Progress 07/01/10 to 06/30/15
Outputs
Target Audience:Stakeholder groups benefiting from this project include industry stakeholder groups (turf, ornamental and arborists), and homeowners and local municipalities affected by BLS, as well as local practitioner organizations such as local Master Gardeners groups. Academic stakeholders include both undergraduate and graduate students in Plant Biology and Biotechnology at Rutgers University, in which the disease description and project results were presented. Scientific advancements on the subject matter were presented to scientists at national and regional professional society meetings (American Phytopathological Society), as well as two international symposia (Bangkok, Thailand and Beijing, China). Changes/Problems:Problems with Goal 1. A sensitive, real time PCR protocol was successfully developed for detection of X. fastidiosa in oak leaves. However, the protocol was not cost effective and was technically challenging for untrained personnel. In addition, real time PCR results were limiting in terms of extrapolating additional information in support of other project goals. Therefore, we chose to revert back to conventional PCR methods for detection purposes. Subsequent results using conventional PCR supported goals 2 and 3. Problems with Goal 2. The bulk of experimental workload over the five year project focused on satisfying Goals 1 and 3. Attempts to identify alternative plant hosts, as part of satisfying Goal 2, failed in most non-oak tested plants. One exception was confirmation of X. fastidiosa identified in Japanese knotweed located in a New Jersey locale where bacterial leaf scorch of oak was prevalent. Identifying additional plants host that serve as potential pathogen reservoirs remains a future priority, as the experimental focus switches to characterizing the disease from the epidemiological perspective. No attempt was made to genetically characterize the pathogen in insect vectors, primarily due to the lack of a collaborator with entomological skills. Like alternative plant hosts, however, pathogen characterization within insect vectors remains a priority for future studies. Problems with Goal 4. To date, insufficient information has been generated to understand disease epidemiology fully, and therefore begin to develop alternative, effective means for slowing the spread and progression of oak leaf scorch of oak. Goal 4 remains the penultimate goal the project. Future goals that focus on epidemiological studies are expected to provide new insights into developing disease management strategies for reducing the rate of disease spread. What opportunities for training and professional development has the project provided?Research related to this project was used to fulfill part of a PhD dissertation for G. Behringer, who successfully defended his PhD in Sept 2015. Former postdoctoral researcher and Research Associate, Dr. N. Patel, worked part time on the project. She received important mentoring experience while supervising undergraduate research during the course of the project. Six undergraduate students gained valuable experience-based education training while working on the project. These students gained valuable experiences in laboratory practices and methodologies in molecular biology and microbiology, as well as practical field experience (sample collection) and experimental design. How have the results been disseminated to communities of interest?Dissemination of results was conducted through extension and outreach presentations and lectures given each year to stakeholder groups. Information was also provided through published extension information fact sheets or postings on websites. Project results were disseminated to the scientific community through oral and poster presentations at professional scientific meetings, and seminars at national and international scientific symposia. Project results were presented as part of undergraduate and graduate level education in the forms of classroom lectures, undergraduate and graduate research, and outreach undergraduate recruitment presentations. Portions of project results have been published as abstracts in professional journals, and three manuscripts describing project results are currently in draft form in preparation for submission for publication to scientific journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
A sensitive, real time polymerase chain reaction (PCR) protocol was developed to detect the presence of X. fastidiosa in oak leaf tissue. The protocol was technically challenging and more expensive to conduct compared with conventional PCR protocols. While results from real time PCR were sensitive from the standpoint of detection, the potential of providing additional scientific information was less than that for conventional PCR. Therefore, a previously developed detection protocol based on conventional PCR was refined for detection purposes to satisfy Goal 1, and for experimental purposes in efforts to achieve Goals 2 and 3. This refined conventional PCR protocol centered on amplification of several genetic loci specific to X. fastidiosa, with sequence analysis of the loci for pathogen verification. Attempts to achieve goal 2 focused mostly on identifying plant host species other than oak that are suspected to be infected with the BLS pathogen. Attempts to identify the pathogen in alternative plants hosts resulted in verified detection within Japanese knotweed plants that were displaying BLS-like symptoms, and were in close physical proximity to infected oak. The refined conventional PCR protocol provided an effective tool for comparative genetic analyses of X. fastidiosa populations recovered from oak within the northeastern US. Using a multilocus sequence analysis (MLSA) approach, northeastern oak populations of X. fastidiosa were determined to be taxonomically related, but genetically distinct, to strains previously recovered from oak in the southern US. Results also verified that the northeastern populations were more distantly related to X. fastidiosa strains previously recovered from alternative host plant species located in different regions throughout the US and South America. To better understand the distribution patterns of the pathogen (Goal 3), deeper genetic analysis of the X. fastidiosa population infecting northeastern oak was conducted by sequencing the genome of a single X. fastidiosa subsp. multiplex isolate, RNB1. Comparative analysis was conducted using the genome of RNB1 and 18 other X. fastidiosa genomes available in the database in an effort to identify sequences unique to the northeastern oak isolate. Using a reciprocal BLASTp approach, nine open reading frames (orfs) were identified inRNB1 for which homologues were not present in any of the other X. fastidiosa genomes. Three of these orfs encoded extended repeat regions within the predicted protein product. Based on gene ontology assignments, each of the three were suspected to encode proteins related to pathogen virulence. Further analysis of the repeat regions, consisting of a poly L repeat in one orf, and poly PELE or poly QA repeats for the other two orf, indicated repeat lengths varied between X. fastidiosa strains, as well as populations in northeastern oaks. The level of uniqueness of these orf sequences indicate their potential as selective markers for both characterizing and tracking spread of X. fastidiosa populations between oak stands over both short and long distances, and well as potential spread among other plant host species and insect vectors. In addition to unique orf sequences described above, prophage sequences within RNB1 were evaluated for their potential as sources for X. fastidiosa population-specific markers. Prophages sequences were identified and compared between all 19 X. fastidiosa genomes. Findings indicate a number of conserved prophage sequences shared between all 19 genomes, as well as sequences unique to specific strains. Overall, results from this study provide a better understanding of X. fastidiosa populations infecting oak in the northeastern United States. In addition, results provide essential genetic tools to extend the study into the epidemiology of bacterial leaf scorch, in efforts to devise effect control of the disease.
Publications
- Type:
Other
Status:
Published
Year Published:
2014
Citation:
Behringer, G., Gould, A.B., and Kobayashi, D.Y. 2014. Multilocus characterization of oak leaf scorch isolated in the northeastern and mid-Atlantic United states. Phytopathology: (Abstr - poster presentation)
- Type:
Other
Status:
Published
Year Published:
2015
Citation:
Behringer, G. and Kobayashi, D. 2015. Delineating the bacterial leaf scorch of oak Xylella fastidiosa subspecies: a comparative genomics approach. Phytopathology 105 (Suppl. 2):S3.2 (Abstr - oral presentation)
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Industry stakeholder groups (turf, ornamental and arborists), homeowners and local municipalities affected by BLS Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Mr. Greg Behringer, a 5th year PhD candidate in the Plant Biology Graduate Program, is a contributor to the project and is using objs 1 and 3 as part of his thesis research. He is expected to complete his thesis in 2015.Four undergraduate students (L. West, M. Gattuso, J. Hoang, N. Bailey) recruited from one of two undergraduate programs (Biotechnology and Plant Biology) contributed to the project. All four gained valuable experience in experimental designs and experimental protocols and general research laboratory experience. How have the results been disseminated to communities of interest? Presentations were given to industry stakeholder groups (turf, ornamental and arborists), homeowners and local municipalities affected by BLS. Academic stakeholders include graduate students in Plant Biology exposed to the disease in the course Plant Disease; undergraduate students in Plant Biology, Biotechnology and Agriculture and Food systems. The disease and project were presented to first yr students at the School of Environmental and Biological Sciences, Rutgers University as well as students at county colleges as a recruitment tool for future students. What do you plan to do during the next reporting period to accomplish the goals? Genetic and genomic evaluation of X. fastidiosa subsp. Multiplex willcontinue. A bioinformatics approach will be employed to identify genetic uniqueness to subspecies and to regional strains. Sampling of symptomatic and non-symptomatic plant species located near diseased oak trees will be tested in BLS hot spots to identify and evaluate alternate host species that serve as pathogen reservoirs.
Impacts
What was accomplished under these goals?
Bacterial leaf scorch (BLS) has been a chronic disease affecting red and pin oak in the northeastern United States for over three decades. Thought to originate in the southeastern United States, the disease has spread progressively as far north as Ontario, Canada. The causative agent of BLS on oak, Xylella fastidiosa subsp. multiplex, has been also identified to cause disease on a broad range of plant species, including blueberry, almond, and a number of ornamentals including various shade tree species. BLS has had a significant effect on oak in the northeastern US, including the state of New Jersey. Little is known about the disease, or the pathogen that causes it and, to date, there are few established methods for controlling the disease. We have taken a genetic and genomic approach to better characterize the pathogen in terms of how strains compare geographically, and how they compared between different plant host species. By characterizing strains in this manner, we expect to gain knowledge of how the disease is spread, and how better control methods can be devised. Analyzing genetic diversity among strains of the bacterial pathogen Xylella fastidiosa continued to be the research focus over the past reporting period. A second round of genome sequencing was conducted to improve assembly of the previously generated genome sequence of a New Jersey oak strain of X. fastidiosa subsp. multiplex. Using the improved genome sequence, comparative analyses with other genome sequences of X. fastidiosa were conducted to identify new genetic loci that could be used as unique strain identification markers. The genome also mined for genes unique to New Jersey oak strain, in efforts to identify genetic specificity of X. fastidiosa subsp. multiplex infecting oak, as compared to other multiplex strains that infect other host plants. Results of these analyses address goals 1 and 3. Rigorous sampling of infected oak trees were conducted in the months of June, July and August in an attempt to relate pathogen titer in known infected trees over the course of late spring and summer. In addition, samples were collected from non-oak tree species displaying scorch-like symptoms, including maple trees. To date, few tree samples showed the presence of the bacterium in the early months. Samples from latter months have yet to be processed. When completed, this study will contribute to goal 1 and indirectly to goal 4.
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Extension presentations were given to industry stakeholder groups, especially those associated with turf, ornamental, and treeindustries. Presentations at professional meetings were directed atthe scientific community comprised mainly of plant pathologists and plant-associated microbiologists. Formal classroom teaching in the course General Plant Pathologyaddressed second, third and fourth year undergraduate students (majors and non-majors) at Rutgers University. Classroom teaching in the graduate course, Core Seminar in Plant Biology addressed first and second year graduate students in the Plant Biology Graduate Program at Rutgers University. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The main contributor to this project was Mr. Greg Behringer, a PhD candidate in the Plant Biology Graduate Program at Rutgers University. Goals 1 and 3 of the project are specific components of Mr. Behringer's PhD thesis research. Assisting Mr. Behringer over the reporting period was Ms. Lorabeth West, anundergraduate student majoring in Plant Science, who gained laboratory experience. Also assisting Mr. Behringer was Mr. Remy Koch, a third year undergraduate student majoring in Biotechnology. Mr. Koch gainedvaluable independent research experience while assisting with analysisof the X. fastidiosa genome. How have the results been disseminated to communities of interest? Results of this study have been presented to the scientific community as presentations at professional meetings. Results were also presented as part of undergraduate and graduate course lectures, bringing greater awarenessand interest of plant pathological problems tostudents inplant biology andplant pathology.Extension and outreach presentations updating progress of the project were made atmeetings for turf and ornamental industry stakeholders in the greater New Jersey area. What do you plan to do during the next reporting period to accomplish the goals? The next reporting period will focus on detecting the presence of X. fastidiosa in non-symptomatic host plants, and detection of the bacterium ininsect hosts, addressing goal 2 of the project. A strategy and plans to test chemical therapy of diseased oak trees have been discussed as possible means of controlling the disease, addressing goal 4.
Impacts
What was accomplished under these goals?
We accomplished goal 1 during the previousreporting periodby developing a reliable and sensitive PCR protocolfor pathogen detection. During this reporting period, we usedthe PCR method as a means to fulfillgoal 3 . Using a multilocus sequence analysis approach, we observed little genetic variation among New Jersey oak populations of X. fastidiosa. In contrast, our analysis revealed distinct genetic diversity between these New Jersey populations and other X. fastidiosa subsp. multiplex populations originating from diverse geographical areas. These results provide strong evidence that pathogen populations in the northeastern US are clonal, and suggestsspread throughout the state resulted from introduction of the pathogenseveral years ago. To further characterize the New Jersey strain of X. fastidiosa, we initiated a comparative study of genome sequences between X. fastidiosa strains, including one from New Jersey oak. A number of genes were identified as unique to the New Jersey isolate, as compared to those found within the genome of a southern oak strain; however, functions of the majority of these genes remain unknown. Survey analysis also supported the results of genetic analysis by providing much needed information directed at progression of disease in specific locations. We anticipate that collectively, genetic and disease progression analyses will provide clues to better understand spread of the disease, factors influencing host specialization, and also novel approaches to disease control.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Behringer, G. and Kobayashi, D.Y. 2013. The genetic characterization and radiation of bacterial leaf scorch of oak in New Jersey. Phytopathology 103(Suppl. 2):S2.14
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Gould, A., Hamilton, G., Vodak, M., Grabosky, J., and Lashomb, J. 2013. Street-tree incidence and severity of bacterial leaf scorch of oak in the New Jersey urban forest. Phytopathlogy 103(Supp.. 2):S2.51
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: The five goals for this project are as follows: 1) develop a real time PCR protocol that is a reliable and sensitive method for Xylella fastidiosa detection; 2) identify insect vectors and potential alternative plant hosts that serve as reservoirs for spread of the pathogen; 3) better understand the distribution of the pathogen throughout New Jersey and the northeastern US region; 4) develop and test possible methods for control of disease caused by X. fastidiosa; and 5) transfer any information gained about the disease to stakeholders groups. The previous reporting period focused on refining methods to utilize standard PCR as a method for detection of the disease (goal 1). Efforts over this reporting period focused on improving the detection method (modification of goal 1). The improved method facilitated determination of the genetic diversity and distribution of X. fastidiosa strains in oak (goal 3) and alternate host species (goal 2) throughout the northeastern US. PARTICIPANTS: Dr. Donald Kobayashi (PI) had responsibility for managing all scientific and technical aspects of project. Dr. Ann Gould (co-PI) had responsibility for extension and outreach components of this project. Dr. Peter Oudemans is a collaborator for this project and is responsible for geographically mapping the location of oak samples collected throughout the state of NJ and maintaining the GIS database in which the information is stored. His group continues to be involved in scouting the greater New Jersey area for potential appearance of blueberry leaf scorch, an emerging disease caused by X. fastidiosa subsp. multiplex. Dr. Nrupali Patel is a collaborating Associate Research Scientist who is involved in refining technical aspects of the project, and oversees analysis of big leaf maple samples and testing of blueberry samples. Mr. Gregory Behringer is currently a PhD candidate in Plant Biology who processes and analyzes oak leaf scorch disease samples and is currently involved with genetic and genomic diversity of strains in the northeastern and mid-Atlantic United States. TARGET AUDIENCES: Extension presentations were directed at industry stakeholder groups, including members of the turf, ornamental and tree industries. Other interest groups include Master Gardeners of Mercer and Somerset counties of New Jersey, and other extension specialists and county agents within the greater New Jersey area. Presentations at professional meetings were directed at fellow plant pathologists. Undergraduate student population in the course General Plant Pathology are typically comprised of 2nd to 4th year students in both science and non-science majors. First year graduate students comprise the population in Seminar in Microbial Biology and Introduction to Plant Biology within the graduate programs in Microbial Biology and Plant Biology, respectively. Principles of Plant Pathology is comprised of both first and second year graduate students in Plant Biology, as well as other related programs such as Microbial Biology, and Ecology and Evolution. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We improved our standard PCR protocol for Xylella detection by developing a new method for consistent extraction of high quality Xylella DNA from plant tissue. This improved method allowed for genetic characterization of X. fastidiosa strains through multilocus sequence analysis (MLSA). Using nine different primer sets, we performed MLSA on a subset of 50 samples collected from oak trees displaying leaf scorch symptoms throughout the northeastern and mid-Atlantic states. Phylogenetic analysis based on these sequences verified that strains from these areas belong to the X. fastidiosa subsp. multiplex, and are closely related to strains originating in the southern United States. Surveying of potential alternative plant hosts was initiated in central New Jersey. Samples were collected from various plant species displaying leaf scorch symptoms in areas known to harbor oak leaf scorch. Among potential alternative hosts, Japanese knotweed was confirmed positive for the presence of X. fastidiosa. A strain of X. fastidiosa subsp. multiplex was successfully cultured from a diseased oak in central New Jersey, and a draft of the genome sequence of this strain was generated. Analysis of the genome is ongoing. Detection of X. fastidiosa in Big Leaf maple leaves collected in western states was continued using methods similar to those used of oak leaf scorch. Putative positive results have been obtained, but results have been inconsistent.
Publications
- Behringer, G., Gould, A.B., Kobayashi, D. 2012. Characterizing Xylella fastidiosa subsp. multiplex in symptomatic northeastern and mid-Atlantic oak trees. Phytopathology 102:S4.11.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Project goals: 1) develop a real time PCR protocol that is a reliable and sensitive method for Xylella fastidiosa detection; 2) identify insect vectors and potential alternative plant hosts that serve as reservoirs for spread of the pathogen; 3) better understand the distribution of the pathogen throughout New Jersey and the northeastern US region; 4) develop and test possible methods for control of disease caused by X. fastidiosa; and 5) transfer any gained about the disease to stakeholders groups. In the previous reporting period, a real time PCR protocol was successfully developed to detect X. fastidiosa in oak leaf tissue, providing a reliable sensitive method for pathogen detection in infected oak (goal 1). The focus of the project over the past year was to refine methods to utilize standard PCR, improving upon supply costs and technical difficulties. To better understand the distribution of X. fastidiosa (goal 3), leaf and branch samples from oak and other hardwood trees were collected throughout the greater NJ area during May through Oct of 2011, and processed for detection of the presence of the bacterium using standard and real time PCR protocols. Attempts to culture the bacterium were conducted each time fresh leaf samples were collected. Cumulative findings of the project were reported in outreach presentations at stakeholder group meetings: 3/8/2011 Central Jersey Turf and Ornamentals Institute: Bacterial Leaf Scorch 3/9/2011 Somerset Master Gardeners: Ornamental Diseases of Invasive or Regulatory Significance 3/17/2011 New Jersey Chapter: ISA: Two Diseases of Invasive Significance 3/29/2011 DelMarVA-NJ Conference: BLS Update 4/9/2011 Certified Tree Experts: Tree Diseases 11/5/2011 Mercer County Master Gardeners: Ornamental Diseases Findings were also reported as part of a lecture in the Rutgers University undergraduate course: 11:776:302 General Plant Pathology: "Vascular diseases" PARTICIPANTS: Dr. Ann Gould (PI) had responsibility for extension and outreach components of this project, and facilitated sample collection throughout the northeastern US. Dr. Donald Kobayashi (co-PI) had responsibility for managing all scientific and technical aspects of project. Dr. Peter Oudemans is a collaborator for this project and is responsible for geographically mapping the location of oak samples collected throughout the state of NJ and maintaining the GIS database in which the information is stored. His group is also scouting the greater New Jersey area for potential appearance of blueberry leaf scorch, an emerging disease caused by X. fastidiosa. Dr. Melinda Moy seved an hourly consultant for conducting real time PCR. Mr. Gregory Behringer is currently a graduate student in Plant Biology who processed and analyzed disease samples. He is now involved in working with oak leaf scorch as part of his PhD thesis project. Ms. Lorabeth West is an hourly helper assisting with technical aspects of the project. TARGET AUDIENCES: Extension presentations were directed at industry stakeholder groups, including members of the turf, ornamental and tree industries. Other interest groups include Master Gardeners of Mercer and Somerset counties of New Jersey, and other extension specialists and county agents within the greater New Jersey area. Undergraduate student population in the course General Plant Pathology are typically comprised of 2nd to 4th year students in both science and non-science majors. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
In the past, detection of X. fastidiosa in oak was only possible after symptoms had developed during the month of August. During the last reporting period, the presence of X. fastidiosa was detected in leaf tissue as early as two months before disease symptoms appeared. This achievement, combined with the ability to detect X. fastidiosa in woody tissue, has provided the capability to survey trees not only before symptom development, but even before leaf development. This capability will help to better understand spread of the pathogen within and between trees. The refined standard PCR protocol has allowed amplification of ITS regions from oak samples testing positive for scorch disease over past 2 years. Amplified DNA regions have been sequenced and analyzed, verifying: 1) all samples previously designated positive for Xylella infection; and 2) stored extraction samples contain sufficient DNA for use in additional studies, including Xylella strain typing and phylogenetic analysis. The scope of this study was broadened by successful detection of X. fastidiosa in Big Leaf maple leaves collected in northern California, and by the successful culturing of the bacterium from oak samples obtained from central New Jersey locations. This latter success has led to the isolation of chromosomal DNA and construction a DNA library needed for genome sequencing. The combined achievements over the past year are now providing the tools needed to develop a better understanding of the epidemiology of oak leaf scorch within and beyond the state of New Jersey. Such information is necessary to evaluate distribution of the disease and spread to other hardwood species, especially as it pertains to global warming.
Publications
- Zhang, J., Lashomb, J., Gould, A., and Hamilton, G. 2011. Cicadomorpha insects associated with bacterial leaf scorch infected oak in Central New Jersey. Environ. Entomol. 40(5): 1131-1143.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Specific goals of this project are to: 1) develop a real time PCR protocol that is a reliable and sensitive method for Xylella fastidiosa detection; 2) identify insect vectors and potential alternative plant hosts that serve as reservoirs for spread of the pathogen; 3) better understand the distribution of the pathogen throughout New Jersey and the northeastern US region; 4) develop and test possible methods for control of disease caused by X. fastidiosa; and 5) transfer any knowledge gained about the disease to stakeholders groups. Advancement of our understanding of oak leaf scorch disease has been slowed by two major impediments. The first has been the inability to effectively detect the pathogen in infected oak trees. The second has been the difficulty of growing the oak leaf scorch strain of X. fastidiosa in culture. Efforts regarding this project over the past year have focused on resolving these issues. In an effort to address the pathogen detection problem, a protocol using real time PCR with ITS primers specific for Xylella fastidiosa was refined and used to evaluate a collection oak samples obtained over the previous 2 years from various locations throughout the northeastern US. Results from this study were used to complete a database that also contained disease symptom information, geographical location of sample collection and date of collection. The same PCR protocol was also used to test for the presence of X. fastidiosa in maple and elm tree samples displaying symptoms of bacterial leaf scorch. Despite the refinement of the PCR protocol, detection of X. fastidiosa in oak and related tree species has remained restricted to leaf samples displaying disease symptoms. We have been evaluating different protocols for extracting X. fastidiosa DNA from woody tissue for PCR detection. Optimization of such a protocol will allow testing for the presence of the pathogen in suspected infected tissue prior to symptom development. We also have been working to develop a new culture medium to improve growth of the oak leaf scorch strain in culture. The project involved part time efforts of a PhD level scientist, an incoming first year graduate student and summer assistance from a rising senior undergraduate student. PARTICIPANTS: Dr. Ann Gould (PI) had responsibility in overseeing the logistics of sample collection. Dr. Donald Kobayashi (co-PI) had responsibility of overseeing all technical aspects of project, including disease sample processing, and the optimization and utilization of real time PCR. Dr. Peter Oudemans is a collaborator for this project and is responsible for geographically mapping the location of oak samples collected throughout the state of NJ and maintaining the GIS database in which the information is stored. Dr. Melinda Moy is an hourly consultant who refined the real time PCR protocol. Mr. Gregory Behringer is currently a graduate student and a former hourly worker for the project who assisted Dr. Moy in processing and analyzing disease samples. His overall experience with the project confirmed his interest in pursuing a graduate degree in Plant Biology, and is now continuing his work with oak leaf scorch disease and Xylella fastidiosa as part of his thesis project. Mr. Mario Cornejo, was an undergraduate student who assisted Mr. Behringer with processing disease samples. He obtained valuable laboratory research experience while working on the project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Positive results for X. fastidiosa using real time PCR were obtained for all samples showing oak leaf scorch symptoms. These included samples that previously tested positive, as well as some that tested negative, using ELISA as a detection method. These findings verify that the real time PCR protocol is much more sensitive as a pathogen detection method for this disease. In addition to New Jersey samples, others that tested positive for the disease ranged from southern states such as Virginia, to those from northern states such as Maine, Vermont, New Hampshire, and Michigan, and as far west as California. Culturing the oak leaf scorch strain from infected tree tissue freshly obtained in New Jersey has not yet been successful, although we are continuing to investigate different modifications of growth medium. Detection of X. fastidiosa from woody tissue has been more successful. Use of a pressure cycler (barocycler, Pressure Biosciences Inc.) has allowed for the effective extraction of nucleic acids from woody tissue that test positive for X. fastidiosa using the real time PCR protocol. The development of this protocol will now allow the sampling of woody tissue prior to development leaf symptoms from trees known to be infected with X. fastidiosa. Such sampling can provide valuable data regarding growth and movement of the pathogen within trees and pathogen population densities in different oak tissues prior to symptom production in leaves. This project provided general laboratory experience and molecular and microbiological skills for an incoming graduate student, who has continued to work on this project as part of his graduate thesis. It has also provided valuable experience for an undergraduate student who wishes to continue graduate studies after completing his undergraduate degree requirements.
Publications
- No publications reported this period
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