Source: CORNELL UNIVERSITY submitted to
DEVELOPMENT, IMPLEMENTATION, AND OUTREACH FOR EMERGING TECHNOLOGIES OF PLANT PATHOGEN DETECTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0216629
Grant No.
2009-55605-05184
Project No.
NYC-153570
Proposal No.
2008-04222
Multistate No.
(N/A)
Program Code
20.2
Project Start Date
Jan 1, 2009
Project End Date
Dec 31, 2013
Grant Year
2009
Project Director
Perry, K. L.
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
PLANT PATHOLOGY
Non Technical Summary
The long-term goal of the project is to develop and implement the use of highly sensitive and rapid plant pathogen detection technologies. Integral to the project are education and extension outreach activities to inform the public about the importance to our food supply of disease monitoring and control practices. One objective is to refine and implement the use of an array for the detection of pathogens of potato, tomato, pepper and eggplant. The ten cooperators represent governmental, industry, and academic institutions, including the National Plant Diagnostic Network. Another objective is to develop a rapid and ultrasensitive nanomechanical resonator system for the diagnostic detection of plant pathogens; the proof of principle will be established for the detection of high consequence pathogens including Xylella fastidiosa, and Phytophthora capsici. A third objective is to broaden extension and education efforts to K-12 students, undergraduates, and growers about agricultural pests and the future use of nanobiotechnology to monitor and detect pathogens. The methods will primarily focus on DNA-based technologies. The outcomes/impacts will include an improvement in the detection of plant pathogens and an increase science literacy and citizenship in grades three and four.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121499116025%
2124099116025%
2127299201025%
2127299116025%
Goals / Objectives
The long-term goal of the project is to develop and implement the use of highly sensitive, rapid and accurate plant pathogen detection technologies. The objectives of the project are: 1) To refine and implement the use of an array for the detection of pathogens of solanaceous crops in cooperating labs and diagnostic clinics across North America, 2) To develop a rapid and ultrasensitive nanomechanical resonator array system for the diagnostic detection of plant pathogens; the proof of principle will be established for the detection of high consequence pathogens including Xylella fastidiosa, and Phytophthora capsici, and 3) To broaden extension and education efforts to K-12 students, undergraduates, and growers about agricultural pests and the future use of nanobiotechnology to monitor and detect pathogens. The outputs of the project will be: a) experiments to refine a macroarray for pathogen detection, b) experiments to improve array sensitivity, c) experiments to test the efficacy of detection for soil and water samples, d) distribution to and testing of the array in cooperating laboratories, e) cooperation for disease testing of National Plant Diagnostic Network samples, f) experiments to optimize a nanomechanical resonator, g) testing of mass labeling in detection with the nanomechanical resonator, h) nanotechnology-based lesson integration into K-12 eductional programs, i) undergraduate and graduate education on plant biosecurity and nanobiotechnology for plant pathogen detection through coursework and summer research, and j) research training in a Summer Research Experience for Undergraduates Program.
Project Methods
The project will be conducted through two key experimental approaches. The first is to perform hybridization analyses for the detection of plant pathogens. A membrane based macroarray will be employed the oligonucleotide probes for specific pathogens. A nanobiotechnological approach for pathogen detection will involve the use on nanomechanical resonators. Results will be analyzed by quantifying the extent of hyrbridization of nucleic acids from plant and environmental samples. Efforts will include a delivery of science-based knowledge to K-12 students through innovative curricula and in class presentations. An undergraduate training program in research will be implemented. Outputs will be evaluated through a comparison of methods and experimental sensitivities in pathogen detection. Science literacy and citizenship in student grades three to four will be increased.

Progress 01/01/09 to 12/31/13

Outputs
Target Audience: Plant disease diagnosticians, Growers, Extension educators and other extension personnel, Teachers, Industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provided training for 7 graduate students and 21 undergraduates. How have the results been disseminated to communities of interest? Every year, results from the project were presented to industry, academic and grower group representatives at the annual meeting of the American Phytopathological Society. Presentations to grower groups were made periodically over the four year course of the project. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Virus detection using the solanaceous virus macroarray; pathogen detection using the solanaceous fungal/oomycete macroarray in soil and in irrigation water. The array for the detection of viruses was refined and utilized over the course of the project. The current version of the virus macroarray has ~1000 probes for the detection of approximately 125 viruses. Additional probes for the detection of 16 viruses described in the past four years have been designed. Improvements in the array include internal control probes for host plant housekeeping genes, internal RNA controls for all steps from sample extraction and cDNA synthesis to virus detection, and a labeled internal control for hybridization and probe detection. The utility of the array has been demonstrated through the detection of newly reported pathogens: Spinach latent virus in tomato, a new member of the genus Ilarvirus in tomato, a chimeric Tospovirus in tomato new to North America. A proof of principle for the development of crop specific arrays was accomplished with the development and deployment of a macroarray able to detect all recognized viruses present in grapevine. Over the course of the project, the Smart lab expanded the use of the macroarray to identify pathogens in soil. The array has probes for the detection of 43 fungal and oomycete pathogens of solanaceous crops, including 12 members of the Fusarium solani species complex. We are now able to use the macroarray using both a chemiluminescent and a chromogenic assay (which requires x-ray film and film developing facilities). This work was done by a visiting scientist who brought the technology back to her lab, and the arrays are now being used in Malaysia. The array is working well to detect pathogens in the rhizosphere, and we have submitted a publication on the utility of the array compared with a more traditional tRFLP method. While the array was more sensitive to detect pathogens, the tRFLP could identify a broader range of microbes (as would be expected). Objective 2: Phytophthora capsici and other pathogen detection in irrigation water, decontamination strategies to remove pathogens from the water; detecting airborne sporangia prior to disease development Irrigation water is known to be a source of plant pathogens. We have tested 20 surface irrigation sources from NY for the presence pathogens, and have identified both human and plant pathogens. We have found that the presence of human pathogens including E. coli and Salmonella are associated with rain events, while the presence of plant pathogens (predominantly oomycetes) is quite constant. That is, the oomycetes are always present. There was a correlation between the type of irrigation source and the number of oomycetes present, with irrigation ponds having fewer oomycetes than creeks or streams. The manuscript discussing this work is currently in preparation and will be submitted to Applied and Environmental Microbiology in February. During the course of the irrigation water survey, we realized that the majority of irrigation sources in New York are very turbid. Because of this turbidity, many sanitation practices (such as chlorination) are not effective. We tested UV light as an appropriate sanitation method for surface irrigation water using a system that was designed to sterilize turbid apple cider. The UV system was effective in removing 99.9% of human and plant pathogens from irrigation water. This work is published ahead of print in Applied and Environmental Microbiology (doi:10.1128/AEM.02964-13) and will appear in print in early 2014. We have developed methods for detection of Pseudoperonospora cubensis airborne sporangia using a roto-rod air sampling system. Spore traps are set out in or near cucumber fields and rods are collected twice weekly. DNA is extracted from the rods and used in a real-time PCR system to detect the pathogen. The system is still being validated, but we expect to publish the detection method in 2014. Objective 3: Broadening extension and education efforts to K-12 students, undergraduates, and growers about agricultural pests To address the training of students in K-12, over a four year period, elementary students have been involved in collecting soil samples to utilize in array experiments to determine the pathogens that are present in soil, to learn about pathogens in soil, and learning how implementing cultural controls will reduce the risk of disease in the garden at their school. Two hundred students in the fall and 200 students in the spring participate. A 4th grade science camp allowed students to be educated about agricultural pests, covering pathogens (both plant pathogens and food-borne human pathogens), and how pathogens can be detected; approximately 15 students per year attend the camp. Undergraduates were trained in the diagnostics of plant diseases and pathogens through summer internships and academic year laboratory research experience. Twelve undergraduates attended 8 to 10 week training programs providing laboratory training. Additionally, nine undergraduates received research training during the academic year.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Jones, L.A., Worobo, R.W. and Smart, C.D. (2014) Ultraviolet light inactivation of human and plant pathogens in unfiltered surface irrigation water. Applied and Environmental Microbiology in press.
  • Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Summers, C.F., Park, S. Dunn, A.R., Rong, X., Everts, K.L., Kleinhenz, M.D., McSpadden Gardener, B., and Smart, C.D. (2014) Fungal and oomycete pathogen detection in the rhizosphere of organic tomatoes grown in cover crop-treated soils. Applied Soil Ecology submitted
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Jones, L.A., Worobo, R.W., and Smart, C.D. (2013) Ultraviolet treatment of surface irrigation water for improved plant health and food safety. Phytopathology 103:S2.69
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Summers, C.F. and Smart, C.D. (2013) Fungal and oomycete pathogen detection in the rhizosphere of organic tomatoes grown in cover crop treated soil. Phytopathology 103:S2.141
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Jones, L.A., Worobo, R.W., and Smart, C.D. (2013) Pathogens in Surface Irrigation Water. 2013 Proceedings: 58th New Jersey Agricultural Convention and Trade Show. Atlantic City, NJ (p. 114-116).
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Thompson, J., Fuchs, M., McLane, H., Toprak-Celebi, F., Fisher, K., Potter, J., and Perry, K. L. 2014. Multiplex detection of grapevine viruses using a randomly primed RT-PCR/Macroarray platform. Phytopathology (accepted for publication 20 August, 2013).
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Lin, Y-H, Abad, J., Maroon-Lango, C. J., Perry, K. L., and Pappu, H.R. 2014. Molecular characterization of domestic and exotic Potato virus S (PVS) isolates and a global analysis of the PVS genomic sequences. Archives of Virology (accepted for publication 4 September 2013).
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Vargas, J. A., Bush, E., and Perry, K. L., 2013. Spinach latent virus infecting tomato in Virginia, USA. Plant Disease 97:1663. http://dx.doi.org/10.1094/PDIS-05-13-0529-PDN
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Celebi-Toprak, F., Thompson, J., Perry, K. L., and Fuchs, M., 2013. Arabis mosaic virus in grapevines in New York State. Plant Disease 97:849.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Thompson, J., Fuchs, M., McLane, H., Toprak-Celebi, F., Potter, J., Vargas, J. A., and Perry, K. L., 2013. A routine crop-specific diagnostic macroarray for profiling viral infections in grapevine. Phytopathology 103, 145.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Smart, C.D., Hansen, Z.R. and Tancos, M.A. (2013) Detecting downy mildew spores before symptoms develop. Proceedings of the 2013 York Fruit and Vegetable Expo, Syracuse, NY. On-line at http://www.hort.cornell.edu/expo/proceedings/2013/Vine%20Crops/Vine%20Crops%20Smart%20Downy%20Mildew.pdf


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: We continue to expand the use of the macroarray to identify pathogens in soil. In a field study that examined the impacts of a single-season application of cover crops on pathogen populations in the tomato crop rhizosphere, the solanaceous fungus/oomycete macroarray and T-RFLP analyses were employed. We were able to detect multiple plant pathogens from the rhizosphere of tomato plants using the macroarray, an approach that has not previously been used for such a field study. We are now able to use the macroarray using a chromogenic assay rather than a chemiluminescent assay. Three visiting scientists were trained on the use of macroarrays for plant disease diagnostics, and these scientists returned to their institutions in Malaysia, Denizli Turkey, and Houghton NY. A total of four undergraduates were trained in plant disease diagnostic methods during this past year. Two presentations relevant to the virus macroarray work were made and published. A surface water pathogen survey was conducted in over thirty actively used surface water irrigation reservoirs in New York throughout over two growing seasons of 2010 and 2011 and analyzed in 2012. Over 1000 individual oomycete isolates have been collected and characterized from the survey. Water quality parameters, pH and turbidity, were recorded for each sample. These parameters dictate which method of water treatment can be used effectively. An ultraviolet (UV) processing unit developed for food safety applications could be a solution for contaminated surface irrigation water. The processing unit has shown promising results and is continuing to be evaluated for irrigation applications. Decontamination studies with zoospores of the plant pathogen Phytophthora capsici show promising results. We are developing methods to detect sporangia of two serious airborne pathogens, Phytophthora infestans (causes late blight of tomato and potato), and Pseudoperonospora cubensis (causes cucurbit downy mildew), prior to a disease outbreak. To accomplish this goal, we have used solar-powered spore traps with two vertical spinning rods that can sample 62 liters of air/minute, to collect wind dispersed spores within an area (see photo below). Rather than staining, counting and differentiating spores based on morphology, we used the spore trap for total DNA extraction and polymerase chain reaction (PCR) assays. Using spore traps combined with PCR and we were able to detect airborne inoculum of both pathogens before symptoms appeared. The K-12 (4th grade) training includes sections covering pathogens (both plant pathogens and food-borne human pathogens), and how pathogens can be detected. In total, Dr. Smart spent just over 1,000 contact hours per year working in K-12 outreach. PARTICIPANTS: The following are currently part of the investigative team: Dr. Keith Perry, Principal Investigator; Dr. Christine Smart, Co-Principal Investigator; Dr. Harvey Hoch, Co-Principal Investigator; Dr. Harold Craighead, Co-Principal Investigator; Dr. Jeremy Thompson, research associate, virus macroarray research; Mr. Jose Vargas, virus macroarray research; Ms. Heather McLane, technician, virus macroarray testing; Mr. Thomas Kartika, undergraduate researcher, methods development; Ms. Carly Summers, graduate student, array development and diagnostics; and Ms. Lisa Jones, graduate student, array development and diagnostics. TARGET AUDIENCES: Plant disease diagnosticians, Growers, Extension educators and other extension personnel, Teachers, Industry . PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Two new findings were observed in tomato using the solanaceous plant virus macroarray. The viruses detected were spinach latent virus (genus Ilarvirus) in Virginia crops, and an undescribed Ilarvirus from Pennsylvania that most closely resembles tomato necrotic spot virus. A grapevine specific macroarray was designed and printed that contained a total of 1578 virus-specific 60-70mer oligonucleotide probes pertaining to 38 of the most important grapevine viruses. Using this array, two previously unreported viruses were shown to be established in field planted vines, arabis mosaic virus and grapevine virus E (GVE). Vines of the native Concord vines were shown to be commonly co-infected by grapevine leafroll associated virus-3 and GVE. We are now able to use the macroarray using a chromogenic assay rather than a chemiluminescent assay. Surface irrigation water survey results indicate that there is an exposure risk of crops to both human and plant pathogens through irrigation water. E. coli and Salmonella species were found frequently throughout surface irrigation water surveys in 30% and 38% of the samples, respectively. Additionally, twenty-eight species of oomycetes have been identified, many of these species can be pathogenic to plants.

Publications

  • Jones, L.A., Worobo, R.W. and Smart, C.D. 2012. Oomycete and bacterial pathogens in New York surface irrigation water: Survey results and ultraviolet treatment. Phytopathology 102:S60
  • Jones LA, Worobo RW, and Smart CD. 2012. Ultraviolet Treatment of Pathogens in Surface Irrigation Water. Abstracts of the Human Pathogens on Plants Workshop p17-18.
  • Krenz, B., Thompson, J., Fuchs, M., and Perry, K. L. 2012. Complete Genome Sequence of a New Circular DNA Virus from Grapevine. Proceedings of the 17th Congress of the international council for the study of virus and virus-like diseases of the grapevine (ICGV), October 7-11, Davis, CA.
  • Krenz, B., Thompson, J., Fuchs, M., and Perry, K. L. 2012b. Complete Genome Sequence of a New Circular DNA Virus from Grapevine. Journal of Virology 86:7715.
  • Smart, C.D. and Davis, R.M. 2012. Diagnostic methods for identifying tomato diseases. In Tomato Health Management. R.M. Davis, K. Pernezny, and J.C. Broome (eds) APS Press St. Paul MN pp. 135-144.
  • Summers, C.F., Smart, C.D., McSpadden Gardener, B.B., Everts, K.L., Dunn, A.R. and Park, S. 2012. The impact of mixed-species cover crops on rhizosphere pathogens of organically managed tomato crops in New York, Ohio, and Maryland. Phytopathology 102:S115
  • Tancos, M.A., Small, I.M., Fry, W.E., and Smart, C.D. 2012. Early detection of airborne inoculum from wind-disseminated oomycetes. Phytopathology 102:S118
  • Thompson, J., Fuchs, M., Fisher, K., and Perry, K. L. 2012. Multiplex detection of grapevine viruses using a randomly primed RT-PCR/Macroarray platform. Proceedings of the 17th Congress of the international council for the study of virus and virus-like diseases of the grapevine (ICGV), October 7-11, Davis, CA.
  • Thompson, J., M. Fuchs, K. Fisher, and Perry, K. L. 2012b. Macroarray detection of grapevine leafroll associated viruses. Journal of Virological Methods 183:161-169.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: We held several events and outreach efforts where the plant disease diagnostics project was discussed including; 1) a field day at our Phytophthora blight research farm in Geneva where we discussed the importance of early pathogen detection in soil and in the rhizosphere of plants (about 25 breeders and industry partners in attendence; 2) an organic vegetable production/pest control field demonstration where we explained the use of the diagnostic technology developed as part of this grant (a 2 hour session with 12 organic growers); 3) Geneva City School District Summer Science Camp where we involve third and fourth-grade elementary students in collecting soil samples to utilize in array experiments to determine the pathogens that are present in soil. The soil was sampled from the students' school garden and the results of the sampling were discussed with students. Elementary students were able to learn about pathogens in soil, and we worked with them to implement cultural controls that would reduce the risk of disease in the garden at their school; 4) Undergraduate Summer Scholar program, where 3 students in the Smart lab were involved in research projects focused on the detection and identification of pathogens using array-based, PCR, and culture methods; 5) Presentations and written articles for commercial growers, industry representatives and extension educators on vegetable and fruit production (Dunn et al., 2011; Jones et al., 2011; Smart et al., 2011a, 2011b, 2011c). Additional array and PCR training in plant virus diagnostics was provided in 2011 for two undergraduates as summer interns. Presentations on the plant virus diagnostics were made an both national and international meetings (Webster, et. al., 2011a), two of which were devoted to array and deep sequencing approaches to plant pathogen (virus) diagnosis. PARTICIPANTS: Dr. Keith Perry, Principal Investigator; Dr. Christine Smart, Co-Principal Investigator; Dr. Harvey Hoch, Co-Principal Investigator; Dr. Harold Craighead, Co-Principal Investigator; Ms. Heather McClane, technician, array development and diagnostics; Ms. Amara Dunn, graduate student, Phytophthora capsici Ms. Lisa Jones, graduate student, Phytophthora capsici Dr. Jaime Benitez, postdoctoral fellow, array and nanobead detection method development. TARGET AUDIENCES: Plant disease diagnosticians, Growers, Extension educators and other extension personnel, Teachers, Industry PROJECT MODIFICATIONS: For the nanobiotechnological efforts in the project, greater emphasis has been given to magnetic bead purification strategies for pathogen detection than to the development of nanomechanical resonators. This is because sample preparation and the concentration of pathogen targets has proved to be a greater priority and critical need for the project as a whole and the array-based detection component in particular.

Impacts
One goal in 2011 has been to employ the fungal/oomycete macroarray to detect and identify pathogens in the soil and rhizosphere surrounding tomato roots, and in soil from contaminated pepper fields. A limiting factor has been obtaining a robust PCR amplification from soil and irrigation water. To address this issue of sensitivity, antibody-coated magnetic beads have been fabricated to concentrate the pathogen from water samples. The antibody currently being used is specific for the genus Phytophthora, and multiple species of Phytophthora are trapped from irrigation water. Specificity is provided by PCR following the immunocapture, methods that will discriminate Phytophthora capsici. An addition improvement in the use of the fungal/oomycete macroarray has been use of a hromogenic dye for pathogen detection (Wong and Smart, 2011). In work on plant virus diagnostics, the macoarray proved informative for two diagnostic samples submitted. A tomato sample from Florida was shown to harbor a tospovirus new to North America. Originally identified as an isolate of Groundnut ringspot virus (GRSV), the virus was shown to be a previously undescribed reassortant (chimera) of GRSV and Tomato chlorotic spot virus (Webster, et. al., 2011b). Recognition of this new pest prompted efforts to determine its distribution and control the thrips vectors. A second sample was a laboratory isolate of Potato virus x (PVX) from arabidopsis that showed an expanded host range. The array revealed the presence of a second virus Pepper ringspot virus that facilitated the systemic plant infection by PVX (Jaubert, et al., 2011). An additional contribution of the array was the detection of Potato virus M in the widely distributed weedy species Solanum dulcamara (bittersweet nightshade) (Perry and McClane, 2011). This is a potential reservoir for this virus that can impact potato production. A final application of the array was the development of more effective probes for the detection of Potato virus Y. Variability was observed in both the position of probe sequences in the virus genome and in the efficacy of plus- versus minus-sense probes.

Publications

  • Dunn, A.R., Smart, C.D., Mazourek, M.M. and Lange, H.W. (2011) Update on Phytophthora Resistant Variety Trials. Proceedings of the 2011 Fruit and Vegetable Expo, Syracuse, NY.
  • Jones, L.A., Worobo, R.W. and Smart, C.D. (2011) Priliminary Results of Irrigation Water Sampling. Proceedings of the 2011 Fruit and Vegetable Expo, Syracuse, NY.
  • Mello, A. F. S., Olarte, R. A., Gray, S. M. & Perry, K. L. (2011). Transmission Efficiency of Potato virus Y strains PVYO and PVYN-Wi by Five Aphid Species. Plant Disease 95, 1279-1283.
  • Perry, K. L., and McClane, H. (2011). Potato virus M in Bittersweet Nightshade (Solanum dulcamara L.) in New York State. Plant Disease 95:619.
  • Smart, C.D., Dunn, A.R., Jones, L.A. and Lange, H.W. (2011a) Prevention is Key: The best way to control Phytophthora blight is to stop the pathogen from showing up on your farm in the first place. American Vegetable Grower, June 2011 p. 10
  • Smart, C.D., Dunn, A.R., Jones, L.A. and Lange, H.W. (2011b) Management Strategies for Phytophthora Blight. Proceedings of the Mid-Atlantic Fruit and Vegetable Convention (p. 81-82).
  • Smart, C.D. and Dillard, H.R. (2011c) Phytophthora Blight: A New Disease of Snap Beans in NY. Proceedings of the 2011 Fruit and Vegetable Expo, Syracuse, NY.
  • Webster, C. G., Reitz, S. R., Frantz, G., Mellinger, H., Perry, K. L. & Adkins, S. (2011a). A novel M RNA reassortant of Groundnut ringspot virus and Tomato chlorotic spot virus infecting vegetables in Florida. Phytopathology 101, S189.
  • Webster, C. G., Reitz, S. R., Perry, K. L. & Adkins, S. (2011b). A natural M RNA reassortant arising from two species of plant- and insect-infecting bunyaviruses and comparison of its sequence and biological properties to parental species. Virology 413, 216-225.
  • Wong, M., and Smart, C. D. 2012. A new protocol using a chromogenic assay in a plant pathogen DNA macroarray detection system. Plant disease (in press).


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Objective 1 is to refine and implement the use of an array for the detection of pathogens of solanaceous crops. Two arrays designed in the first part of this project were combined to create a multipathogen diagnostic macroarray with probes for viruses and fungi/oomycetes. The effectiveness of the virus detection array has been demonstrated by its use in identifying a new pathogen introduction to North America. The use of the fungi/oomycete macroarray was expanded to identify pathogens in soil and water by implementing soil DNA extraction methods and streamlining the hybridization process. Two cooperating diagnostic laboratories made on-site visits to train in the use of the array. Work is in process to compare the sensitivity of the macroarray with that of a microarray for the detection of plant viruses (Bagewadi et. al., 2011). Objective 2 is to develop a rapid and ultrasensitive nanomechanical resonator array system for the diagnostic detection of plant pathogens with the proof of principle to be established for the detection of high consequence pathogens including Xylella fastidiosa, and Phytophthora capsici. The resonator array system was not conducive to efficient sample processing, therefore a shift has been made to achieve ultrasensitive detection of P. capsici from irrigation water using a magnetic bead capture system and two capture strategies. One strategy uses a monoclonal antibody that binds to most all Phytophthora species. The other is nucleic acid-based and targets fungal/oomycete ribosomal gene sequences. PCR-based strategies are being used to detect prokaryotic plant pathogens in irrigation water. These assays are also being used to determine the efficacy of irrigation water decontamination strategies, including the inactivation of the pathogens using ultraviolet irradiation. Objective 3 is to broaden extension and education efforts to K-12 students, undergraduates, and growers about agricultural pests and the future use of nanobiotechnology to monitor and detect pathogens. Presentations have been made and ongoing discussions continued with growers affected by vegetable diseases caused by water-borne pathogens. Strategies are being developed to modify practices such that pathogen-free irrigation water can be used. Elementary students are being educated about agricultural pests through an elementary school science camp. PARTICIPANTS: The following are part of the investigative team: Dr. Keith Perry, Principal Investigator; Dr. Christine Smart, Co-Principal Investigator; Dr. Harvey Hoch, Co-Principal Investigator; Dr. Harold Craighead, Co-Principal Investigator; Dr. Alexandre Mello, postdoctoral fellow, Dr. Jeremy Thompson, research associate, and Ms. Lisa Jones, graduate student, array development and diagnostics; Dr. Madhukar Varshney and Jaime Benitez, postdoctoral fellows, array and nanomechanical resonator development. The following individuals from cooperating diagnostic labs visited Cornell labs to receive training in the design, fabrication and use of the macroarray: Dr. Chet Sutula, Agdia, Inc.; Dr. Ioannis Tzanetakis, University of Arkansas. TARGET AUDIENCES: Plant disease diagnostians, Growers, Extension educators and other extension personnel, Teachers, Industry PROJECT MODIFICATIONS: Of the three objectives in this project, objective 2 has been modified based on the initial experimental work with the nanomechanical resonator. While this method provides sensitivity, there were logistical problems associated with the processing of field samples. In project years two and three, the technical approach has been shifted to the use of a magnetic bead trapping strategies to concentrate and enhance the detection of the target pathogen P. capsici and other Phytophthora sp.

Impacts
Using the multipathogen detection array, a virus-like disease of tomato in Florida was shown to be due to a newly introduced tospovirus, Groundnut ringspot virus (GRSV) (Perry and Lu, 2010;Webster et al., 2010a, 2010b). Although probes for this virus were not present on the array, the virus was detected with oligonucleotides designed to hybridize with multiple members of the tospovirus group. This demonstrates the power of the array in detecting emerging pathogens. Irrigation water is known to be a source of plant pathogens and implicated in the etiology of some vegetable diseases. Twenty surface irrigation sources on NY vegetable farms have been tested for the presence pathogens using the macroarray, PCR, and traditional baiting methods to recover and culture pathogens. The detected pathogens include P. capsici, Xanthomonas campestris, and Pseudomonas syringae. A more direct and comprehensive assessment of P. capsici has led to an understanding of the population structure and fungicide resistance of this pathogen associated with pepper and other hosts (Camp et al., 2010a). Regarding the extension and educational efforts, elementary students have been involved in collecting soil samples that were utilized in array experiments to determine the pathogens that are present in soil; 200 students the fall and 200 students in the spring participate. The soil was sampled from an elementary school garden plot and the results of the sampling were discussed with students. Elementary students were able to learn about pathogens in soil, and we worked with them to implement cultural controls that would reduce the risk of disease in the garden at their school (Camp et al., 2010b). A new section was added to our 4th grade science camp covering pathogens (both plant pathogens and food-borne human pathogens), and how pathogens can be detected; approximately 15 students per year attend the camp. We also discussed how human pathogens can get on food and how to prevent this contamination. The utility of the macroarray detection system has been explained to multiple grower groups, industry consultants at extension educator meetings, academics at invited university seminars, and national and international scientific meetings. During the 2010 funding period, three undergraduate summer interns from different universities were hosted for a ten-week training program.

Publications

  • Bagewadi, B., Fischer, K., Henderson, D. C., Jordan, R. L., Wang, D., Perry, K. L., Melcher, U., Hammond, J., and Fauquet, C. M. 2010. Virus Fishing with Chips: Plant Virus Microarrays and Next Generation Sequencing. Phytopathology 100:S154.
  • Camp, A.R., Milgroom, M.G., Meitz, J.C., McLeod, A., Fry, W.E., McGrath, M.T., Dillard, H.R. and Smart, C.D. (2010a) Phytophthora capsici in New York State: Resistance to mefenoxam and population structure. Phytopathology 100:S20
  • Camp, A.R., Lange, H.W., Reiners, S. and Smart, C.D. (2010b) Sowing the Seeds of Science. Phytopathology 100:S20
  • Perry, K. L., and Lu, X. 2010. A tospovirus new to North America: Virus detection and discovery through the use of a macroarray for viruses of solanaceous crops. Phytopathology 100:S100.
  • Webster, C., Adkins, S., Perry, K. L., Liu, X., Horsman, L., Frantz, G., and Mellinger, C. (2010a) Groundnut ringspot virus detected in tomato in south Florida. in: University of Florida Institute of Food and Agricultural Sciences Pest Alert 04/01/10. http://entomology.ifas.ufl.edu/pestalert/groundnut_ringspot.htm.
  • Webster, C. G., Perry, K. L., Liu, X., Horsman, L., Frantz, G., Mellinger, C., and Adkins, S. (2010b) First report of Groundnut ringspot virus infecting tomato in south Florida. Plant Management Network. doi:10.1094/PHP-2010-0707-01-BR.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: This project has three objectives: 1) to refine and implement the use of an array for the detection of pathogens of solanaceous crops in cooperating labs and diagnostic clinics across North America, 2) to develop a rapid and ultrasensitive nanomechanical resonator array system for the diagnostic detection of plant pathogens; the proof of principle will be established for the detection of high consequence pathogens including Xylella fastidiosa, and Phytophthora capsici, and 3) to broaden extension and education efforts to K-12 students, undergraduates, and growers about agricultural pests and the future use of nanobiotechnology to monitor and detect pathogens. Two arrays have been combined to create a multipathogen diagnostic array with ~1000 probes for the detection of viral and fungal/oomycete pathogens. These probes have sequences represented in approximately 125 viruses and 43 fungal and oomycete pathogens of solanaceous crops, including 12 members of the Fusarium solani species complex. Improvements in the system include the introduction of three internal controls. The first is a dilution series of spotted rDNA-specific oligonucleotide probes; while in theory this provides information on the combined relative efficiency of the chemical labeling, hybridization and visualization, in practice it is a strong indicator of labeling efficiency. The second is a dilution series of spotted oligonucleotide probes specific to a non-plant RNA virus; an in vitro transcribed RNA is added to the sample RNA prior to reverse transcription, providing an internal control for all steps in the array from reverse transcription onward. The third control is the spotting of oligonucleotides specific to a synthetic RNA; a labeled synthetic DNA standard is added to the hybridization solution immediately prior to hybridization, provides information specifically on the relative efficiency of the hybridization step. In preliminary work for both pathogen detection with both the array and a nanomechanical resonator, an RNA concentration and purification scheme has been developed using paramagnetic particles and oligonucleotide probes to specifically bind, concentrate, pathogen RNAs (or DNAs). The specificity of the probes has been tested against total RNA from healthy and infected plants and using non-specific oligonucleotide probes. Quantification and qualitative studies has been performed using real-time PCR and nylon membrane macroarray techniques. Soil samples have been collected from fields known to be infested with P. capsici. These samples are being tested with the macroarray and real-time PCR. We have done preliminary tests to detect P. capsici, Xanthomonas campestris, and Pseudomonas syringae in irrigation water. Future studies will test multiple field soils and irrigation sources. Additional studies will be performed to attempt to inactivate the pathogens in irrigation water using ultraviolet irradiation. Detection strategies will then be utilized to determine if the water is pathogen-free following irradiation. PARTICIPANTS: Dr. Keith Perry, Principal Investigator; Dr. Christine Smart, Co-Principal Investigator; Dr. Harvey Hoch, Co-Principal Investigator; Dr. Harold Craighead, Co-Principal Investigator; Dr. Alexandre Mello, postdoctoral fellow, array development and diagnostics; Dr. Madhukar Varshney, postdoctoral fellow, array and nanomechanical resonator development. TARGET AUDIENCES: Plant disease diagnostians, Growers, Extension educators and other extension personnel, Teachers, Industry PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The last step in the array protocol is to visualize hybridization. A modification has been introduced so that colorometric detection (rather than chemiluminescent) can be used. This will make the detection method more user-friendly in clinics, other labs and other countries not set up for x-ray film development or with a camera imaging system. Regarding the extension, outreach, and training components of the project, the utility of the macroarray detection system has been explained to multiple grower groups, industry consultants and at ten extension educator meetings. Additionally, it has been presented during invited seminars at three universities, and at one national and one international scientific meetings. We have also included macroarray detection strategies in five hand-outs and fact sheets. The implications of contaminated irrigation water have been discussed with growers that have the problem. We are now working with these growers to identify a strategy to modify practices such that pathogen-free irrigation water can be used. Elementary students have been involved in collecting soil samples to utilize in array experiments to determine the pathogens that are present in soil. The soil was sampled from a school garden plot and the results of the sampling were discussed with students. Elementary students were able to learn about pathogens in soil, and we worked with them to implement cultural controls that would reduce the risk of disease in the garden at their school. Lastly, three summer 2009 undergraduate interns from different universities were hosted for a ten-week training.

Publications

  • Smart, C. D., Lange, H. W., Camp, A. R., Reiners, S., Dinnard, H. R., and Gu, J. (2009) Look out for Phytophthora blight on beans. Proceedings of the 2009 Fruit and Vegetable Expo, Syracuse, NY (page 97).