Progress 10/01/10 to 09/30/15
Outputs
Target Audience:The target audiences were the grape and wine industries of New York State and the rest of the U.S. This includes nursery businesses, vineyard and winery owners. Another audience was cooperative extension who helps facilitating the dissemination of information. Thirdly and audience was the scientific community that is interested in plant pathogens, plant diseases and plant-microbe interactions. Changes/Problems:The only major change in the approach was the development of the sensitive method for detection of the crown gall pathogen. This was possible because of a postdoctoral associate hired in the laboratory that had expertise in magnetic capture hybridization and real-time pcr. The development of the method had positive effects on the rest of the research by allowing us to verify more precisely the presence or absence of the pathogen. What opportunities for training and professional development has the project provided?Results from the research are sought after by grape educators across the US. The results give producers a clearer picture of where and how the pathogen survives and how it affects grapevines. The research is also leading to the development of a biological control for grape crown gall disease. How have the results been disseminated to communities of interest?The results have been delivered to academic audiences through publication in refereed journals and listed. They have been provided to grower audiences through oral presentations at grower meetings in the Finger Lakes Region of NY, the Lake Erie Region of NY, to the Ohio grape industry and to the California grape industry. A fact sheet on the biology of crown gall and considerations for production and maintenance of clean plants was prepared together with the USDA, APHIS, NCPN program. What do you plan to do during the next reporting period to accomplish the goals?Although this is a final report for this project the research will continue. The main goals will be to further determine sources of the pathogen in the environment and how these affect the production and maintenance of clean plants. In addition we will further demonstrate the significance of necrosis by A. vitis in success of graft take and on grape root development. The research will also continue on developing the biological control for grape crown gall and understanding the mechanism of disease inhibition.
Impacts
What was accomplished under these goals?
A highly sensitive method was developed for detection of the grape crown gall pathogen in grapevines and in other environmental sources. This allows testing of propagation material as well as determining sources of the pathogen in the environment. Through utilization of the detection method it was determined that the pathogen survives in wild grapevines. These are a major weed plant in New York and in many regions of the US. Therefore wild grapevines can provide a significant source of crown gall inoculum. We made significant progress in understanding the mechanism by which A. vitis cause necrosis of grapevines and its significance in root development and in graft take. Genes required to cause necrosis include those encoding specific polyketide and nonribosomal peptide synthases as well as a pptase and other regulatory networks. The ability to cause necrosis appears to affect graft take by different strains of A. vitis. This particular aspect of the research is continuing. Significant progress was also made in determining the mechanism by which strain F2.5 is able to inhibit grape crown gall. The PKS and NRPS genes required for necrosis were also required for crown gall inhibition. However the pptase gene is not required for gall inhibition. It was determined that F2.5 inhibits crown gall on grape but not on other plants such as tobacco. F2.5 inhibits transformation of grape by tumorigenic A. vitis strains but does not inhibit their growth on grape. In addition F2.5 inhibits grape crown gall by A. vitis but does not inhibit the formation of galls on grape by A. tumefaciens strains. It is most significant that the pptase mutant of F2.5 does not cause necrosis but still maintains gall inhibition ability. Thus its potential as a biological control for grape crown gall could be developed.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Kaewnum, S., Zheng, D., Reid, C. L., Johnson, K. L., Gee, J. C. and Burr, T. J. 2013. A host-specific biological control of grape crown gall by Agrobacterium vitis strain F2/5; its regulation and population dynamics. Phytopathology 103:427-435.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2015
Citation:
Zheng, D. and Burr, T. J. 2015. Inhibition of grape crown gall by Agrobacterium vitis strain F2/5 requires two non-ribosomal peptide synthetases and one polyketide synthase. Mol Plant Microbe Int. (accepted).
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2015
Citation:
Johnson, K. L., Cronin, H, Reid, C. L. and Burr, T. J., 2015. Distribution of Agrobacterium vitis in grapevines and its relevance to pathogen elimination. Plant Disease (accepted).
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Johnson, K. L., Zheng, D., Kaewnum, S. Reid, C. L. and Burr T. J. 2013. Development of an MCH-real-time PCR assay for detection of tumorigenic Agrobacterium vitis in grapevines. Phytopathology 103:633-640.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: The target audiences include the grape and wine industry of NY as well as the industries of other grape growing regions. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Results from this work are directly relevant to the grape and wine industries. We are often asked to speak to stakeholders about the results and to how our work directly impacts management of the disease on a commercial scale. How have the results been disseminated to communities of interest? The results have been disseminated by presentation so stakeholder audiences and through publications. We have also reached audiences and will continue to do so through the website Appelation Cornell. I the spring of 2015 I will be presenting the information to NY growers and to grower audiences in Ohio and in California. What do you plan to do during the next reporting period to accomplish the goals? We will continue to work on all of the objectives. This will include determining if vines propagated from shoot tips or meristems are free of the pathogen. These experiments are underway using cuttings from vines with crown gall as the initiating plant material. Research will also continue on determining environmental sources of the pathogen. This will include repeating work on wild grapevines as well as initiating testing of soils and water. In addition research has been initiated to determine how different pathogenic and nonpathogenic strains at different concentrations affect graft take and also crown gall formation at graft unions. We will also continue to evaluate the necrosis minus variant as a biological control that would likely be applied during grafting to prevent crown gall.
Impacts
What was accomplished under these goals?
The overall aim of this research is to generate the knowledge required for development of effective management strategies for crown gall disease on grapevines. This disease is a limiting factor globally to vineyard production. The pathogen is able to survive systemically in vines and therefore development of a sensitive method to index grapevines was necessary. We have accomplished that goal and are now using the method to study the presence of the pathogen in the environment and to evaluate strategies for producing crown gall free grapevines. Towards objective one we have determined that the pathogen can be detected in tips of grape shoots and are currently evaluating whether propagation of vines from shoots or meristems can be used as a way of producing clean vines. The detection method is also being used to study the presence of the pathogen in environmental sources such as wild grapevines and soil as these may provide sources for contaminating vines that were previously clean. To date we have determined the pathogen can be detected in wild grapevines. The pathogen can also cause a necrosis of grapevines. We have recently shown that when present at the graft union the pathogen can have a detrimental effect on graft take. This research is currently being done with a diverse group of strains of the pathogen and by inoculating with different populations. Biological control has shown promise in controlling crown gall on different host plants including grapevines. We have shown that a nonpathogenic strain is able to block infection by the pathogenic strain. A problem with this strategy is that even the nonpathogenic strain causes necrosis that can affect graft take and root development. Therefore we have knocked out a gene that is required for necrosis and the new variant still is able to inhibit crown gall. We determined that this new variant is not harmful to graft unions and are now testing it at different concentrations and against different strains of the pathogens. We will be testing it under field conditions.
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: New York and US wine and grape industries Local and US grape nurseries National Grape and Wine Initiative Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Results have been disseminated to communities primarily through meetings with growers and on website http://grapesandwine.cals.cornell.edu/cals/grapesandwine/appellation-cornell/issue-9/loader.cfm?csModule=security/getfile&PageID=1053091 What do you plan to do during the next reporting period to accomplish the goals? One question we will ask is whether A. vitis survives in wild grape species and also in other environmental samples such as in association with vineyard weeds, water and/or soil. We see no issues in doing this research now that we have the MCH real-time method. We will also begin examining whether it is possible to generate A. vitis – free vines from meristems with the aid of hot water treatments and antibiotics. These questions go hand in hand in that if the bacterium is ubiquitous on the environment it may not be worth the effort to develop pathogen-free vines. Another component of the research will be to further elucidate the mechanism of biological control by F2.5 and determine how it can be employed commercially. A goal for the next testing period will be to test selected mutants including the PPT mutant by application to grape grafting sites. The goal will be to determine if graft take is affected and whether such applications will control crown gall at the graft unions.
Impacts
What was accomplished under these goals?
Agrobacterium vitis is a bacterium that cause of crown gall disease on grape, a serious disease that occurs worldwide. It survives systemically in grapevines and therefore is spread in propagation material. In 2012 we developed of a rapid and sensitive method for indexing cuttings that employs technologies of magnetic capture hybridization and real-time PCR. In 2013 we determined the distribution of A. vitis in vines using this method. We also used the method to determine the efficacy of a hot water treatment for reducing the pathogen in grape cuttings. It was determined the bacterium resides throughout dormant canes. It can be detected in nodes as well as internodes. It was also detected in green shoots and in shoot tips and meristems. This differs from previous reports that were done without having the benefit of the MCH, real-time method. A specific nonpathogenic strain of A. vitis, F2.5, is able to inhibit the development of crown gall specifically on grapes. We determined that while inhibiting grape transformation by tumorigenic strains, F2.5 does not inhibit their growth at wounds on grape. We have identified specific genetic loci involved in biological control. One that is presumably unique to F2.5, called Avs, was determined to be required for biological control. In addition a gene encoding a nonribosomal peptide synthase and polyketide synthase were also shown to be associated with biological control. In 2013 further research was done on certain mutants of F2.5 that were grape necrosis negative but maintained biological control capability. One involves a PPTase gene that when knocked out results in a necrosis-minus grape phenotype that is still biological control positive. A biological control field trial was established in Geneva in 2009. Treatments included treating the roots and crowns of grapevines with water or with F2.5 prior to planting. The treatments were replicated in a field at NYSAES that was previously planted to grapevines and had crown gall. Annual disease evaluations are being made in this field experiment. This research has greatly enhanced our knowledge of the biology of A. vitis and crown gall disease of grapevines. Utilization of the MCH real-time method has demonstrated that the bacterium is much more prevalent in vines than previously expected. This will be particularly important as we determine how pathogen-free plants may be developed and the potential or reinfection from the bacterium that may be harbored in the environment. We have also made significant progress in understanding the mechanism of biological control by F2.5 which will enhance its implementation as a component of a disease management program. In particular necrosis negative variants of F2.5 have great potential for use in nurseries and vineyards. The research results are presented regularly in verbal and written forms to members of the wine and grape industries of the US as well as in other countries.
Publications
- Type:
Websites
Status:
Published
Year Published:
2013
Citation:
Johnson, K. .L, Zheng, D., Kaewnum, S., Reid, C. L. and Burr, T. J. 2013. Development of an MCH-real-time PCR assay for detection of tumorigenic Agrobacterium vitis in grapevines. Phytopathology: Posted online on 16 Jan 2013.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Agrobacterium vitis is the cause of crown gall disease on grape, a serious disease that occurs worldwide. Some nonpathogenic strains of A. vitis, such as F2.5, are able to inhibit the development of crown gall specifically on grapes. A. vitis survives systemically in grapevines and therefore is spread in propagation material. In 2012 we focused on development of a rapid and sensitive method for indexing cuttings that is based on enrichment of bacteria present in woody grape node tissue followed by magnetic capture hybridization technology and real-time PCR. Bacterial enrichment is accomplished using nutrient broth. The detection method is 1000 times more sensitive than previously used methods. Controlled experiments show the method can detect as low as 10 cells of the pathogen in a sample. A. vitis was isolated from about 70 percent of cuttings taken from vineyards showing crown gall on some parts of the vines. Currently further verification of the method is being done to limit the possible numbers of false positive reactions. The method will be used to assay for the pathogen in tissue culture plants as well as in soil and water. Biological control of grape crown gall provided by strain F2.5 is also studied in our lab. It was determined that the F2.5 biological control is specific to grape in that F2.5 and does not prevent A. vitis from causing galls on plants such as sunflower, tomato and tobacco. Certain gene mutations in F2.5 knocked out biological control including different quorum sensing related genes and caseinolytic protease genes. We have compared the F2.5 genome sequence to that of tumorigenic strain S4 to identify sequences in F2.5 that might be involved in biological control. In 2012 the focus was on loci involved in siderophore production that are also involved in the biological control process. A locus unique to F2.5 called Avs was determined to be required for biological control. In addition a gene encoding a nonribosomal peptide synthase and polyketide synthase were also shown to be associated with biological control. We also identified certain mutants of F2.5 that were grape necrosis negative but maintained biological control capability. One involves a PPTase gene. These findings are particularly interesting and useful in that necrosis is detrimental to root development and graft take. Therefore a necrosis negative mutant that maintains biological control could be quite useful for commercial development. A biological control field trial was established in Geneva in 2009. Treatments included treating the roots and crowns of grapevines with water or with F2.5 prior to planting. The treatments were replicated in a field at NYSAES that was previously planted to grapevines and had crown gall. In 2012 additional vines in the trial expressed crown gall and several more were infected in the control treatment compared to the F2.5 treatment. Annual disease evaluations will be made in this field experiment for at least another two years. PARTICIPANTS: Desen Zheng, Research Associate. Responsible for a majority of the molecular biology on A. vitis. Primary goals are to identify mechanisms of grape root necrosis and biological control. Kameka Johnson, Postdoctoral Associate. Responsible for development of indexing method for A. vitis in grapevines. Cheryl Reid, Technician, Cornell. Coordinates all of the vine indexing and assists in the biological control research. She has also coordinated the biological control field experiments. TARGET AUDIENCES: New York and US wine and grape industries Local and US grape nurseries National Grape and Wine Initiative PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This research has greatly enhanced our knowledge of the biology of A. vitis and crown gall disease of grapevines. It has resulted in development of a method for efficient indexing grape material for presence of the crown gall pathogen. Previous methods required weeks for completion and were not highly sensitive. This method will be instrumental in producing and maintaining clean plants that can be generated and distributed in commercial businesses. We have also made significant progress in understanding the mechanism of biological control by F2.5 which will enhance its implementation as a component of a disease management program. In particular necrosis negative variants of F2.5 have great potential for use in nurseries and vineyards. The research results are presented regularly in verbal and written forms to members of the wine and grape industries of the US as well as in other countries.
Publications
- Zheng, D., Hao, G., Cursino, L., Zhang, H. and Burr, T. J. 2012. LhnR and upstream operon LhnABC in Agrobacterium vitis regulates the induction of tobacco hypersensitive responses, grape necrosis and swarming motility. Molecular Plant Pathology 13, 641 to 652
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Agrobacterium vitis is the cause of crown gall disease on grape, a serious disease that occurs worldwide. Some nonpathogenic strains of A. vitis, such as F2.5, are able to inhibit the development of crown gall specifically on grapes. A. vitis survives systemically in grapevines and therefore is spread in propagation material. In 2011 we indexed cuttings from the Foundation Plant Service in California for A. vitis. Cuttings from a commercial nurseryman in NY were also indexed. We also focused on developing a more rapid and sensitive method for indexing cuttings that is based on enrichment of bacteria present in woody grape node tissue followed by DNA extraction and PCR using primers designed to the virD2 gene. Bacterial enrichment is accomplished with nonselective bacterial broth media. A. vitis was isolated from a small number of the cuttings demonstrating that symptomless propagation material may carry the bacterium internally. Further refinements of the indexing procedure are being done utilizing Magnetic Capture Hybridization and Immuno Capture Hybridization as tools. Comparisons of the different methods are being done to determine the most efficient, sensitive, timely and cost-effective process for indexing. Biological control of grape crown gall provided by strain F2.5 is also studied in our lab. It was determined that the F2.5 biological control is specific to grape in that F2.5 and does not prevent A. vitis from causing galls on plants such as sunflower, tomato and tobacco. Certain gene mutations in F2.5 knocked out biological control. It was shown that mutations in genes involved in the caseinolytic protease system, clp, affected the ability of F2.5 to provide biological control. In addition to clpA which is a proposed ATP dependent chaperone, clpP, the protease was also mutated resulting in loss of biological control. We have compared the F2.5 genome sequence to that of tumorigenic strain S4 to identify sequences in F2.5 that might be involved in biological control. In 2011 it was determined that in addition to the clp protease genes, loci involved in siderophore production are also involved in the biological control process. Two siderophore loci are found in the S4 genome whereas the same two are found in the F2.5 genome along with a third. The unique locus was determined to be required for biological control. Questions regarding why F2.5 is effective as a control on grapevines but does not prevent A. vitis from causing gall on other plants and the role of iron regulation in the biological control are being studied. A biological control field trial was established in Geneva in 2009. Treatments included treating the roots and crowns of grapevines with water or with F2.5 prior to planting. The treatments were replicated in a field at NYSAES that was previously planted to grapevines and had crown gall. Of 60 control vines one is showing symptoms of crown gall. Of 60 vines treated with F2.5, seven have crown gall. Annual disease evaluations will be made in this field experiment for at least another two years. PARTICIPANTS: Desen Zheng, Research Associate. Responsible for a majority of the molecular biology on A. vitis. Primary goals are to identify mechanisms of grape root necrosis and biological control. Supaporn Kaewnum, Postdoctoral Associate. Responsible for development of indexing method for A. vitis and for research on the mechanism of biological control of F2.5. Kameka Johnson, Postdoctoral Associate. Responsible for development of indexing method for A. vitis in grapevines. Cheryl Reid, Technician, Cornell. Coordinates all of the vine indexing and assists in the biological control research. She has also coordinated the biological control field experiments. TARGET AUDIENCES: New York and US wine and grape industries Local and US grape nurseries National Grape and Wine Initiative PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This research has greatly enhanced our knowledge of the biology of A. vitis and crown gall disease of grapevines. It has resulted in development of methods to produce crown gall free vines and for improving methods for indexing grape material for presence of the crown gall pathogen. The traditional method of indexing grape cuttings by first callusing them required a minimum of 8 weeks to complete. The current method we are refining requires 4 to 8 days. Crown gall outbreaks can be quite unpredictable and having the proper detection and management tools will have a great positive impact on US viticulture. They will be essential for developing sources of clean planting material. We have also made significant progress in understanding the mechanism of biological control by F2.5 which will enhance its implementation as a component of a disease management program. Research results are presented regularly in verbal and written forms to members of the wine and grape industries of the US.
Publications
- No publications reported this period
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Agrobacterium vitis is the cause of crown gall disease on grape, a serious disease that occurs worldwide. The bacterium causes a root necrosis on grape and a hypersensitive response on plants like tobacco. Some nonpathogenic strains, such as F2.5, are able to inhibit the development of crown gall specifically on grapes. A. vitis survives systemically in grapevines and therefore is spread in propagation material. In 2010 grapevine cuttings from California to be used for a mother block at UC Davis were indexed in our laboratory. In addition cuttings from a commercial nurseryman in NY were also indexed. In both cases the cuttings were callused in the greenhouse and attempts to isolate the pathogen from the callus tissues were done. Agrobacterium vitis was isolated from a small number of the cuttings showing again that symptomless propagation material may be contaminated. Further research is being done to improve the rapidity and sensitivity of indexing methods for this pathogen. This involves isolating from placing node tissue in an enrichment broth, Yeast Mannitol Broth, and then isolating total DNA from bacterial growth in the broth and doing PCR with primers for genes virD2. Further improvements and testing of the indexing protocol are in progress. Biological control of grape crown gall provided by strain F2.5 is also studied in our lab. It was determined that the F2.5 biological control is specific to certain plants such as grape and does not prevent A. vitis from causing galls on plants such as sunflower, tomato and tobacco. It was shown that mutations in the clp protease system affected the ability of F2.5 to provide biological control. In addition to clpA, a proposed chaperone, clpP, the protease was also mutated resulting in loss of biological control. In 2010 we had the genome of F2.5 sequenced and began comparing the sequence to that of pathogenic A. vitis strain S4. Genomic regions that differed and appeared to possibly play a role in biological control were mutated in F2.5 and tested for biological control. To date these have included mutants in auxin transport and siderophore associated genes. Mutants showing lack of biological control are being complemented. The goal of this research is to develop a biological control for crown gall of grapevines that can be implemented on a commercial scale. PARTICIPANTS: Desen Zheng, Research Associate. Responsible for the majority of the molecular biology on A. vitis. Primary goals are to identify mechanisms of hypersensitive response and biological control. Supaporn Kaewnum, Postdoctoral Associate. Responsible for development of indexing method for A. vitis and for research on the mechanism of biological control of F2.5. Cheryl Reid, Technician, Cornell. Responsible for all of the vine indexing and has assisted in the sequencing and analysis project for studying movement of A. vitis between vineyards. TARGET AUDIENCES: New York and US wine and grape industries Local and US grape nurseries National Grape and Wine Initiative PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The research has greatly enhanced our knowledge of the biology of A. vitis and crown gall disease of grapevines. It has also resulted in development of methods to produce crown gall free vines and for improving methods for indexing grape material for presence of the pathogen. The traditional method of testing callused cuttings for A. vitis required a minimum of 8 weeks to complete. The current method we are testing requires 4 to 8 days. Crown gall outbreaks can be quite unpredictable and having the proper detection and management tools will have a great positive impact on US viticulture. We have also made significant progress in understanding the mechanism of biological control by F2.5 which will make it possible to implement this as a component of a disease management program. Research results are presented regularly to members of the wine and grape industries of the US.
Publications
- Savka, M. A., Le, P. T. and Burr, T. J. 2010. LasR receptor for detection of long chain quorum sensing signals. Identification of N acyl homoserine lactones encoded by the avsI locus of Agrobacterium vitis. Current Microbiol 62: 101-110.
- Krastanova, S. V., Balaji, V., Holden, M. R., Sekiya, M., Xue, B., Momol, E. A., and Burr, T. J. 2010 Resistance to crown gall disease in transgenic grapevine rootstocks containing a truncated virE2 gene of Agrobacterium. Transgenic Research 19:949-958.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Agrobacterium vitis is the cause of crown gall disease on grape, a serious disease that occurs worldwide. The bacterium causes a root necrosis on grape and a hypersensitive response on plants like tobacco. Some nonpathogenic strains, such as F2.5, are able to inhibit the development of crown gall specifically on grapes. A. vitis survives systemically in grapevines and therefore is spread in propagation material. In 2009 grapevine cuttings from California to be used for a mother block at UC Davis were indexed in our laboratory. In addition cuttings from a commercial nurseryman in NY were also indexed. In both cases the cuttings were callused in the greenhouse and attempts to isolate the pathogen from the callus tissues were done. No Agrobacterium vitis were isolated from any of the cuttings. Further research is being done to improve the rapidity and sensitivity of indexing methods for this pathogen. This involves giving cuttings a freezing treatment followed by collecting xylem extracts by flushing with water. Extracts are cultured and DNA isolated from total bacterial growth and tested with primers for genes virD2. Research from this work was published. Biological control of grape crown gall provided by strain F2.5 is also studied in our lab. It was determined that the F2.5 biological control is specific to certain plants such as grape and does not prevent A. vitis from causing galls on plants such as sunflower, tomato and tobacco. We hypothesize F2.5 is either affecting tumorigenic A. vitis in a specific manner that makes it avirulent on grapevine or is interacting directly with grape wounds making them resistant to crown gall infection. It was shown that mutations in the clp protease system affected the ability of F2.5 to function as a biological control. Additional mutants were generated in 2009 and tested in the same manner. It was also found that clpA specifically is required for biological control. clpA is likely to encode a chaperone that recruits specific proteins in the bacterium for proteolysis or modification is a key protein in the biological control mechanism. Although clpA is essential for biological control related genes clpS which is thought to deliver proteins to ClpA and ClpP1, a predicted protease, are not required for biological control. We also discovered in 2009 that A. vitis has the ability to move across surfaces by a phenomenon known as swarming. Swarming by A. vitis is also associated with the production of a surfactant material. The mechanism of swarming is unknown but may be involved in the ability of the bacterium to invade plant tissues, as is the case for other bacteria that infect mammalian tissues. PARTICIPANTS: Desen Zheng, Research Associate. Responsible for the majority of the molecular biology on A. vitis. Primary goals are to identify mechanisms of hypersensitive response and biological control. Cheryl Reid, Technician, Cornell. Responsible for all of the vine indexing and has assisted in the sequencing and analysis project for studying movement of A. vitis between vineyards. TARGET AUDIENCES: New York and US wine and grape industries Local and US grape nurseries National Grape and Wine Initiative PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The research has greatly enhanced our knowledge of the biology of A. vitis and crown gall disease of grapevines. It has also resulted in development of methods to produce crown gall free vines and continues to improve methods for indexing grape material for presence of the pathogen. Crown gall outbreaks can be quite unpredictable and having the proper detection and management tools will have a great positive impact on US viticulture. By identifying an effective biological control and understanding how it functions will make it possible to implement this as one component of a disease management program. Research results are presented regularly to members of the wine and grape industries of the US.
Publications
- Bini, F., Kuczmog, A., Putikony, P., Otten, L., Bazzi, C., Burr, T. J. and Szegedi, E., 2008. Novel pathogen-specific primers for the detection of Agrobacterium vitis and Agrobacterium tumefaciens. Vitis 43 (3), 147-153.
- Sule, S., Cursino, L., Zheng, D., Hoch, H.C. and Burr, T. J. 2009. Surface motility and associated surfactant production in Agrobacterium vitis. Letters in Applied Microbiology 49:596-601
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Agrobacterium vitis is the cause of crown gall disease on grape, a serious disease that occurs worldwide. The bacterium causes a root necrosis on grape and a hypersensitive response on plants like tobacco. Some nonpathogenic strains, such as F2.5, are able to inhibit the development of crown gall specifically on grapes. A. vitis survives systemically in grapevines and therefore is spread in propagation material. In 2008 more than 100 samples of grape cuttings from a mother block of grapevines were screened for the presence of A. vitis. Although none of the cuttings were positive for the pathogen some of the vines were previously shown to harbor the pathogen. Therefore indexing techniques are in question and need further refinement. We assisted in development of a new indexing method and began testing it on commercial samples. This is based on a technique known as multiplex PCR. Briefly, cuttings are given a freezing treatment followed by collecting xylem extracts by flushing with water. Extracts are cultured and DNA isolated from total bacterial growth and tested with primers for genes pehA, virD2 and virF. Initial tests showed clear presence of the proper PCR products. The publication from this research is in press. The distribution of A. vitis in infected vines was also investigated. Sections of dormant canes from known infected mother block vines were indexed. No A. vitis was detected from any of the cane sections of vines with crown gall on trunks in 2008. Again this was surprising and reflects an inefficiency of the indexing method. Biological control of grape crown gall by strain F2.5 was also investigated further. Mutants associated with a putative clp protease system in A. vitis were compared for biological control on grape. Mutation in a clpA homolog abolished biological control whereas a clpS homolog immediately upstream had no effect. F2.5 was effective when mixed with pathogen prior to inoculation of grapevines but control was lost when pathogen was inoculated to wounds prior to F2.5. Biological control is specific to certain plants such as grape and does not prevent A. vitis from causing galls on plants such as sunflower. Therefore it appears F2.5 is either affecting the pathogen in a specific manner that makes in avirulent on grapevine or is interacting directly with grape wounds making them resistant to crown gall infection. PARTICIPANTS: Desen Zheng, Research Associate. Desen does the majority of the molecular biology on A. vitis. He is working on determining mechanisms of hypersensitive response and biological control. Cheryl Reid, Technician, Cornell. Cheryl does all of the vine indexing and has assisted in the sequencing and analysis project for studying movement of A. vitis between vineyards. Thomas Zabadal, Professor, Michigan State University. Dr. Zabadal cooperates in the preparation of crown gall free vines and in conducting field experiments. TARGET AUDIENCES: NY and US wine and grape industries Local and US grapevine nurseries National Grape and Wine Initiative PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The research has resulted in the production of crown gall free vines through improvement of methods for indexing of grape plant material. As we improve on indexing methods it will be possible to further expand the research to more commercial nurseries. Crown gall outbreaks can be quite unpredictable and having the proper detection and management tools will have a great positive impact on US viticulture. The research is also providing further insight into the biology of crown gall and to the diversity of A. vitis and how the pathogen is spread in and between vineyards. By identifying an effective biological control and understanding how it functions will make it possible to implement this as one component of a disease management program. Research results are presented regularly to members of the wine and grape industries of the US.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Agrobacterium vitis is the cause of crown gall disease on grape, a serious disease that occurs worldwide. The bacterium causes a root necrosis on grape as well. Some nonpathogenic strains, such as F2.5, are able to inhibit the development of crown gall specifically on grapes. A. vitis survives systemically in grapevines and therefore is spread in propagation material. As a means of preventing spread of the pathogen in propagation material mother blocks of clean vines were established. Cuttings from the blocks were indexed and one of the blocks was found to be contaminated with the pathogen after four years. In 2007 forty six isolates were collected from a vineyard directly adjacent to the mother block that is infected with crown gall. These are being compared to isolates from the mother block. The genomic region between 16S and 23S rDNA genes was amplified and sequenced to determine the relatedness of isolates from the two sources. At this time isolates from both plantings
appear identical suggesting that the pathogen spread from the infected vineyard into the mother block. Additional isolates have been obtained and are under investigation. The distribution of A. vitis in infected vines was also investigated. Sections of dormant canes from known infected mother block vines were indexed. No A. vitis was detected from any of the cane sections of vines with crown gall on trunks in 2007. This a surprising result but may be associated with the extremely dry growing season and general lack of disease. It may also reflect an inefficiency of the indexing method. The mechanisms of biological control was also investigated further. Mutants of F2.5 that differ in their ability to cause a hypersentitive response on tobacco were evaluated for biological control activity. Some of these enhance biological control whereas other mutations reduced the biological control effectiveness.
PARTICIPANTS: Luciana Cursino Parent, Postdoctoral Associate, Cornell, hired January 2006. Dr. Parent works half time on crown gall and is studying aspects related to biological control of A. vitis by a non pathogenic strain of the same bacterium. She has been testing mutants of F2.5 for their biological control effectiveness. She is also leading the effort for sequencing isolates of A. vitis to study their movement between vineyards. Cheryl Reid, Technician, Cornell. Cheryl does all of the vine indexing and has assisted in the sequencing and analysis project for studying movement of A. vitis between vineyards. Thomas Zabadal, Professor, Michigan State University. Dr. Zabadal cooperates in the preparation of crown gall free vines and in conducting field experiments.Thomas Zabadal, Professor, Michigan State University. Dr. Zabadal cooperates in the preparation of crown gall free vines and in conducting field experiments.
TARGET AUDIENCES: NY and US wine and grape industries Local and US grapevine nurseries National Grape and Wine Initiative
Impacts
The research is leading to the production of crown gall free vines that can be used by growers for management of the disease. It is also helping to explain the diversity of A. vitis and how the pathogen is spread in and between vineyards. By understanding how the biological control strain of A. vitis functions it will be possible to implement this as one component of a disease management program. The research is presented regularly to the wine and grape industries of the US.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
Agrobacterium vitis is the cause of crown gall disease on grape, a serious disease that occurs worldwide. The bacterium causes a root necrosis on grape as well. Some nonpathogenic strains, such as F2.5, are able to inhibit the development of crown gall specifically on grapes. A. vitis survives systemically in grapevines and therefore is spread in propagation material. As a means of preventing spread of the pathogen in propagation material we generated crown gall free vines via tissue culture to plant in mother blocks. Cuttings from the blocks were assayed for A. vitis over the past six years. In 2006, one of the blocks consisting of different varieties and rootstocks, remains free of the pathogen whereas the other has become contaminated. Means by which the pathogen spread to the contaminated mother block are being investigated. Isolates from the mother block and from an adjacent block with crown gall have been collected and genetic fingerprints are being compared. A
more rapid procedure for producing large numbers of pathogen free vines is being tested using green shoots. Thus far vines propagated from green shoots have been found to be free of the pathogen. Field trials utilizing F2.5 were assessed for crown gall. The biological control reduced crown gall by 50 percent in some of the field experiments and no reduction in disease was observed in the others. The effect of applying strains of A. vitis to cut surfaces of cuttings prior to grafting was investigated further in 2006. Nonpathogenic strains of the bacterium negatively affect graft take whereas tumorigenic strains caused crown gall. A strain of A. tumefaciens appeared to have no effect on graft take.
Impacts
The research will lead to the development of crown gall free vines that can be used by growers for management of the disease. In addition possible other means by which the bacterium spreads between vines will be elucidated. An effective biological control for grape crown gall will be developed. The research has also led to the discovery that nonpathogenic strains of A. vitis can be detrimental to graft take and may be an important cause of graft incompatibility.
Publications
- Li, Y., Gronquist, M. R., Hao, G. Holden, M. R., Eberhardc, A., Scottd, R. A., Savka M. A., Szegedi,E., Sule, S., and Burr, T. J. 2006. Chromosome and plasmid-encoded N-acyl homoserine lactones produced by Agrobacterium vitis wildtype and mutants that differ in their interactions with grape and tobacco. Physiol. And Molec. Plant Pathol. 67:284-290.
- Zauner, S., Creasap, J. E., Burr, T. J., and Ullrich, C. I. 2006. Inhibition of crown gall induction by Agrobacterium vitis strain F2/5 in grapevine and Ricinus. Vitis 45:131-139.
- Hao, G. and Burr, T. J. 2006 Regulation of long-chain N-acyl-homoserine lactones in Agrobacterium vitis J. Bacteriol. 2006 188: 2173-2183.
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Progress 01/01/05 to 12/31/05
Outputs
Agrobacterium vitis is the cause of crown gall disease on grape, a serious disease that occurs worldwide. The bacterium causes a root necrosis on grape as well that appears to be a second type of disease induced by this pathogen. Some nonpathogenic strains of the bacterium are able to inhibit the development of crown gall specifically on grapes. In particular we are studying a strain called F2.5. To help determine the mechanism that is associated with biological control, mutants of F2.5 were made. A. vitis survives systemically in grapevines and therefore is spread in propagation material. As a means of preventing this from happening we have initiated crown gall free vines via tissue culture and they have been planted in mother blocks. Cuttings from the blocks were assayed for A. vitis over the past six years. In 2005, one of the blocks remains free of the pathogen whereas the other has become contaminated. In addition, rootstocks C3309 and 101-14Mgt were initiated in
tissue culture for production of pathogen-free vines that will also be placed in a mother block. Eight field trials with F2.5 were applied over the past years. The fields were assessed for crown gall in 2005. In three trials, crown gall was reduced by 50 percent and no reduction in disease was observed in the others. There is no apparent evidence that the bacterial treatment had a negative effect on vines. The effect of applying strains of A. vitis to cut surfaces of cuttings prior to grafting was also investigated. The bacterium at the graft union had a negative effect on graft take.
Impacts
This research has resulted in the development of crown gall free vines that can be used by growers for management of the disease. Since there are no effective chemical controls and biological control is still being developed, this is the most promising method for controlling the disease at this time. The research has also led to the discovery that strain F2.5 prevents infection of susceptible grape cells in wounds and that A. vitis may be an important cause of graft incompatibility.
Publications
- Creasap, J. E., Reid, C. L., Goffinet, M. C., Aloni, R., Ulrich, C. and Burr, T. J. 2005. Effect of wound position, auxin and Agrobacterium vitis strain F2.5 on wound-healing and crown gall development in woody grapevine tissue. Phytopathology 95:362-267.
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Progress 01/01/04 to 12/31/04
Outputs
Agrobacterium vitis is the cause of crown gall disease on grape, a serious disease that occurs worldwide. The bacterium causes a root necrosis on grape as well that appears to be a second type of disease induced by this pathogen. Some nonpathogenic strains of the bacterium are able to inhibit the development of crown gall specifically on grapes. In particular we are studying a strain called F2.5. To help determine the mechanism that is associated with biological control, mutants of F2.5 were made. A. vitis survives systemically in grapevines and therefore is spread in propagation material. As a means of preventing this from happening we have initiated crown gall free vines via tissue culture and they have been planted in mother blocks. Cuttings from the blocks have been assayed for A. vitis over the past five years. In 2004, one of the blocks remains free of the pathogen whereas the other has become contaminated. In addition, additional grape genotypes are being added
to the mother blocks. Six field trials with F2.5 were applied over the past years. Two of the field trials have significantly less crown gall on treated vines. The other field trials show no apparent difference between treated and nontreated vines. There is not apparent evidence that the bacterial treatment had a negative effect on vines. We also studied the sights within grape wounds that become infected to initiate crown gall. Callus cells that are generated following cambium activity were shown to be susceptible to infection. The generation of these cells and increase in infection was stimulated by addition of auxin. When strain F2.5 was applied to the wounds prior to application of pathogenic A. vitis, no infection occurred.
Impacts
This research has resulted in the development of crown gall free vines that can be used by growers for management of the disease. Since there are no effective chemical controls and biological control is still being developed, this is the most promising method for controlling the disease at this time. The research has also led to the discovery that strain F2.5 prevents infection of susceptible grape cells in wounds and that A. vitis may be an important cause of graft incompatibility.
Publications
- Hao, G., Zhang, H., Zheng, D. and Burr, T. J. 2005. A luxR homolog avhR in Agrobacterium vitis affects the development of a grape-specific necrosis and a tobacco hypersensitive response. J. Bacteriol. 187:185-192.
- Creasap, J. E., Reid, C. L., Goffinet, M. C., Aloni, R., Ulrich, C. and Burr, T. J. 2005. Effect of wound position, auxin and Agrobacterium vitis strain F2.5 on wound-healing and crown gall development in woody grapevine tissue. Phytopathology (In Press).
- Burr, T. J. 2004. Grape crown gall biology and strategies for control. Foundation Plant Services, UC Davis, FPS Grape Program Newsletter, October 2004.
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Progress 01/01/03 to 12/31/03
Outputs
Agrobacterium vitis is the cause of crown gall disease on grape, a serious disease that occurs worldwide. The bacterium causes a root necrosis on grape as well that appears to be a second type of disease induced by this pathogen. Some nontumorigenic strains of the bacterium are able to inhibit the development of crown gall specifically on grapes. In particular we are studying a nontumorigenic strains called F25. To help determine the mechanism that is associated with biological control, mutants of the biological control strain were made. Two mutants that are biological control negative will be studied further as a means of discovering how the bacterium functions as a biological control. A. vitis survives systemically in grapevines and therefore is spread in propagation material. As a means of preventing this from happening we have initiated crown gall free vines via tissue culture and they have been planted in mother blocks. Cuttings from the blocks have been assayed
for A. vitis over the past four years and they remain free of tumorigenic forms of the bacterium. Currently vines propagated from the mother blocks is being planted in commercial vineyards. This strategy is providing crown gall free vines to growers. If and how quickly such vines become infected with crown gall will now be determined. Although A. vitis causes necrosis on roots of vines the development of necrosis on woody parts of the vine has not been observed. Because crown gall is often a problem at graft unions we inoculated grafts with both tumorigenic and nontumorigenic strains. The nontumorigenic strains were tested as possible biological controls. Surprisingly, greater than 80% of the grafts treated with the different A. vitis strains did not heal. Buds on the scion variety did not grow. Inspection of the grafts revealed that very little or no callus formed at the graft, as compared to grafts that were treated with water. Therefore it appears that A. vitis may be a cause of
graft incompatibility. Current research is underway to determine if the incompatibility response is specific to A. vitis and if concentration of the bacterium affects development of the response.
Impacts
This research has resulted in the development of crown gall free vines that can be used by growers for management of the disease. Since there are no effective chemical controls and biological control is still being developed, this is the most promising method for controlling the disease at this time. The research has also led to the discovery that A. vitis may be an important cause of graft incompatibility.
Publications
- Zheng, D., Zhang, H., Carle, S., Hao, G., Holden, M. R., and Burr, T. J. 2003. A luxR homolog, aviR, in Agrobacterium vitis is associated with induction of necrosis on grape and a hypersensitive response on tobacco. Molec. Plant Microbe Interact. 7:650-658.
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Progress 01/01/02 to 12/31/02
Outputs
Agrobacterium vitis is the cause of crown gall disease on grape, a serious disease that occurs worldwide. The bacterium causes a root necrosis on grape as well that appears to be a second type of disease induced by this pathogen. Some nontumorigenic strains of the bacterium are able to inhibit the development of crown gall specifically on grapes. In particular we are studying a nontumorigenic strain called F2/5. To study the mechanism that is associated with biological control, mutants of one biological control strain were made. Two mutants that are biological control negative will be studied further as a means of discovering how the bacterium functions as a biological control. Crown gall disease develops at wound sites. It is known that plant wounds release chemicals that turn on the expression of virulence genes in the bacterium thus leading to infection. We found that cells associated with the vascular cambium are most susceptible to transformation by A. vitis.
This was done using a strain of the pathogen that transfers a reporter gene, GUS, that is expressed in the plant cell following transformation. It was also determined that auxin plays a role in transformation. When different concentrations of auxin were added to the wound, significantly different levels of transformation were observed. One possibility is that auxin stimulates the development of cells that are susceptible to infection by A. vitis. Using the GUS reporter gene system, we examined whether strain F2/5 prevents transformation of the cambial-associated grape cells by tumorigenic strains of A. vitis. When F2/5 is applied to wounds on woody rooted grape cuttings that have actively growing shoots, no evidence for transformation, as indicated by GUS expression, was observed.
Impacts
This research will provide a clear understanding of how A. vitis and crown gall disease compromise the normal wound healing process in grape. It will allow the development of an effective biological control for grape crown gall. This will be of great benefit to the grape industry and is likely to expand the geographical region in which Vitis vinifera cultivars can be grown. The research will also provide important insight into bacterial plant interactions and specifically those associated with the development of host specific necrosis.
Publications
- Burr, T. J. 2002. Why crown gall occurs and what can be done to manage it. Wine East. July-August pp. 10-15.
- Creasap, J. E., Reid, C. L., Goffinet, M. C., and Burr, T. J. 2002. Susceptibility of cell types in grape wounds to transformation by Agrobacterium vitis. 62nd Annual NEDAPS Meeting, Bromont, Quebec, Canada. October 2-4, 2002.
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Progress 01/01/01 to 12/31/01
Outputs
Agrobacterium vitis is the cause of crown gall disease on grape, a serious disease that occurs worldwide. The bacterium causes a root necrosis on grape and induces a hypersensitive response on tobacco. Some nontumorigenic strains of the bacterium also inhibit the development of crown gall specifically on grapes. To study the mechanisms that are associated with these reactions, mutants of one biological control strain were made. One of the genes that were disrupted leading to the loss of HR, necrosis and biological control is a homolog of transcriptional regulators belonging to the LuxR family. Therefore these phenotypes appear to be regulated by a common LuxR homolog and are density-dependent responses. Knowing that these responses are regulated in this manner allows one to determine which genes are affected by the LuxR homolog and thus determine the genetic mechanisms involved with each phenotype. These results also indicate that A. vitis produces acyl homoserine
lactone molecules that are called autoinducers. Autoinducers allow bacteria to communicate by sensing their density and to turn on genes under specific density and environmental conditions. Autoinducers bind to the regulators that subsequently interact with DNA promoter regions to induce transcription of specific genes. It was determined using a bioreporter protocol that the nontumorigenic A. vitis strain produces at least three autoinducers. Another mutant that maintained biological control activity but is reduced in grape necrosis and has lost HR induction was disrupted in a gene that appears to be involved in production of long-chain polyunsaturated fatty acids. This mutant was also complimented. The fatty acid may be the elicitor of HR or be a component of the elicitor molecule. Elicitors of the HR often lead to induction of responses in the plant that are related to defense against disease. Crown gall disease develops at wound sites. It is known that plant wounds release
chemicals that turn on the expression of virulence genes in the bacterium thus leading to infection. Cells within the grape wound that are most susceptible to infection were investigated. It was discovered that cells associated with the cambial zone are most susceptible to infection. It also appears that auxin may be essential for the development of these susceptible cells. By identifying cell types that are susceptible to infection and conditions that lead to the formation of such cells, it may be possible to circumvent their development as a way of controlling crown gall.
Impacts
This research will allow the development of an effective biological control for grape crown gall. This will be of great benefit to the grape industry and is likely to expand the geographical region in which Vitis vinifera cultivars can be grown. The research will also provide important insight into bacterial plant interactions and specifically those associated with the development of host specific necrosis and the induction of the hypersensitive response.
Publications
- Herlache, T.C., Zhang, H.S., Reid, C.L., Carle, S.A., Basaran, P., Thaker, M., Burr, A.T., and Burr, T.J., 2001. Mutations that affect Agrobacterium vitis - induced grape necrosis also alter its ability to cause a hypersensitive response on tobacco. Phytopathology 91:966-972.
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Progress 01/01/00 to 12/31/00
Outputs
A nontumorigenic strain of Agrobacterium vitis, inhibited the development of crown gall disease of grapevines in a field experiment that was conducted over three years. Supporting research showed that the strain rapidly colonizes wounds on grape wood. It effectively controls crown gall when applied to wounds on wood at concentrations of at least 1,000,000 cells per ml of inoculation solution. At that concentration it controls disease even when higher concentrations of the pathogen are present at the wounds. To study the mechanism by which the biological control bacterium inhibits crown gall, mutant strains were made that have lost biological control function. Probes made from sequences around the mutation were used to screen a DNA library of the biological control strain. Positive clones were used to compliment the mutants and for one mutant, biological control was restored. Sequencing of regions of the clone is underway in attempts to determine genes that are
associated with the phenotype. Once the mechanism is known, it may be possible to improve biological controls and to genetically engineer plants that will be resistant to the disease. Forty Vitis accessions were screened for susceptibility to A. vitis in 2000. Screening of germplasm from the USDA Clonal Repository at Geneva over the past several years has facilitated the identification of rootstocks and scion cultivars of different Vitis species which vary in their susceptibility to crown gall. It was discovered that some accessions, such as rootstock Courderc 3309, are resistant to a diverse group of pathogenic strains whereas others are susceptible, Kuhlman 149-3 to the same strains. Some accessions, such as Marechal Jouffre, differ with regards to the component of strains that they are resistant to. This work will result in the identification of accessions that can be used for breeding resistance and for determining factors related to the mechanism of infection.
Impacts
(N/A)
Publications
- Szegedi, E., Sule, S., and Burr, T. J. 1999. Agrobacterium vitis strain F2/5 contains tartrate and octopine utilization plasmids which do not encode functions for tumour inhibition on grapevine. J. Phytopathology 147: 665-669.
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Progress 01/01/99 to 12/31/99
Outputs
A PCR-based detection method for Agrobacterium vitis was developed using primer sequences derived from a chromosomal gene and two Ti plasmid genes. Amplification of a product of characteristic size from an rfb chromosomal gene identifies the bacterium as Agrobacterium vitis whereas primers to the Ti plasmid genes, virE2 and virA, identify whether the A. vitis strain is tumorigenic and which type of Ti plasmid it carries. This method will be tested further to determine its sensitivity for detecting the presence of the pathogen in infected plant material. Vitis germplasm was screened for susceptibility to A. vitis. Screening of germplasm from the USDA Clonal Repository at Geneva has facilitated the identification of rootstocks and scion cultivars of different Vitis species which vary in their susceptibility to crown gall. Accession 731, a Vitis hybrid, is highly resistant to the three strains of A. vitis that were used as inoculum and to the one A. tumefaciens strain.
Rootstock 41B was found to be highly susceptible to all strains. V. vinifera cultivar Tarnau and some other accessions as well, were found to be resistant to some strains of A. vitis, but susceptible to others. The most effective biological control for grape crown gall that has been discovered thus far is nontumorigenic A. vitis strain F2,5. The mechanism of biological control is being studied. To determine if the ability of F2,5 to efficiently cause root necrosis is associated with its biocontrol activity we made Tn5 mutants that are altered in their ability to induce grape root necrosis. They were then tested for biological control activity. Presently 9 mutants with altered necrosis phenotype have been identified and at least two of them have also lost biological control. Probes made from sequences around the Tn5 insertions are being used to screen an F2,5 cosmid library. Positive cosmids will be used to compliment the mutants to determine if biological control can be restored. If
so, the specific genes involved will be identified as a way of determining the possible mechanism involved. Once the mechanism is known, it may be possible to improve biological controls and to genetically engineer plants that will be resistant to the disease.
Impacts
(N/A)
Publications
- Burr, T. J., Bazzi, C., Sule, S. and Otten, L. 1998. Crown gall of grape: biology of Agrobacterium vitis and the development of disease control strategies (Feature Article). Plant Disease 82:1288-1297.
- Burr, T. J., Reid, C. L., Adams, C. E., and Momol, E. A. 1999. Characterization of Agrobacterium vitis strains isolated from feral Vitis riparia vines. Plant Disease 83: 102-107.
- Huang, T. C. and Burr, T. J. 1999. Characterization of plasmids that encode streptomycin-resistance in bacterial epiphytes of apple. J. of Applied Microbiology 86: 86:741-751.
- Xue, B. et al. 1999. Transformation of five grape rootstocks with plant virus genes and a virE2 gene from Agrobacterium tumefaciens. INVITRO PLANT 35:226-231.
- Burr, T. J. and Otten, L. 1999. Crown gall of grape: Biology and disease management. Ann. Rev. Phytopathol. 37:53-80.
- Alexandrova, M., Bazzi, C., Stefani, E., Anaclerio, F., and Burr, T. J. 1999. Biological control of Agrobacterium vitis using non-tumorigenic agrobacteria. Vitis 38:31-35.
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Progress 01/01/98 to 12/31/98
Outputs
The genetic diversity of Agrobacterium vitis was determined by comparing DNA fingerprints of the 5 prime end of the 23S rRNA gene and by RAPD analysis. Strains from a worldwide collection formed almost the same identical groups that they did when a previous analysis was done using the IGS region between the 16S and 23S genes. Fingerprint groups were highly correlated with the type of Ti plasmids carried by strains. A. vitis was found to be a common inhabitant of feral Vitis riparia grapevines. All strains were nontumorigenic and formed IGS, DNA fingerprints that were different from all of the tumorigenic strains. Some of the strains inhibited a tumorigenic strain from causing grape crown gall as efficiently as a previously tested biological control agent, strain F2,5. Field experiments were done to determine the effect of using crown gall resistant rootstocks and for testing the efficacy of biological control agents. Grape scions that were grafted onto a resistant
rootstock became less diseased and survived longer that vines grafted onto a susceptible rootstock. The most efficacious biological control agent was found to be strain F2,5. Plant injury was observed when large inoculum doses of A. vitis were infiltrated into cuttings.
Impacts
(N/A)
Publications
- Sule, S. and Burr, T. J. 1998. The effect of resistance of grape rootstocks to crown gall (Agrobacterium spp.) on the susceptibility of scions in grape vine cultivars. Plant Pathology 47:84-88.
- Momol, E. A., Burr, T. J., Reid, C. L., Momol, M. T. Hseu, S. H. and Otten, L. 1998. Genetic diversity of Agrobacterium vitis as determined by DNA fingerprints of the 5'-end of the 23S rRNA gene and Random Amplified Polymorphic DNA. J. of Applied Microbiology 85:685-692.
- Burr, T. J., Bazzi, C., Sule, S. and Otten, L. 1998. Crown gall of grape: biology of Agrobacterium vitis and the development of disease control strategies. Plant Disease 82:1288-1297.
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Progress 01/01/97 to 12/31/97
Outputs
The mechanism by which a nontumorigenic Agrobacterium vitis strain, F2/5, prevents crown gall at wound sites on grape was investigated. Mutants of F2/5 that no longer inhibit growth of pathogenic A. vitis strains in vitro, still prevented them from causing grape crown gall. In addition, pathogenic strains appear to attach to grape cells as efficiently as F2/5 when they are mixed together and applied to grape. Therefore competition for attachment sites on grape also does not appear to be a factor. F2/5 inhibits gall formation by most pathogenic strains on grape, but does not inhibit gall formation on sunflower, tomato or Kalanchoe plants. Therefore the control mechanism appears to be highly specific to grape. Control of a pathogenic strain, CG78, which is not controlled when it is mixed with F2/5 and applied to grape, was greatly enhanced by applying F2/5 to the grape wounds 24 hours before applying CG78 as investigated. Mutants of F2-rape cells as efficiently as F2-
not appear to be a factor. F2-rolled when it is mixed with F2-greatly enhanced by applying F2-. The crown gall susceptibility of several grape rootstocks, Vitis species and clones was determined by inoculating potted vines with a collection of A. vitis strains. Rootstocks such as Courderc 3309 and Riparia-Gloire were highly resistant to gall formation whereas the majority of V. vinifera scion cultivars are highly susceptible. Some Vitis germplasm are susceptible to specific A. vitis strains and resistant to others indicating that susceptibility is determined by genetic determinants of the pathogen and grape host. Polygalacturonase production by A. vitis, encoded by pehA, is a virulence factor. When pehA sequence and polygalacturonase enzyme properties were compared to enzymes from other plant pathogens, they were most like polygalacturonase from Ralstonia solanacearum. The enzyme from A. vitis releases primarily dimers and trimers from polygalacturonic acid and caused less electrolyte
leakage from potato tuber tissue than did the Erwinia carotovora and R. solanacearum polygalacturonases. When crown gall susceptible and resistant grape genotypes were compared, A. vitis causes greater electrolyte leakage from tissues of susceptible germplasm than from resistant, again suggesting that polygalacturonase plays a role in virulence of the pathogen.
Impacts
(N/A)
Publications
- Stover, E. W., Swarz, H. J., and Burr, T. J. 1997. Susceptibility of a diverse collection of Vitis genotypes to crown gall caused by Agrobacterium vitis. J. Amer. Soc. Enol. Vitic 48:26-32.
- Heidenreich, M. C., Corral-Garcia, M. R., Momol, E. A., and Burr, T. J. 1997. Russet of apple fruit caused by Aureobasidium pullulans and Rhodotorula glutinis. Plant Disease 81:337-342.
- Stover, E. W., Swarz, H. J., and Burr, T. J. 1997. Agrobacterium vitis-induced electrolyte leakage in crown gall-suscpetible and resistant grape genotypes. J. Amer. Soc. Enol. Vitic. 48:145-149.
- Burr, T. J., Reid, C. L., Tagliati, E., Bazzi, C., and Sule, S., 1997. Biological control of grape crown gall by strain F2/5 is not associated with agrocin production or competitions for attachment sites on grape cells. Phytopathology 87:706-711.
- Stover, E. W., Swarz, H. J., and Burr, T. J. 1997. Endophytic Agrobacterium in crown gall-resistant and suscpetible Vitis genotypes. Vitis 36:21 -26.
- Herlache, T. C., Hotchkiss, A. T., Burr, T. J., and Collmer, A. 1997. Characterization of the Agrobacterium vitis pehA gene and compariosn of the encoded polygalacturonase with the homologous enzymes from Erwinia carotovora and Pseudomonas (Burkholderia) solanacearum. App. and Env. Microbiol (in press).
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Progress 01/01/96 to 12/30/96
Outputs
The genetic diversity and phylogenic relationship between Agrobacterium vitis strains from several locations in the US and Europe were determined. A. vitis was divided into four main groups based on chromosomal type, as determined by generating DNA fingerprints of PCR-amplified DNA from the IGS region between the 16s snd 23s ribosomal RNA genes. Fingerprint categories correlated highly with the types of Ti plasmids carried by the strains. Ti plasmid types were determined by probing genomic digests with specific probes consisting of IS elements and T-DNA regions. Therefore a high correlation between chromosome type and Ti plasmid was discovered. Factors associated with the mechanism of grape crown gall biological control by the nonpathogenic A. vitis strain, F2/5, were studied. Production of an antibiotic, competition for attachment sites on grape cells and prevention of virulence gene induction in the pathogen were found not to be associated with control. However, no
genetic transformation of grape cells could be detected as determined by GUS, following inoculation of grape with pathogen and F2/5. Sequencing of the pehA gene from A. vitis revealed a predicted protein similar to polygalacturonases of Erwinia carotovora and Ralstonia solanacearum. PehA protein accumulated in the periplasm of A. vitis and released dimers, trimers and monomers from polygalacturonic acid and caused less electrolyte leakage from potato tuber tissue than did the polygalacturonases from the other bacteria.
Impacts
(N/A)
Publications
- STOVER, E. W., BURR, T. J., and SWARZ, H. J., 1996. Transformation of crown gallresistant and susceptible Vitis genotpes by Agrobacterium vitis. Vitis 35(1):29-33.
- BURR, T. J., REID, C. L., SPLITTSTOESSER, D. F., and YOSHIMURA, M. 1996. Effect of heat treatments on grape bud mortality and survival of Agrobacterium vitis invitro and in dormant grape cuttings. Am. J. Enol. and Vitic. 47:119-123 OTTEN, L, DE RUFFRAY, P., MOMOL, E.
- A., MOMOL, M. T., and BURR, T. J. 1996. Molecular characterization of North American Agrobacterium vitis strains and detection of a new type of Ti plasmid. MPMI 9:782-786 HERLACHE, T.
- C., et al. 1997. Characterization of the A. vitis pehA gene and comparison of the encoded polygalacturonase with the homologous enzymes from E. carotovora and Ralstonia solanacearum. App. and Env. Microbiol (in press).
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Progress 01/01/95 to 12/30/95
Outputs
Grape rootstocks C 3309 and Riparia 'Gloire', that are resistant to crown gall, caused by Agrobacterium vitis, harbored systemic populations of the pathogen that are comparable to those detected in the highly susceptible cultivar, Chardonnay. Therefore although certain rootstocks may be resistant to crown gall, they can still maintain a high systemic population of the pathogen that may be transmitted to a crown gall susceptible scion. To determine whether nontumorigenic A. vitis strains from wild grape can acquire Ti plasmids from tumorigenic strains and thus become pathogens, nontumorigenic strains were mated on galls with tumorigenic strains carrying different Ti plasmids. The tumorigenic strains were also mated with a nontumorigenic strain that came from a gall in a vineyard. Transfer of the Ti plasmid to the vineyard strain but not to the wild grape strains was detected. The mechanism by which nontumorigenic A. vitis strain F2-5 provides biological control of grape
crown gall is not related to production of a bacteriocin. It also does not result from F2-5 preventing the tumorigenic strains from attaching to grape cells, or by preventing the induction of virulence genes in the pathogen. Experiments to determine the efficiency of F2-5 under field conditions have been initiated.
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Progress 01/01/94 to 12/30/94
Outputs
Strains of Agrobacterium vitis, the cause of grape crown gall were isolated fromroot samples of Vitis riparia that were collected in different geographic regions of New York. Strains were characterized using standard bacteriological tests and by their reaction to an A. vitis-specific monoclonal antibody. None of the strains were tumorigenic on grape or other indicator hosts. DNA from the strains did not hybridize with a probe consisting of T-DNA from A. tumefaciens strain C58. Twenty eight of the strains were tested as biological controls by coinoculating them with tumorigenic A. vitis at wound sites on grape. Three strains strongly suppressed crown gall. None of these inhibited growth of tumorigenic A. vitis in vitro indicating that the mechanism of biological control is probably not related to antibiotic production. Seventy-six genotypes of grape including sixty of V. riparia were tested for susceptibility to crown gall. Woody stems were inoculated with two A. vitis
and one A. tumefaciens strains. Some V. riparia selections produced no galls and others produced large galls at the majority of wound sites. Previously reported crown gall resistant rootstocks, C 3309 and Riparia Gloire and susceptible cultivar Riesling were planted in soil infested with A. vitis. After three months galls were only observed on Riesling but approximately equal populations of A. vitis were detected systemically in the resistant and susceptible selections. Therefore genotypes resistant to gall formation may harbor s.
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Progress 01/01/93 to 12/30/93
Outputs
The mechanism of biological control of crown gall of grape by the nontumorigenicstrain of Agrobacterium vitis, F2/5 was evaluated. Mutants of F2/5 were generated by Tn5 mutagenesis and they provided the same level of control on grape as wildtype F2/5. F2/5 is not effective for prevening gall formation by A. vitis on hosts other than grape including sunflower, kalanchoe and tomato. It was demonstrated using the GUS reporter gene system that F2/5 prevents transformation of grape by A. vitis, but the mechanism of prevention is unknown. A heat treatment was investigated for eradicating A. vitis from dormant grape cuttings. A treatment of 30 C for 2 hr followed by 50 C for 90 min was tested. None of the treatments completely eradicated the pathogen, but significant reductions were observed. It was possible to reduce the level of pathogen in the cuttings by over 90 percent. No significant effect was observed on bud mortality of dormant cuttings of cultivars
Chardonnay, Niagara, Seyval or Riesling treated at 50 C in February. Sixty-eight wild grapevines (V. riparia) were collected from different regions of New York State and assayed for A. vitis. About 500 strains of A. vitis were isolated and characterized by colony morphology, reaction to a monoclonal antibody, and production of polygalacturonase and endoglucanase. None of the strains were tumorigenic on sunflower, tomato or kalanchoe. The role these strains may play in crown gall disease of grape is being investigated.
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Progress 01/01/92 to 12/30/92
Outputs
A biological control for crown gall of grape, caused by Agrobacterium vitis was evaluated. It is a nontumorigenic A. vitis strain from South Africa called F2/5 that produces an agrocin that is inhibitory to most A. vitis strains in vitro. F2/5 also provided biological control of crown gall on grape when coinoculated with the pathogen or when inoculated prior to the pathogen. F2/5 is effective when applied at concentrations equal to or greater than that of the pathogen. A heat treatment was investigated for eradicating A. vitis from dormant grape cuttings. Treatments of 50 C for 30, 60, and 90 min and 55 C for 30 min were tested. None of the treatments completely eradicated the pathogen. When treated at 50 C for 90 minutes, up to 96 percent of the cuttings were free of the pathogen as compared to 100 percent of the nontreated cuttings being contaminated. All treatments at 50 C had little effect on bud mortality whereas treatments of 55 C often killed a significant
number of buds.
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Progress 01/01/91 to 12/30/91
Outputs
Hot water treatments were tested for eradicating Agrobacterium tumefaciens biovar 3, the cause of crown gall from dormant grape cuttings. Cuttings were submersed in a water bath at 50C for 60 min and 55C for 30 min and then indexed for the presence of the pathogen. No pathogen was recovered using either of these treatments. A treatment of 50C for 30 min greatly reduces the pathogen in dormant cuttings but did not eradicate it. Bud survival and graft take were affected by heat treatments for some cultivar, rootstock combinations. Polygalacturonase, produced by A. tumefaciens biovar 3, was shown to be associated with a tissue decay response on grape as well as attachment of the bacterium to grape roots. A biovar 3 mutant that does not produce the enzyme, was less tumorigenic on grape and developed significantly lower populations at wound sites. The polygalacturonase gene was cloned and used to probe other agrobacteria and rhizobia. No homology was detected with
bacteria other than A. tumefaciens biovar 3.
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Progress 01/01/90 to 12/30/90
Outputs
A hot water treatment was tested for eradicating Agrobacterium tumefaciens biovar 3, the cause of crown gall from dormant grape cuttings. Cuttings were submersed in a water bath at 50C for 30 min and then indexed for the presence of the pathogen. The pathogen was reduced to below detectable levels in some rootstocks and scions, but was still detectable in others. DNA probes were developed for determining the tumorigenic nature of Agrobacterium strains. Two probes consisting of portions of the T-DNA were very effective for determining tumorigenicity. Probes were used in a colony hybridization assay to evaluate tumorigencity.
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Progress 01/01/89 to 12/30/89
Outputs
A monoclonal antibody was developed that is specific for Agrobacterium tumefaciens biovar 3, the cause of crown gall on grapevines. The antibody reacted positively with all strains of the pathogen tested from several viticultural regions worldwide. It does not react with other biovars or species of Agrobacterium, or with related genera or sparophytic bacteria from soil or grapevines. The antibody was used successfully in microtitre ELISA and immunoblot procedures when indexing grapevine propagation material for the presence of the pathogen. Detectable A. tumefaciens biovar 3 were eradicated from dormant, systemicaly infested grape cuttings by submersing then in a 50 C water bath for 30 min. In large commercial trials, the numbers of infested cuttings was greatly reduced, however, some pathogen was still present. Experiments are underway to refine the hot water treatment to make it more effective for commercial usage.
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Progress 01/01/88 to 12/30/88
Outputs
Grape rootstocks 3309 and 101-14 from New York were found to be systemically infested with Agrobacterium tumefaciens biovar 3, the cause of crown gall. When clean rootstocks and scions were planted in soil that was infested with the pathogen, all became systemically contaminate with it. These included 5BB, 101-14, 18-815, 3309, 44-53, Steuben and Pinot Chardonnay. This shows that symptomless rootstocks which normally do not develop crown gall in the field may harbor and spread the pathogen. All detectable A. tumefaciens biovar 3 were eradicated from dormant hardwood cuttings by submersing them in a 50 C water bath for 30 min. These included naturally and artificially-infested cuttings. In vitro tests showed that some strains of biovar 3 may tolerate this temperature when present at high populations. All strains of biovar 1 tolerated the treatment. Bud kill was noted when the cuttings that were treated were not fully dormant. Pectolytic enzymes were shown to be
produced by biovar 3 strains and they may be involved in the root decay incited by this biovar. Enzymes were only detected at acid pH.
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Progress 01/01/87 to 12/30/87
Outputs
Agrobacterium tumefaciens was not detected in sap assays or rhizosphere samples from bacteria-free tissue culture-propagated grapevines, one year after planting in an old vineyard site and a site not previously planted to grapevines. A monoclonal antibody, specific for biovar 3 strains of Agrobacterium was developed. Using immunoblot and microtiter assays the monoclonal antibody was effective for identifying biovar 3 strains of Agrobacterium but did not react with biovars 1 and 2 or with several other bacteria tested. A DNA probe useful for identifying tumorigenic strains of A. tumefaciens was developed. Chromosomal and plasmid DNA was isolated from field strains of A. tumefaciens. Only plasmid DNA from tumorigenic strains hybridized with the DNA probe. Low populations of A. tumefaciens in artificially infested soil did not increase to detectable levels after planting grapevines in the soil. This illustrates that low inoculum levels in soil may not be important
as primary inoculum for crown gall disease.
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Progress 01/01/86 to 12/30/86
Outputs
The highest populations of Agrobacterium tumefaciens biovar 3 were present in the rhizosphere of grapes as compared to oats and to populations in fallow soil. Oats, a nonhost of Agrobacterium, supported a significantly higher level than fallow soil. When noncontaminated tissue culture- propagated grapevines were planted in soil that was artificially infested with biovar 3, they became systemically infested within six months. In addition to being detected systemically in grape and in association with roots in soil, Agrobacterium was detected in association with roots of one- year-old grapevines that were in commercial nursery storages. A.tumefaciens biovar 3 was isolated from dark sunken lesions on grape roots in the field. Upon inoculation, both tumorigenic and nontumorigenic biovar 3 strains caused decay of grape roots. Biovar 1 and 2 strains did not decay grape roots. Biovar 3 strains did not decay the roots of sunflower or beans, but tumorigenic strains caused
tumors on sunflower roots. Streptomycin-resistant Pseudomonas syringae pv. papulans were detected in additional orchards in western New York. Resistant strains were found to overwinter as streptomycin sensitive strains, in apple buds. Resistant strains carry a specific plasmid that can be conjugatively transferred to sensitive strains.
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Progress 01/01/85 to 12/30/85
Outputs
Agrobacterium tumefaciens biovar 3 was detected in vineyard soils where vines had crown gall but was not detected in nonvineyard soils using direct plating methods on selective media. The highest populations of the pathogen were found associated with grape roots. High levels of avirulent strains of biovar 1 were found in nonvineyard and vineyard soils. Tumors of 20 biovar 3 strains from grape synthesized nonpaline and only two synthesized octopine. Fourteen strains of biovar 3, collected worldwide, were inoculated to nine different grape rootstocks and three grape cultivars. Some of the strains infected only certain rootstocks or cultivars, however, none of the hosts were resistant to all 14 strains. Strains that produced the largest tumors on one cultivar did not necessarily do so on the others. Strains of Pseudomonas syringae pv. papulans that are resistant to at least 1000 ppm streptomycin were detected in 11 of 24 'Mutsu' apple orchards in western New York
State. Resistance was suspected because control of blister spot, which is caused by the bacterium, failed after the recommended applications of the antibiotic were applied in 1985. None of the resistant strains was inhibited by the highest level of streptomycin (1000 ppm) tested.
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Progress 01/01/84 to 12/30/84
Outputs
Agrobacterium tumefaciens biovar 3 was recovered from symptomless grapevine cuttings collected from the cultivars Chardonnay and White Riesling in five vineyards in New York State. From some vineyards a high proportion of the isolated bacteria were avirulent and thus characterized as A. radiobacter. By dissecting cuttings into small pieces it was possible to grind them individually and to recover the pathogen from a small percentage of the pieces. This proved to be an unpractical way to index cuttings but illustrated that the pathogen is not evenly distributed within grape tissues. Eighty strains of A. tumefaciens from grape were examined with regard to host range in the greenhouse. Almost all of the strains infected more than one host but none infected all hosts, which included tobacco, sunflower, tomato, Bryophyllum, grape and Datura. Almost all virulent strains infected tobacco (Nicotiana glauca). Thirty grape strains have also been investigated with regard to
relative virulence as determined by tumor size on sunflower and grape. Significant differences in strains have been identified. These data illustrate that much variability exists among grapevine strains with regards to host range and degree of virulence.
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Progress 01/01/83 to 12/30/83
Outputs
Agrobacterium tumefaciens biovar 3 was recovered from dormant, apparently healthy, grape cuttings of five commercially important Vitis vinifera cultivars. Isolations on selective media were made from callus and roots of surface sterilized cuttings potted in a sterile mixture. Approximately 50% of 22 cutting samples yielded virulent strains of the pathogen. From these limited samplings, the pathogen was only recovered from cuttings taken from vines that were obviously infected. This may reflect the absence of the pathogen in apparently healthy vines or the failure of our methods to detect them. Only 2 of 20 vineyard soil samples yielded A. tumefaciens. Recoveries were from soil collected around infected trunks and roots and not from between rows or around healthy vines. Pseudomonas syringae pv. syringae was isolated from infected sour cherry blossoms and fruits. Many of the fruits were also infected with Monilinia fructicola.
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Progress 01/01/82 to 12/30/82
Outputs
Agrobacterium tumefaciens biotype 3 was recovered from symptomless grape cane cuttings taken while vines were dormant in 1982. Attempts to isolate the pathogen directly from cane tissues on a selective medium failed. However, when cuttings were callused and rooted in sterile sand the pathogen was recovered from the callus and root tissues. This finding is significant since it shows that the pathogen does survive systemically in vines and may be disseminated in propagation material. It appears from the above results that A. tumefaciens survives systemically in grapevines and gall initiation is incited following winter or mechanical injury. The exact time when injury is most likely to incite gall formation is not known. Therefore, a series of wounding experiments have been initiated in the vineyard. If a critical wounding period is found, it may be possible to apply bactericides to stop gall development. Experiments were initiated to study the effect of the
bactericide, Gallex (2, 4-xylenol meta-cresol) in killing gall tissue and stopping additional and recurring gall formation. Fresh galls on grapes were thoroughly painted with the material on July 6, 1982.
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Progress 01/01/81 to 12/30/81
Outputs
Agrobacterium tumefaciens biotype 3 was recovered from two of 30 soil samples taken from five vineyards. Recoveries were made using a modification of the selective medium of New and Kerr that substitutes sucrose as the carbon source. The bacterium was only recovered from rhizosphere soil samples taken from vines containing fresh galls on their trunks. When samples were taken early in the season, before new galls developed, virulent strains of the pathogen were not detected. Biotype 3 and intermediate strains were recovered from vineyard soil. Biotype 3 was also detected in grape sap from bleeding vines, collected in the spring. Pathogenic isolates were obtained from sap of seven of 24 infected vines and one of 17 apparently healthy vines. The majority of isolates obtained produced galls on sunflower, tomato, and grapevines when inoculations were made in the greenhouse. Attempts were made to find an effective biological control bacterium for biotype 3. Over 300
candidate isolates were tested on agar plates and six isolates were obtained, which inhibited growth of biotype 3. None of these were effective biological control agents when tested on grapevines. Several isolates of Pseudomonas, that were recovered from dormant apple buds during the 1980-81 winter, were inoculated to Mutsu apple fruit in the orchard. All of the isolates which were characterized as P. syringae pv. papulans in laboratory tests caused blister spot symptoms on fruits.
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Progress 01/01/80 to 12/30/80
Outputs
Pseudonomas syringae py papulans was isolated from up to 50% of dormant Mutsu apple buds. Populations of the pathogen were greatest on the differentiated leaves and shoot portions of the bud and decreased on scales moving out from the center of the bud. The bacterium was rarely recovered from outer bud scales. The pathogen was also rarely recovered from bud surfaces (1 in 500). Eliminating or greatly reducing overwintering bacteria by the application of a bactericide at or shortly after bud break, when the bacteria would be exposed, may control the disease. P syringae pv syringae was also detected in the dormant apple buds. Agrobacterium tumefaciens biotype 3 isolates were obtained from diseased grapevines and vineyard soils. This is the first report of this biotype being recovered from soil. A selective medium was developed which allowed growth of biotype 3 and inhibited a high percentage of other soil bacteria. No A. tumefaciens was recovered from sap or wood
tissue from symptomless grape canes. A project to look for biological control agents, which are effective against biotype 3, was started. Over 150 bacteria from grape roots and soils have been screened of which five inhibit growth of biotype 3 in culture.
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Progress 01/01/79 to 12/30/79
Outputs
Pseudomonas syringae pv. papulans was found to overwinter in dormant apple buds and in infected fruit on the orchard floor. Epiphytic populations of the pathogen were determined using a selective medium which we developed. Populations of the pathogen on fruits and leaves varied greatly between apple cultivars. 'Mutsu' fruits were found to be susceptible to the pathogen starting 2-2 1/2 weeks after petal fall. Streptomycin applied at this time was effective in controlling the disease. Agrobacterium tumefaciens was isolated from several cultivars of grapevines. The pathogen was identified as biotype 3 of the bacterium. Biological control using isolate 84 was ineffective in the greenhouse. An apparently new bacterial leaf spot of grapevine was found on Long Island. The pathogen was identified as a Xanthomonas spp.
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Progress 01/01/78 to 12/30/78
Outputs
Isolates of Pseudomonas syringae pv. papulans were obtained from several 'Mutsu'apple orchards in New York. Fruits exhibited typical blister spot symptoms. Isolates were characterized using diagnostic tests in the laboratory and were differentiated for typical P. syringae isolates. Blossoms and fruits were inoculated weekly from bloom to early September in two 'Mutsu' orchards to determine when fruits were most susceptible to blister spot. They were most susceptible from about June 20 to the end of July in 1978. The natural inoculum was also measured by plating out blossom and fruit washes from two 'Mutsu' orchards at weekly intervals during the season. Inoculum reached a maximum near the end of June with approximately 4 x 10 3 colony-forming units/fruit. Very low levels of inoculum were detected at bloom. Agrobacterium tumefaciens was isolated from grapevines in the state, thus confirming its presence. Isolates were identified using greenhouse inoculation tests.
Biological control field and greenhouse trials, using A. radiobacter isolate 84 were initiated. Studies on other bacterial pathogens were started including Xanthomonas pruni, Pseudomonas syringae, and a Xanthomonas sp. isolated from grapevines.
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