Abstract
Agrobacterium vitis, the primary cause of grape crown gall disease, is known to survive internally in grapevines and to spread in propagation material. In this study, we showed that the bacterium can be detected in dormant grape buds and on surfaces of leaves collected from commercial vineyards. Using a highly selective and sensitive method based on magnetic capture hybridization (MCH) together with real-time PCR, we detected A. vitis in as much as 90% of dormant bud samples and in up to 40% of leaf samples from individual vineyards. The highest percentages of detection occurred in samples collected from vineyards with high incidences of crown gall. A. vitis was also detected in 22% of wild grapevines (Vitis riparia) collected in New York and in 25% of feral grapevines that included V. californica in California. Several of these vines were growing more than 2 km from commercial vineyards, demonstrating that wild grapevines can serve as a significant inoculum reservoir. The specificity of the MCH and real-time PCR assay used to detect tumorigenic A. vitis in the environment was further demonstrated by the finding that 69 nontumorigenic strains from regions across the United States did not amplify a virD2 PCR product.
- bacterial endophytes
- bacterial epiphytes
- crown gall
- magnetic capture hybridization
- PCR detection
- wild grapevines
Agrobacterium vitis is the major cause of crown gall disease on grape worldwide (Burr et al. 1987). The bacterium survives systemically in grapevines, and significant outbreaks are often reported after injury to trunks and canes from cold winter temperatures or graft wounds. A. vitis can survive in soil for at least two years in association with plant debris (Burr et al. 1995). In some countries, such as Turkey, A. vitis is a quarantine pathogen. When crown gall is detected in a nursery, all vines within a given row in which an infected vine is identified, regardless of their infection status, are eradicated. In addition, growing grapevines is prohibited for five years at the infected site according to quarantine regulations (www.tarim.gov.tr). Therefore, understanding pathogen biology and effective indexing of propagation material is significantly important for disease management.
PCR is a preferred technique for detecting microorganisms and allows amplification of target DNA from mixed DNA samples. Samples collected from different environmental sources may contain minute traces of undesirable compounds, such as plant-derived phenolics, that may inhibit the nucleic acid amplification process, resulting in a false negative test. An efficient method for isolation of target DNA devoid of PCR inhibitors, called magnetic capture hybridization (MCH), was introduced by Jacobsen (1995) to separate a specific DNA target from a complex DNA mixture. MCH is based on conjugation of paramagnetic streptavidin-coated beads with a biotin-labeled oligonucleotide probe for capture of single-stranded DNA from crude DNA preparations. The Burr laboratory recently adapted MCH in combination with real-time PCR for the identification of tumorigenic A. vitis in grape tissue based on detection of a region of the virD2 gene that is conserved in tumorigenic strains of the bacterium (Johnson et al. 2013). The method has proved to be highly specific and sensitive for the detection of the pathogen in grapevine tissues (Johnson et al. 2016). virD2 primers had been developed previously and were used for specific detection of tumorigenic strains of A. tumefaciens (Haas et al. 1995). In that study, however, a virD2 product was amplified in 1 of 29 nontumorigenic strains, and it was hypothesized that this strain had resulted from a deletion of its T-DNA. Therefore, to further verify the specificity of the virD2 primer set for selectively identifying tumorigenic strains of A. vitis, and for its use in the MCH method, 69 nontumorigenic A. vitis strains previously collected from different geographic regions of the United States were examined for the presence of virD2.
We previously used the MCH real-time PCR method to determine the presence of tumorigenic A. vitis in dormant grape canes, on grape shoot tips, and in meristem tissues as well as in wild grapevines (Johnson et al. 2013, 2016). In the present study, we employed MCH real-time PCR to further our knowledge of environmental sources of A. vitis by investigating its presence in dormant grape buds and on leaves of grape, as well as on leaves of weeds and cover crops collected from vineyards. Indexing of wild grapevines, that is, of V. riparia collected in New York and of feral wild grapes from California, was also done.
Materials and Methods
Analyses of nontumorigenic A. vitis strains for the presence of virD2
The accuracy and utility of the MCH method depends on being able to specifically detect a diversity of tumorigenic A. vitis strains. Previously, it has been shown that the 5′ end of the virD2 gene is conserved in a representative set of tumorigenic strains that included strains of A. vitis as well as other Agrobacterium species (Haas et al. 1995, Johnson et al. 2013). It was also reported that 0 of 5 (Johnson et al. 2013) and 1 of 29 (Haas et al. 1995) nontumorigenic Agrobacterium strains were PCR positive for virD2, and it was suggested that such cases may result from tumorigenic strains losing their T-DNA. Therefore, to examine nontumorigenic A. vitis strains for the presence of virD2 and to further confirm the specificity of the MCH method, an additional 69 nontumorigenic A. vitis strains previously isolated from California, New Mexico, New York, and Washington State were investigated for the presence of virD2 in this paper. The strains were isolated from galls, sap, roots, or callus tissues from both cultivated and wild grapes. They are maintained in the Cornell Agrobacterium collection in the Burr laboratory and were previously confirmed as A. vitis by ELISA with an A. vitis-specific monoclonal antibody (which identifies both tumorigenic and nontumorigenic strains) and were verified as being nontumorigenic by inoculating grapes as well as other plants, including sunflower and tobacco, in the greenhouse and observing them for crown gall development.
The strains were grown on potato dextrose agar for 48 hr at 28°C. DNA templates were prepared by boiling a 107 cfu/mL bacterial suspension for 10 min and then incubating it on ice for 5 min. Conventional PCR with the primer set VirD2For1/VirD2Rev1 in a total volume of 25 μL, which included 5 μL 5× Buffer Green, 2 μL MgCl2, 1 μL of each virD2for1 and virD2.rev1 primers, 0.125 μL Go Taq Flexi DNA Polymerase (Promega), 0.5 μL dNTPs, 0.5 μL DMSO, 6.875 μL water, and 8 μL of DNA from the sample. A. vitis strain S4 was used as a positive control and sterile HPLC water as a negative control. The PCR products were visualized by electrophoresis on 1% agarose gels stained with GelRed (Biotium, Inc.).
Assaying grape buds and leaves for A. vitis
Dormant buds (three per sample) were excised from grape canes that had been collected during the winter of 2015 and 2016. In 2015, buds were harvested from the interspecific hybrid cultivar Vignoles (Seibel 6905 × V. vinifera cv. Pinot noir); V. vinifera cvs. Chardonnay, Cabernet franc, and Riesling; and from wild grapes (V. riparia). The wild grapevines were located adjacent to and also further removed (>2 km) from vineyards. These included V. riparia vines growing in New York state parks, at a national wildlife refuge, and along roadways. In 2016, buds were sampled from a different Riesling vineyard. For each cultivar and wild grapevine, three buds from each of 20 individual canes were tested. For the 2016 Riesling vineyard, 20 vines from each of two separate blocks were sampled. During field sampling, individual buds were excised from dormant canes, placed in 1.5 mL Eppendorf tubes, and processed on the same day of collection. Buds were crushed in 3 mL 1× PBS (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4) in extraction bags, and the resulting tissue suspensions were transferred to sterile, disposable, polypropylene centrifuge tubes to which an equal volume of 2× nutrient broth plus cycloheximide (NBC) was added (1 μL of a 100 mg/mL stock solution was added per mL of the total volume). The tubes were shaken at 165 rpm at room temperature for three days, after which 2 mL of the bacterial culture, along with any plant cells, was centrifuged at 10,000 g for 4 min. The supernatant was discarded, and the cells in the resuspended pellet were used in the MCH procedure as described below.
Grape leaves of infected and healthy cvs. Vignoles, Chardonnay, Cabernet franc, and Riesling, and of wild grapevines, were sampled from June to August. The infection status of the wild grapes was unknown. Ten leaves from each of 20 individual vines were collected in plastic bags and carried to the laboratory. The same number of grape leaves of Cabernet franc growing in a greenhouse was also sampled for presence of A. vitis. These vines were inoculated with A. vitis for experiments that are not included in this paper.
The leaves were processed by adding 10 mL 1× PBS buffer to each plastic bag and submersing the bags in a sonication bath (Bransonic Ultrasonic Cleaner, Fisher Scientific), and then underwent a mild sonication for 3 min to help remove bacteria from the leaf surfaces. The resulting solution was added to an equal volume of NBC in a polypropylene centrifuge tube for the enrichment step as described for buds. Cells were collected by centrifugation and used in MCH real-time PCR.
Analyses of leaves from vineyard weeds and cover crops for A. vitis
In 2015, leaves from different weeds growing in asymptomatic and diseased vineyards were collected to determine if A. vitis could be detected from their surfaces. Ten leaves were collected from each plant species, including dandelion (Taraxacum sp.), clover (Trifolium sp.), and broadleaf plantain (Plantago major), and processed as described for grape leaves. All 2015 weed and cover crop samples were assayed with the same protocol used for grape leaves harvested in 2015.
In 2016, leaves of cover crops were collected in two commercial and one research vineyard. A cover crop research trial with fescue (Festuca sp.), alfalfa (Medicago sativa), chicory (Cichorium intybus), and tillage radish (Raphanus sativus) was planted in one of the commercial vineyards of cultivar Noiret, an interspecific hybrid of V. labrusca and V. vinifera. Leaves were collected from cover crops and Noiret vines. In a second commercial vineyard, leaves were collected from clover (Trifolium sp.) and ryegrass (Lolium perenne) that were used as cover crops, and from combined species of broadleaf weeds and combined wild grasses as well as from cultivar Riesling. Ryegrass samples were collected from two separate blocks in the vineyard. From the research vineyard, leaves were collected from combined broadleaf weeds and combined wild grasses. Because these samples were used in another study to determine the presence of ice nucleation-active bacteria, 500 mL distilled water was added to 10 g of leaf tissue, sonicated as described above, and 20 mL of the leaf wash solution was added to an equal volume of NBC. After enrichment as described above, bacteria were collected by centrifugation for subsequent MCH real-time PCR to determine the presence of A. vitis. All samples were collected in the spring of 2016.
Assays of wild grapevines for A. vitis
A total of 41 wild grapevines (V. riparia) were collected in the fall and winter of 2014 to 2015 and assayed for the presence of A. vitis. These vines were sampled from locations not previously sampled, and from sites adjacent to and removed (>2 km) from commercial vineyards in New York.
In addition, a total of 87 wild grapevines were sampled from the Napa Valley region of California. These included feral grapes that represented either wild species (true V. californica), seedling vines that grew following seed dissemination from vineyards, or hybrids between V. californica and cultivated grapes (scion or rootstocks). For these samples, dormant canes were collected, and again a combined sample from each that included six nodes was assayed with the MCH method for detecting A. vitis in dormant canes (Johnson et al. 2016).
MCH
MCH was performed as previously described by Johnson et al. (2013). This method employs an enrichment step, followed by incubation of total DNA with paramagnetic streptavidin-coated beads to which the biotinylated virD2 capture probe (VITIS) is attached.
Real-time PCR
Real-time PCR was performed on the MCH-extracted DNA from the bud, leaf, cane, and shoot samples using the aforementioned VirD2For1/VirD2Rev1 primer set (Johnson et al. 2013). DNA from A. vitis strain S4 was used as a positive control, and buffer only and sterile HPLC water were used as negative controls for each reaction. Real-time PCR reaction contained 12.5 μL SYBR Green mix (Bio-Rad) and 100 nM VirD2For1/VirD2Rev1 in a total volume of 25 μL. Positive reactions were identified by the presence of a cycle threshold (Ct) curve and specific melt peaks observed between 84 and 88°C.
Results
Presence of tumorigenic A. vitis in dormant grape buds
Of the 20 Vignoles vines sampled, four were positive for tumorigenic A. vitis in dormant buds in the first sampling in December 2014. For the sampling done in April 2015, 8 of 20 vines were positive, including the four that were positive in the December sampling (Table 1). For Chardonnay vines, the same single vine was positive for tumorigenic A. vitis in both samplings. For Riesling, only one vine was positive in the first sampling, whereas the same vine and three additional vines were positive in the second. For wild grapes from New York, one bud was positive in the first sampling, and none were positive in the second sampling (Table 1).
Detection of tumorigenic Agrobacterium vitis in dormant buds and on the surface of grape leaves.
In 2015 to 2016, dormant buds of Riesling were sampled in two vineyards that had not been sampled the previous year, one with a high incidence of crown gall and one with a low incidence. For the high-incidence vineyard, 18 of 20 vines sampled were positive for A. vitis. For the low-incidence vineyard for which two blocks were sampled, of 20 vines sampled, block A had 11 positive vines, and block B had nine (Table 1). These results showed that A. vitis can be detected in dormant grape buds in two different years. They also suggested that vineyards with a high disease incidence are more likely to have a higher proportion of buds carrying the pathogen than vineyards with low levels of crown gall, and that infected buds may provide an overwintering source of the pathogen. Further research is warranted to confirm that the level of bud infestation is correlated with the crown gall disease incidence in a vineyard.
Presence of A. vitis on surfaces of leaves of grapes, weeds, and cover crops
Leaves from the cultivars Vignoles, Chardonnay, Riesling, and Cabernet franc (from vineyard and greenhouse vines) were assayed for epiphytic A. vitis. In addition, leaves from wild V. riparia vines, weeds, and cover crops were also assayed. Leaves from 3 of 20 Vignoles vines were positive in the first sampling (Table 1) and one of the three vines was also positive in the second sampling. For Riesling vines from a vineyard with a high incidence of crown gall, leaves from 8 of 20 vines were positive in each sampling (Table 1). For Chardonnay, there was only one positive leaf sample in both samplings. A. vitis was not detected on leaves from any of the wild grape samples (data not shown).
Analyses of leaves from Cabernet franc vines grown in the greenhouse indicated that 3 of 20 vines tested positive for tumorigenic A. vitis in the first sampling and none in the second. These results conclusively showed that A. vitis can be detected on the surface of grape leaves in both vineyards and the greenhouse. Additional research will be needed to determine the environmental conditions that contribute to the dynamics of epiphytic populations of the pathogen on grape.
The 2015 vineyard weeds, including dandelion (Taraxacum sp.), clover (Trifolium sp.), and broadleaf plantain (Plantago major) were all negative for A. vitis in both samplings. These weed samples were taken from vineyards that were symptomatic or asymptomatic for crown gall. Similarly, in 2016, samples collected from alfalfa, chicory, fescue, and tillage radish, as well as from Noiret leaves, were all negative for A. vitis in analyses of both samplings. In another commercial vineyard, none of the grape or clover samples, but one of the rye samples, was positive for A. vitis. In addition, none of the mixed grass samples, but one of the mixed broadleaf leaves was positive for A. vitis. In the research vineyard, A. vitis was not detected in the mixed broadleaf or mixed grass samples. Although A. vitis was detected in leaves of a rye cover crop, additional research is needed to determine the significance of persistence of the pathogen on different plant species in vineyards and their possible significance for disease development in vineyards.
Detection of A. vitis in wild grapevines
A. vitis was detected in 9 of 41 wild grapevines (V. riparia) distant from commercial vineyards in New York (Table 2). Among these, A. vitis was detected in 8 of 38 vines distant from commercial vineyards as compared with 1 of 3 vines collected directly adjacent to the vineyards (Table 2). A total of 87 wild vines were assayed from California, all from Napa Valley. Of these, 18 were distant from commercial vineyards, of which seven tested positive for the pathogen. These results showed that wild grapevines comprise a significant source of A. vitis and that even vines more than 2 km away from commercial vineyards can harbor this pathogen.
Detection of tumorigenic Agrobacterium vitis in wild grapevines in New York and California.a
Absence of virD2 in nontumorigenic A. vitis strains
We observed no PCR amplification of the virD2 virulence gene from all 69 nontumorigenic A. vitis strains tested. This result further confirmed that nontumorigenic A. vitis strains do not carry a virD2 gene, increasing the level of confidence that the MCH real-time PCR–based detection method specifically identifies tumorigenic strains of the bacterium.
Discussion
Crown gall limits vineyard production in several viticultural regions of the world. The disease is associated with vine injuries that are primarily caused by freezing temperatures or grafting wounds. Grape crown gall is difficult to manage because the causative agent, A. vitis, persists in healthy-appearing grape tissues, including those used for propagation. Although plant injury is required for initiation of crown gall infection, other predisposing factors, such as the timing of injury and vine physiology, are not fully understood. In addition to being present in symptomless propagation material, A. vitis also persists in grape root debris in the soil (Burr et al. 1995).
Previous knowledge of the distribution of A. vitis in cultivated and wild grapevines was based on detection methods that were not highly sensitive and whose detection thresholds were unknown (Bauer et al. 1994, Stover et al. 1997). Recently, our knowledge of environmental sources and prevalence of A. vitis has been significantly advanced by employing a detection method based on MCH in combination with real-time PCR (Johnson et al. 2013, 2016). MCH allows the specific capture of target DNA from a plant sample of mixed components and enables the elimination of nontarget DNA and inhibitors prior to the real-time PCR step (Jacobsen 1995). This technology has now been used effectively for the specific and sensitive detection of different bacteria in diverse environments (Chen et al. 1998, Amagliani et al. 2006, Rodriguez et al. 2012). It is an ideal technology to use for detection of A. vitis because the virD2 target has a highly conserved 5′-end sequence and plays a central role in tumorigenesis (Tinland et al. 1995).
Crown gall disease in commercial vineyards is generally managed by cultural methods that focus on proper site selection and implementation of practices that help limit freeze injury of vines. There are no effective chemical controls for grape crown gall, and although various biological controls are being investigated, none has currently been developed for commercial use (Filo et al. 2013). Therefore, understanding the distribution of the pathogen in host plants as well as its persistence in environmental niches is critical to the development of effective strategies for disease management. In this paper, we documented the detection of A. vitis in dormant buds of grapes and from grape leaf surfaces as well as from other vineyard plant species during the growing season. In addition, we expanded our knowledge of the presence of tumorigenic A. vitis in wild grapevines that are proximal and distant from commercial vineyards. We also observed that wild grapevines in New York and California frequently harbor tumorigenic strains of A. vitis and therefore may provide a significant inoculum source of the pathogen.
Plant-pathogenic bacteria frequently survive epiphytically on leaves of host plants as well as nonhosts, including cover crops and weeds. An example of a well-studied epiphytic bacterial plant pathogen is Pseudomonas syringae pv. syringae, which survives on the surface of several host plants (Wilson and Lindow 1993). Epiphytic bacterial communities from different parts of grapevine, including fruits, leaves, and bark, were reported by Martins et al. (2013). Pseudomonas sp. was reported as the most prevalent genus epiphytic on grape leaves, followed by Sphingomonas sp., Curtobacterium sp., and Bacillus sp. We therefore explored whether A. vitis could be detected on surfaces of grape leaves and also of leaves of vineyard cover crops and weeds. It is conceivable that A. vitis would spread from galls on grapevines to leaves and surrounding plant species after rain, irrigation, or spray events. This hypothesis is supported by the fact that, in this study, we discovered that A. vitis can live epiphytically on grapevine leaves and is detected at a higher frequency in vineyards with high levels of crown gall. For example, a Riesling block with severe crown gall infection yielded 8 of 20 leaf samples that tested positive for tumorigenic A. vitis. In contrast, tumorigenic A. vitis was not detected on leaves of wild grape, which do not exhibit crown gall disease symptoms. On the basis of these findings, we hypothesize that the severity of infection in a vineyard is positively correlated with the presence of A. vitis on grape leaves and possibly on surrounding plant species as well. Whether epiphytic colonization of grape, other plant species, or both, by A. vitis affects subsequent persistence of the pathogen in vineyards and disease development warrants further research.
Conclusion
An understanding of the environmental sources of A. vitis is critical for the development of strategies to manage crown gall disease. Here, we identified previously unknown environmental niches that harbor A. vitis, including dormant grape buds and the surfaces of grape leaves. We also demonstrated the presence of the pathogen in wild grapevines, that is, in V. riparia, in New York and, for the first time, also in feral grapevines in California. We further enhanced the confidence in the specificity of the MCH system for detecting tumorigenic A. vitis by determining that a diverse collection of nontumorigenic strains does not possess the target gene virD2.
Acknowledgments
This research supported D. Canik Orel through the 2219 program of TUBITAK. It was also partially funded by USDA Federal Capacity Funds and by USDA-APHIS-NCPN. The authors thank Justine Vanden Heuvel for access to the cover crop research vineyard trial.
- Received September 2016.
- Revision received November 2016.
- Accepted November 2016.
- ©2017 by the American Society for Enology and Viticulture
Literature Cited
Vol 68 Issue 2
