on resistant cultivar Williams 82. Mutations in two predicted functional domains of avrXg1 resulted in gained virulence on Williams 82 and an increase in bacterial population number on susceptible cultivars. Expression of avrXg1 in race 1, that is predicted to confer a nonspecific HR, led to virulence on susceptible cultivars Spencer and PI 520733. Expression of avrXg1 in race 2, that is predicted of carrying avrBs3-like genes, resulted in gained virulence and fitness of pathogen on both resistant and susceptible cultivars. The results demonstrate multifunctions for avrXg1 dependent on pathogen and plant genetic backgrounds.

Insight Into the Microbial Co-occurrence and Diversity of 73 Grapevine (Vitis vinifera) Crown Galls Collected Across the Northern Hemisphere.

Crown gall (CG) is a globally distributed and economically important disease of grapevine and other important crop plants. The causal agent of CG is Agrobacterium or Allorhizobium strains that harbor a tumor-inducing plasmid (pTi). The microbial community within the CG tumor has not been widely elucidated and it is not known if certain members of this microbial community promote or inhibit CG. This study investigated the microbiotas of grapevine CG tumor tissues from seven infected vineyards located in Hungary, Japan, Tunisia, and the United States. Heavy co-amplification of grapevine chloroplast and mitochondrial ribosomal RNA genes was observed with the widely used Illumina V3-V4 16S rRNA gene primers, requiring the design of a new reverse primer to enrich for bacterial 16S rRNA from CG tumors. The operational taxonomic unit (OTU) clustering approach is not suitable for CG microbiota analysis as it collapsed several ecologically distinct Agrobacterium species into a single OTU due to low interspecies genetic divergence. The CG microbial community assemblages were significantly different across sampling sites (ANOSIM global R = 0.63, p-value = 0.001) with evidence of site-specific differentially abundant ASVs. The presence of Allorhizobium vitis in the CG microbiota is almost always accompanied by Xanthomonas and Novosphingobium, the latter may promote the spread of pTi plasmid by way of acyl-homoserine lactone signal production, whereas the former may take advantage of the presence of substrates associated with plant cell wall growth and repair. The technical and biological insights gained from this study will contribute to the understanding of complex interaction between the grapevine and its microbial community and may facilitate better management of CG disease in the future.

Autoaggregation of Xylella fastidiosa cells is influenced by type I and type IV pili.

Autoaggregation of widely dispersed Xylella fastidiosa cells into compact cell masses occurred over a period of hours following 7 to 11 days of growth in microfluidic chambers. Studies involving the use of mutants defective in polarly positioned type I (fimA-negative), type IV (pilB-negative), or both type I and IV (fimA- and pilO-negative) pili revealed the importance and role of pili in the autoaggregation process.

A gene cluster in Agrobacterium vitis homologous to polyketide synthase operons is associated with grape necrosis and hypersensitive response induction on tobacco.

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.

Isolation of Streptomycin-Resistant Isolates of Erwinia amylovora in New York.

Streptomycin is currently the only antibiotic registered for the control of fire blight, a devastating disease of apple (Malus), pear (Pyrus), and other rosaceous plants caused by the bacterium Erwinia amylovora. Resistance of E. amylovora to streptomycin was first identified in California pear orchards in 1971 and is currently endemic in many parts of the United States. The Northeast remains the only major U.S. apple-growing region without streptomycin-resistant isolates of E. amylovora. In 2002, during a routine survey for streptomycin resistance, isolates from two neighboring orchards in Wayne County, NY were found to be highly resistant to streptomycin at a concentration of 100 µg/ml. This constitutes the first authenticated report of streptomycin resistance in New York State. Infected trees were shipped at the same time from a single nursery in Michigan. Resistance was caused by the acquisition of the strA-strB gene pair, inserted into the ubiquitous nontransmissible E. amylovora plasmid pEA29. Previously, streptomycin-resistant E. amylovora populations from Michigan were described with a similar mechanism of resistance, although the strA-strB genes are not unique to Michigan. These findings illustrate how unintentional movement of nursery material could undermine efforts to prevent the spread of antibiotic-resistant E. amylovora.

Twitching motility among pathogenic Xylella fastidiosa isolates and the influence of bovine serum albumin on twitching-dependent colony fringe morphology.

Fourteen Xylella fastidiosa isolates from grapevines exhibiting Pierce's disease symptoms in California, Texas, and South Carolina were examined for type IV pilus-mediated twitching motility, a phenotype previously observed in a Temecula isolate from California. All isolates except one from South Carolina (SC 19A97) exhibited colonies with a peripheral fringe on PW agar, a feature indicative of twitching motility; however, when individual cells of SC 19A97 were examined at higher magnifications twitching motility was observed. The presence and width of colony peripheral fringes were related to the amount of bovine serum albumin (BSA) present in the medium; no or low levels of BSA (0-1.8 g L(-1)) permitted development of the widest fringe, whereas higher levels (3.5-6.0 g L(-1)) severely limited, and in many instances prevented, peripheral fringe development. The growth rate of the wild-type Temecula isolate in PW broth with different concentrations of BSA was similar for all tested concentrations of BSA; however, growth was significantly reduced in medium without BSA.

Characterization of the Xylella fastidiosa PD1311 gene mutant and its suppression of Pierce's disease on grapevines.

Xylella fastidiosa causes Pierce's disease (PD) on grapevines, leading to significant economic losses in grape and wine production. To further our understanding of X. fastidiosa virulence on grapevines, we examined the PD1311 gene, which encodes a putative acyl-coenzyme A (acyl-CoA) synthetase, and is highly conserved across Xylella species. It was determined that PD1311 is required for virulence, as the deletion mutant, ΔPD1311, was unable to cause disease on grapevines. The ΔPD1311 strain was impaired in behaviours known to be associated with PD development, including motility, aggregation and biofilm formation. ΔPD1311 also expressed enhanced sensitivity to H2 O2 and polymyxin B, and showed reduced survival in grapevine sap, when compared with wild-type X. fastidiosa Temecula 1 (TM1). Following inoculation, ΔPD1311 could not be detected in grape shoots, which may be related to its altered growth and sensitivity phenotypes. Inoculation with ΔPD1311 2 weeks prior to TM1 prevented the development of PD in a significant fraction of vines and eliminated detectable levels of TM1. In contrast, vines inoculated simultaneously with TM1 and ΔPD1311 developed disease at the same level as TM1 alone. In these vines, TM1 populations were distributed similarly to populations in TM1-only inoculated plants. These findings suggest that, through an indirect mechanism, pretreatment of vines with ΔPD1311 suppresses pathogen population and disease.

Grape Cultivar and Sap Culture Conditions Affect the Development of Xylella fastidiosa Phenotypes Associated with Pierce's Disease.

Xylella fastidiosa is a xylem-limited bacterium in plant hosts and causes Pierce's disease (PD) of grapevines, which differ in susceptibility according to the Vitis species (spp.). In this work we compared X. fastidiosa biofilm formation and population dynamics when cultured in xylem saps from PD-susceptible and -resistant Vitis spp. under different conditions. Behaviors in a closed-culture system were compared to those in different sap-renewal cultures that would more closely mimic the physicochemical environment encountered in planta. Significant differences in biofilm formation and growth in saps from PD-susceptible and -resistant spp. were only observed using sap renewal culture. Compared to saps from susceptible V. vinifera, those from PD-resistant V. aestivalis supported lower titers of X. fastidiosa and less biofilm and V. champinii suppressed both growth and biofilm formation, behaviors which are correlated with disease susceptibility. Furthermore, in microfluidic chambers X. fastidiosa formed thick mature biofilm with three-dimensional (3-D) structures, such as pillars and mounds, in saps from all susceptible spp. In contrast, only small aggregates of various shapes were formed in saps from four out of five of the resistant spp.; sap from the resistant spp. V. mustangensis was an exception in that it also supported thick lawns of biofilm but not the above described 3-D structures typically seen in a mature biofilm from the susceptible saps. Our findings provide not only critical technical information for future bioassays, but also suggest further understanding of PD susceptibility.