Progress 07/01/19 to 06/30/23
Outputs
Target Audience:Our target audience comprises the academic scientific community, including graduate and undergraduate students of UGA, UF, and other peer institutions, members of the Region 8 FS-Forest Health and Protection Program, members of State agencies such as the Georgia Forestry Commission or the Florida Forest Service, as well as national and regional forestry industry stakeholders, including members of the UGA Southern Pine Health Research Cooperative and members of the UF Forest Biology Research Cooperative. Changes/Problems:Because of the travel and social distancing limitations imposed by the pandemic, we had to adjust our timeline, especially for the two hands-on training sessions. The first training was postponed to October 2021, and the second training session was postponed to February 2023. Despite the delay, both sessions were eventually successfully completed. What opportunities for training and professional development has the project provided?In addition to the two hands-on workshops previously described (see the accomplishments section, Objective #3), this project has allowed for the training of four undergraduate students and two graduate students. Three of the undergraduate students, Brianna B., Katy D., and Robert C., were at the University of Florida and have been trained in working with fungal cultures, spore count estimation, DNA extraction, PCR techniques, and the use of different spore trap prototypes. The last undergraduate student, Julia L., was at the University of Georgia and was trained in working with fungal cultures and the general concepts of LAMP. Julia L. was also trained in outreach skills and helped with the organization and the delivery of the second hands-on workshop in February 2023. One of the two graduate students, Mr. Meinecke, successfully graduated with an MS in Forestry and Natural Resources at the University of Georgia in December 2021. In addition to his MS classwork, Mr. Meinecke had been trained in working with the pathogen Fusarium circinatum, and in the use of LAMP, including the design of LAMP primers and probes. The student also had the opportunity to travel to South Africa to perform project-related experiments, with the great benefit of exploring different work environments and expanding his professional network. One of the two research chapters in his thesis focused entirely on the design and validation of the LAMP assay for the detection of F. circinatum and he is the first author of the three main products we published/prepared (i.e., the scientific publication, the extension fact sheet, and the operational protocol). Mr. Meinecke was also one of the organizers and instructors of both the hands-on training workshops. The other graduate student, Ms. Bonilla, was a rotating Ph.D. student in the Integrated Plant Sciences program at the University of Georgia. During her 6-week rotation in Dr. Villari's laboratory, Ms. Bonilla was trained in the use of LAMP technology and helped in the organization and delivery of the first hands-on training workshop in October 2021. How have the results been disseminated to communities of interest?Results have been extensively disseminated to communities of interest through the delivery of presentations (see list of products) at many regional, national, and international meetings, through the publication of a manuscript (Meinecke et al. 2023, https://doi.org/10.1094/PDIS-04-22-0972-SR) in Plant Disease, which is one of the journals of the American Phytopathological Society of America, and through the publication of a joint UGA/UF extension fact sheet (Meinecke et al. 2022; WSFNR-22-62A). We also prepared an operational protocol for the preparation of samples and the in-field use of LAMP technology for the detection of the causal agents of pitch canker and brown spot needle blight, which will be publicly shared via the protocol.io platform. To fulfill Objective #3 of the project, we offered two hands-on training workshops in October 2021 and February 2023, respectively, which were attended by a total of 39 participants from five states (AL, FL, GA, NC, and TX) and which comprised university graduate and undergraduate students, postdoctoral associates, USDA FS and state agencies personnel, and employees of diagnostic clinics and private industries. Finally, we were interviewed on the use of LAMP technology by the nationally renewed environmental educator Dr. Nick Fuhrman (known to the public as "Ranger Nick"), and the interview, in addition to being available on YouTube (https://www.youtube.com/watch?v=4WE1CP5x6Cc), was aired three times on television, once on the Georgia Public Broadcasting state network, and twice nationally on RFD-TV, for a combined audience of approximately 2 million. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We successfully developed an affordableLAMP-based protocol for the detection of the pitch canker pathogen that can be used directly at point-of-care. The assay is well suited to facilitate diagnostic and surveillance efforts both in the laboratory and in the field, having the potential to help reduce the spread and impact of the disease worldwide. We also developed a low-cost spore trap system that can be coupled with the LAMP assay and provided two hands-on training opportunities for forest health practitioners, diagnostic clinics, and other stakeholders. • Objective 1 We designed a LAMP primer set in an insertion encoding for five genes putatively involved with pathogenicity, which has been identified as species-specific for F. circinatum in previous genome comparison studies. To test the specificity of the primer set, the graduate student working on the project was hosted by the Forestry and Agricultural Biotechnology Institute at the University of Pretoria, South Africa, to have access to the large Fusarium fungal collection of Dr. Brenda Wingfield. The specificity of the primer set was successfully validated with 45 F. circinatum isolates and other 14 non-target Fusarium species. We also compared the LAMP primer binding site sequences against all available genomes of F. circinatum and closely related Fusarium species. The in silico comparisons support earlier wet-lab testing and indicate that the primers should detect all regional varieties of F. circinatum and should not detect non-target Fusarium species, except for a closely related new Fusarium species, F. pilosicola, which has been recently described. The specificity of the primer set was also successfully tested both on infected and healthy pine tissues, to make sure the assay is neither inhibited by nor cross-reacts with the host plant. We then proceeded with establishing the sensitivity of the assay. We successfully determined the limit of detection in terms of picograms of F. circinatum DNA per reaction volume and the number of F. circinatum spores per reaction. Under standard reaction conditions, the minimum amount of DNA that could be detected is 100 pg per 25 uL reaction volume, which is comparable to the sensitivity of a commonly used qPCR method specific to the pathogen, while the minimum detectable number of F. circinatum spores per reaction volume is 10. Finally, we demonstrated the compatibility of the LAMP assay with a simplified DNA extraction method that eliminates the need for pipettes. This method uses simple mechanical lysis by steel beads, filter paper dipsticks, and a series of pre-prepared buffers to capture host DNA, wash it, and directly transfer it to reaction vessels. All results regarding Objective 1 of the project have been published in a peer-reviewed article in a scientific journal (Meinecke et al. 2023, https://doi.org/10.1094/PDIS-04-22-0972-SR). • Objective 2 One of the objectives of this project was to minimize the number of steps for DNA extraction for on-site testing. We needed a material for building the spore traps that was optimal for the hydrophobicity and electrostatic properties of F. circinatum spores and we were able to identify that a synthetic membrane used for sealing culture plates (Breathe Easy ®, Sigma-Aldrich) commonly fulfilled our needs. The use of the membrane for our scope involves placing the membrane directly into a centrifuge tube and performing a rapid DNA extraction (X-Tract-N-Amp, Sigma-Aldrich) as per the manufacturer's instructions. The advantage of using this extraction protocol is that it requires less time and fewer steps compared to other standard DNA extraction protocols. We then verified that samples can be amplified by PCR using F. circinatum-specific primers, indicating this as a suitable material for direct DNA extraction and amplification. The membranes are also transparent, which allows visualization through light or electron microscopy. We also tested the minimum amount of spores on the membrane that could be successfully extracted and amplified using PCR, and found that we could detect as low as 10 spores with this method, which would be a satisfactory threshold for field applications. Next, we developed 3D-printed "cassettes" to replace the glass microscope slides, as these are fragile and may shatter, resulting in sample loss and potential injuries. We tested these cassettes in the field using the synthetic membranes by placing four traps on two different sites in Alachua County, Florida. Cassettes were replaced weekly for the duration of the test, which was 12 weeks, between June and September 2020. DNA was extracted using the new protocol and half of the volume of each sample was used for real-time PCR whereas the other half was sent to the Villari lab for testing using the LAMP assay. For the conventional PCR tests, we used three different primer sets: one to test whether fungal DNA was present on the membranes, one to test whether Fusarium sp. DNA was present, and one to test whether F. circinatum DNA was present. Results showed that we were able to detect fungal DNA from the spore trap membranes and hence fungal material from the environment was being retained by the traps. Increased specificity using Fusarium-specific primers, as well as F. circinatum-specific primers also showed that the membranes were able to capture Fusarium species in some traps and F. circinatum in one of the traps tested. This suggests that while F. circinatum was detected in at least one trap, there were possibly low amounts of F. circinatum in the environment to be retained in the membranes and detected in sufficient quantities by the conventional PCR test. Testing of the DNA with the LAMP assay also resulted in all negative results, confirming that when/if present, the DNA of F. circinatum was below the limit of detection for the assay. These results are currently being drafted in a manuscript that will be submitted for publication in a scientific journal in the coming months. In the last two years, the original design of the 3D printed cassettes for the traps was improved even further and Printing codes for the best-performing 3D printed cassettes will be made available by including them in an outreach publication that is currently being drafted. • Objective 3 We successfully provided two hands-on training opportunities to forest health practitioners and diagnostic clinics for the implementation of the integrated detection system. The first hands-on training workshop was held in October 2021, at the Austin Cary Learning Center in Gainesville, FL. Sixteen attendees were present at the workshop, including students and postdocs from the University of Georgia and the University of Florida and industry professionals from Rayonier, Inc. The second hands-on training workshop was held in February 2023 at the Warnell School of Forestry and Natural Resources Flinchum's Phoenix in Athens, GA. The event had 23 attendees including students and postdocs from the University of Georgia, the University of Florida, and Auburn University, and employees of the USDA Forest Service, the Georgia Forestry Commission, the Texas A&M Forest Service, and Bartlett Tree Experts. Both training courses were two-day events and had a similar format that included direct lectures, a field trip, and two hands-on learning activities to train attendants about building low-cost spore traps and how to perform the LAMP assay. After both workshops, we administered a brief questionnaire to obtain feedback from the attendees, who reported being overall satisfied with the learning experience and rated all activities with high grades (4 or 5, where 5 is the highest score). Finally, we published a joint UGA/UF extension fact sheet on pitch canker disease and produced an operational protocol for the preparation of samples and the in-field use of LAMP technology for the detection of the causal agents of pitch canker and brown spot needle blight.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Meinecke, C. D., L. De Vos, N. Yilmaz, E. T. Steenkamp, M. J. Wingfield, B. D. Wingfield, and C. Villari. 2023. A LAMP assay for rapid detection of the pitch canker pathogen Fusarium circinatum. Plant Disease. https://doi.org/10.1094/PDIS-04-22-0972-SR
PDF
- Type:
Other
Status:
Other
Year Published:
2022
Citation:
Meinecke, C. & Villari, C. Improving point-of-care diagnostics for forest tree diseases. Poster presented at the Plant Center at the University of Georgia 2022 Fall Retreat. December 12, 2019. Young Harris, Georgia.
- Type:
Other
Status:
Other
Year Published:
2022
Citation:
Meinecke, C. Villari, C. and Brawner J. New fights with old foes: progress in the understanding and management of two emerging native pine diseases. Seminar at the The Jones Center at Ichauway. November 2022. Newton, Georgia.
- Type:
Other
Status:
Other
Year Published:
2023
Citation:
Smith, J.A. 2023. The Tree Disease Diagnosis Process. Arboriculture Short Course. FL Chapter ISA. Orlando, FL. May 17. 2023
- Type:
Other
Status:
Other
Year Published:
2023
Citation:
Smith, J.A. 2023. Common Southeast U.S. Tree Diseases & How to I.D. Them. Arboriculture Short Course. FL Chapter ISA. Orlando, FL. May 17. 2023
- Type:
Other
Status:
Other
Year Published:
2023
Citation:
Smith, J.A. 2023. Updates on new and emerging disease issues on trees. Arborversity. FL Chapter ISA. Palm Coast, FL. April 5, 2023.
- Type:
Other
Status:
Other
Year Published:
2023
Citation:
Smith, J.A. 2023. You dont need a PhD to diagnose a sick tree. Arborversity. FL Chapter ISA. Clearwater, FL. April 7, 2023
- Type:
Other
Status:
Other
Year Published:
2023
Citation:
Quesada, T. and Smith, J.A. Air monitoring and early detection of airborne pathogens. Workshop presentation at Monitoring forest diseases using spore traps and LAMP, February 2023, Athens, Georgia
- Type:
Other
Status:
Other
Year Published:
2023
Citation:
Villari, C., Meinecke C., and Eshleman R. Field-portable molecular diagnostics using LAMP How does it work? Workshop presentation at Monitoring forest diseases using spore traps and LAMP, February 2023, Athens, Georgia
- Type:
Other
Status:
Other
Year Published:
2023
Citation:
Meinecke C., Quesada, T. and Smith, J.A, and Villari C. Field-ready forest disease diagnostics using LAMP. Workshop presentation at Monitoring forest diseases using spore traps and LAMP, February 2023, Athens, Georgia
|
Progress 07/01/21 to 06/30/22
Outputs
Target Audience:Our target audience comprises the academic scientific community, including graduate and undergraduate students, members of the Region 8 FS-Forest Health and Protection Program, as well as national and regional forestry industry stakeholders, including members of the UGA Southern Pine Health Research Cooperative and members of the UF Forest Biology Research Cooperative. Changes/Problems:Because of the travel and social distancing limitations imposed by the pandemic, we were not able to hold the firsthands-on training session we planned to organize in Fall 2020. The first training was postponed to October 2021,and the second training event will be held in October 2022. What opportunities for training and professional development has the project provided?To date, this project has allowed for the training of two undergraduate students and two graduate students. The undergraduate students, Brianna Benitez and Katy Deitz, both at the University of Florida, have been trained in working with fungal cultures, spore count estimation, DNA extraction, and PCR techniques. In addition, one undergraduate student, Robert Casper, will be starting in the second week of July as part of the University of Florida IFAS Undergraduate Research Internship Program. He will be trained in fungal culture sterile techniques, DNA extraction, PCR and field testing of new spore trap prototypes. One of the two graduate students, Colton Meinecke, successfully graduated with a MS in Forestry and Natural Resources at the University of Georgia in December 2021. In addition to his MS classwork, Mr. Meinecke had been trained on working with the pathogen Fusarium circinatum, and in the use of LAMP, including the design of LAMP primers and probes. The student also had the opportunity to travel to South Africa to perform project-related experiments, with the great benefit of exploring different work environments and expanding his professional network. One of the two research chapters in his thesis focused entirely on the design and validation of the LAMP assay for the detection of F. circinatum and he is the first author of the corresponding manuscript we submitted. The other graduate student, Kayla Bonilla, was a rotating PhD student within the Integrated Plant Sciences program at the University of Georgia. During her 6-week rotation in Dr. Villari's laboratory, Ms. Bonilla was trained in the use of LAMP technology, and together with Mr. Meinecke, they helped in the organization and delivery of the hands-on training workshop in October 2021. How have the results been disseminated to communities of interest?The results obtained so far have been extensively disseminated to communities of interest through the delivery of presentations (see list of products), the submission of a manuscript to a scientific journal, and the production of an extension fact sheet. To fulfill Objective #3 of the project, we also offered a hands-on training workshop in October 2021, which was attended by students of the University of Florida and by forestry industry stakeholders. What do you plan to do during the next reporting period to accomplish the goals?• Field testing of spore traps with new frames and synthetic membranes. Tests comparing the glass slides used in the original spore trap prototype and the 3D-printed cassettes with the adhesive membrane have already started in the current reporting period, but we will further test the newer version and adhesive material, as well as complete the testing of the collected samples with the LAMP assay. • Second workshops for stakeholders and forestry professionals. The second workshop on the use and implementation of this detection system is scheduled for October 2022 and will be held in Athens, Georgia. • Prepare and submit a second peer-reviewed journal manuscript. We plan to prepare and submit a second peer-reviewed journal manuscript on the development and validation of the spore trapping system. • Production of the YouTube tutorial. We will finalize the filming and post-production of the YouTube tutorial for the use of the LAMP technology.
Impacts
What was accomplished under these goals?
We successfully developed an affordable and ready-to-use LAMP-based protocol for the detection of the pitch canker pathogen that can be used directly at point-of-care. The assay is well suited to facilitate diagnostic and surveillance efforts both in the laboratory and in the field, having the potential to help reduce the spread and impact of the disease worldwide. We also developed a low-cost spore trap system that can be coupled with the LAMP assay and provided a hands-on training opportunity for the forestry industry and other stakeholders. •Objective 1 We designed a LAMP primer set in an insertion encoding for five genes putatively involved with pathogenicity, which has been identified as species-specific for F. circinatum in previous genome comparison studies. To test the specificity of the primer set, the graduate student working on the project was hosted by the Forestry and Agricultural Biotechnology Institute at the University of Pretoria, South Africa, to have access to the large Fusarium fungal collection of Dr. Brenda Wingfield. The specificity of the primer set was successfully validated with 45 F. circinatum isolates and other 14 non-target Fusarium species. We also compared the LAMP primer binding site sequences against all available genomes of F. circinatum and closely related Fusarium species. The in silico comparisons support earlier wet-lab testing and indicate that the primers should detect all regional varieties of F. circinatum and should not detect non-target Fusarium species, except for a closely related new Fusarium species, F. pilosicola, which has been recently described. The specificity of the primer set was also successfully tested both on infected and healthy pine tissues, to make sure the assay is neither inhibited by nor cross-reacts with the host plant. We then proceeded with establishing the sensitivity of the assay. We successfully determined the limit of detection in terms of picograms of F. circinatum DNA per reaction volume and the number of F. circinatum spores per reaction. Under standard reaction conditions, we found the minimum amount of DNA that could be detected by this assay is 100 pg per 25 uL reaction volume, which is comparable to the sensitivity of a commonly used qPCR method specific to the pathogen. Similarly, we found the minimum detectable number of F. circinatum spores per reaction volume to be 10. Finally, we demonstrated the compatibility of the LAMP assay with a simplified DNA extraction method that eliminates the need for pipettes. This method uses simple mechanical lysis by steel beads, filter paper dipsticks, and a series of pre-prepared buffers to capture host DNA, wash it, and transfer it to reaction vessels simply. •Objective 2 We initially established a collaboration with a material science engineer, Dr. Andrew, University of Florida, to identify the best material to be used for building the spore traps based on the hydrophobicity and electrostatic properties of F. circinatum spores. However, this collaboration has fallen through due to other obligations on both sides. One of the objectives of this project is to minimize the number of steps for DNA extraction for on-site testing. In this regard, we finalized testing the use of a synthetic membrane (Breathe Easy ®, Sigma-Aldrich) commonly used for sealing culture plates. Tests involved placing the membrane directly into a centrifuge tube and performing a rapid DNA extraction (X-Tract-N-Amp, Sigma-Aldrich) as per the manufacturer's instructions. The advantage of using this extraction protocol is that it requires less time and fewer steps compared to other standard DNA extraction protocols. The samples were then amplified by PCR using F. circinatum-specific primers, indicating this as a suitable material for direct DNA extraction and amplification. The membranes are also transparent, which allows visualization through light or electron microscopy. We then tested the minimum amount of spores on the membrane that could be successfully extracted and amplified using PCR, and found that we can detect as low as 10 spores with this method, which would be a satisfactory threshold for field applications. Next, we developed 3D printed "cassettes" to replace the glass microscope slides, as these are fragile and may shatter, resulting in sample loss and potential injuries. We tested these cassettes in the field using the synthetic membranes by placing four traps on two different sites: Austin Cary Forest and the Millhopper sites, both located in Alachua County, Florida. Cassettes were replaced weekly for the duration of the test, which was 12 weeks, between June and September 2020. DNA was extracted using the new protocol and half of the volume of each sample was used for real-time PCR whereas the other half was sent to the Villari lab for testing using the LAMP assay. For the conventional PCR tests, we used three different primer sets: one to test whether fungal DNA was present on the membranes, one to test whether Fusarium sp. DNA was present, and one to test whether F. circinatum DNA was present. Results showed that we were able to detect fungal DNA from the spore trap membranes and hence fungal material from the environment was being retained by the traps. Increased specificity using Fusarium-specific primers, as well as F. circinatum-specific primers also showed that the membranes were able to capture Fusarium species in some traps and F. circinatum in one of the traps tested. This suggests that while F. circinatum was detected in at least one trap, there were possibly low amounts of F. circinatum in the environment to be retained in the membranes and detected in sufficient quantities by the conventional PCR test. Testing of the DNA with the LAMP assay has not been completed yet. In the past year, we have developed modifications to the 3D printed cassettes through collaboration with Dr. Paul Dominguez-Gutierrez, from the College of Medicine, who has been involved in the design and improvement of the trapping prototypes. He has designed several models that will be tested in the field this summer by undergraduate student Robert Casper, who is funded through the UF/IFAS Undergraduate Research Internship program. He will be also testing the effectiveness of Kapton® tape as another alternative adhesive. These tapes are polyamide films with a silicone adhesive that is alcohol-soluble and could potentially improve DNA extraction efficiency. •Objective 3 We held the first hands-on training workshop in October 2021, at the Austin Cary Learning Center in Gainesville, FL. The workshop was a two-day event that included direct lectures, a field trip, and two hands-on learning activities to train attendants about building low-cost spore traps and how to perform the LAMP assay. Sixteen attendees were present at the workshop, including students and postdocs from the University of Georgia and the University of Florida and industry professionals from Rayonier, Inc. After the workshop, we administered a brief questionnaire to obtain feedback from the attendees. In general, they were interested mainly in learning more about pitch canker disease and the detection of pathogens using LAMP and expressed that the workshop met or exceeded their expectations. Among the topics they enjoyed most were learning about LAMP techniques and pitfalls, the hands-on aspect of the workshop, and the field experience of spore trap setup. Attendees also rated all oral presentations with high grades (4 or 5, where 5 is the highest score). As additional suggestions/feedback, attendees mentioned that some of the terminology used might need to be simplified if attendees include a more general audience but overall mentioned that the workshop was very "useful and practical". Finally, we finalized and submitted an extension publication on pitch canker disease, and have advanced the script for the video tutorial on the use of the LAMP assay.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
Meinecke, C., De Vos, L., Yilmaz, N., Steenkamp, E., Wingfield, M., Wingfield, B., Villari, C. A LAMP assay for rapid detection of the pitch canker
pathogen Fusarium circinatum. Submitted to Plant Disease
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Meinecke, C. (2021). New tools to diagnose ad identify emerging diseases in commercially managed loblolly pine. Masters thesis, University of Georgia.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Villari, C. (2021). Forefront technologies for in-field applications: phenotyping of resistance and diagnostics. Invited Seminar at ProForest - Forest Health and Resilience Fall Seminar Series. November 2021, Gainesville, Florida
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Villari, C., Meinecke, C., & Hamilton, J. (2021). LAMP and the advance of field-portable molecular diagnostics in forest pathology. Invited oral presentation at Plant Health 2021, American Phytopathological Society annual meeting, August 2021, Virtual event.
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Villari, C., Meinecke, C., Mohamed Niyas, A., Bonilla, K. (2021) Field-portable molecular diagnostics using LAMP How does it work? Workshop presentation at Monitoring pitch canker disease using spore traps and LAMP, October 2021, Gainesville, Florida
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Villari C. (2022). Modern diagnostic approaches in forest pathology: needs and research directions. Invited oral presentation at the 33rd Annual Meeting of the Tree Protection Co-Operative Programme (TPCP) and the DSI NRF Center of Excellence in Plant Health Biotechnology (CPHB), May 2022, Pretoria, South Africa
- Type:
Other
Status:
Submitted
Year Published:
2022
Citation:
Meinecke, C., McCarty, E., Quesada, T., Smith, J., Villari, C. Pitch Canker (Fusarium circinatum): A Fungal Disease of Southern Pine Forests. Fact Sheet submitted to the Warnell School of Forestry & Natural Resources,
University of Georgia, Outreach Committee
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Quesada, T., Benitez, B., Smith, K, Dominguez-Gutierrez, P., Misth, J. (2021) Using a low-cost air monitoring system to detect airborne pathogens Workshop presentation at Monitoring pitch canker disease using spore traps and LAMP, October 2021, Gainesville, Florida
|
Progress 07/01/20 to 06/30/21
Outputs
Target Audience:Our target audience comprises the academic scientific community, including graduate and undergraduate students, members of the Region 8 FS-Forest Health and Protection Program, as well as national and regional forestry industry stakeholders, including members of the UGA Southern Pine Health Research Cooperative and members of the UF Forest Biology Research Cooperative. Changes/Problems:Because of closures due to the Covid19 epidemic, the 2020 field trials were delayed by two months, which may have affected our ability to capture and detect F. circinatum from the environment. In 2021, we started the field trials earlier and we anticipate improved detection of F. circinatum. Because of the travel and social distancing limitations imposed by thepandemic, we were also not able to hold the first hands-on training session we planned to organize in Fall 2020. The first training event has been postponed to October 2021, and the second training event will be held in 2022. What opportunities for training and professional development has the project provided?To date, this project has allowed for the training of two undergraduate students and one graduate student. The undergraduate students, Brianna Benitez and Katy Deitz, both at the University of Florida, have been trained on working with fungal cultures, spore count estimation, DNA extraction, and PCR techniques. The graduate student, Colton Meinecke, is pursuing an MS in Forestry and Natural Resources at the University of Georgia, and in addition to his MS classwork, he has been trained on working with the pathogen Fusarium circinatum, and in the use of LAMP, including the design of LAMP primers and probes. The student had also the opportunity to travel to South Africa to perform project-related experiments, with the great benefit of exploring different work environments and expanding his professional network. How have the results been disseminated to communities of interest?The results obtained so far have been extensively disseminated to communities of interest through the delivery of presentations (see list of products). In December 2020, we discussed our progress on this project with an ad-hoc Advisory Board whose members were selected among the major stakeholders. The Advisory Board provided useful feedback which we incorporated into our operations. We are planning on meeting with the Advisory Board again by the end of 2021. What do you plan to do during the next reporting period to accomplish the goals?•Collaboration with material science engineer team.We plan to revisit the selection of the material of the synthetic adhesive membrane, performing tests to evaluate potential materials that would be selective for the fungal spores. •Field testing of spore traps with new frames and synthetic membranes.Tests comparing the glass slides used in the original spore trap prototype and the 3D-printed cassettes with the adhesive membrane have already started in the current reporting period, but we still need to test a second year of collection, as well as complete the testing of the collected samples with the LAMP assay. •Workshops for stakeholders and forestry professionals.The first workshop on the use and implementation of this detection system is scheduled for October 2021, and the second one for May 2022. •Prepare and submit two peer-reviewed journal manuscripts.We plan to prepare and submit two peer-reviewed journal manuscripts, one on the development and validation of the LAMP assay, and one on the development and validation of the spore trapping system. •Production of the YouTube tutorial.We will finalize the filming and post-production of the YouTube tutorial for the use of the LAMP technology. •Publication of a Fact Sheet.We willfinalize and submit the outreach publication on pitch canker, the disease caused by F. circinatum.
Impacts
What was accomplished under these goals?
•Objective 1- Design and validate a LAMP assay to specifically detect F. circinatum. We designed six LAMP primer sets in a 12,000 base pair insertion encoding for five genes putatively involved with pathogenicity, and which has been identified as species-specific for F. circinatum in previous genome comparison studies. After preliminary tests performed with both target and non-target species, we selected one of the six primer sets as the most promising and discarded the other five. Preliminary tests were performed with F. circinatum isolates and other closely related species from the collection of Co-PI Dr. Quesada. To finalize the specificity testing of the selected primer set, the graduate student working on the project was hosted by the Forestry and Agricultural Biotechnology Institute at the University of Pretoria, South Africa, to have access to the large Fusarium fungal collection of Dr. Brenda Wingfield. The specificity of the primer set was successfully validated with 45 F. circinatum isolates and other 14 non-target Fusarium species. We also compared the LAMP primer binding site sequences against all available genomes of F. circinatum and closely related Fusarium species. This collection of genomic data includes genotypes and species for isolates that were not readily available for direct testing. Thein silicocomparisons support earlier wet-lab testing and indicate that the primers should detect all regional varieties of F. circinatum and should not detect non-target Fusarium species, except for a closely related new Fusarium species, F. pilosicola, which has been recently described. The specificity of the primer set was also successfully tested both on infected and healthy pine tissues, to make sure the assay is neither inhibited by nor cross-reacts with the host plant. We then proceeded with establishing the sensitivity of the assay. We successfully determined the limit of detection in terms of picograms of F. circinatum DNA per reaction volume and number of F. circinatum spores per reaction. Under standard reaction conditions, we found the minimum amount of DNA that could be detected by this assay is 100 pg per 25 uL reaction volume. This is an acceptable limit considering the specific target region is present as only a single copy per cell. Similarly, we found the minimum detectable number of F. circinatum spores per reaction volume to be 10. Finally, we demonstrated the compatibility of the LAMP assay with a simplified DNA extraction method that eliminates the need for pipettes. This method uses simple mechanical lysis by steel beads, filter paper dipsticks, and a series of pre-prepared buffers to capture host DNA, wash, and transfer to reaction vessels simply. While it requires more buffers than the X-Tract-N-Amp method we use with the spore trap membranes, the dipstick method may be employed when testing diseased host tissue. •Objective 2- Identify the best low-cost spore trap, sampling strategy and in-field DNA extraction method to be coupled with the LAMP assay.We established a collaboration with a material science engineer, Dr. Jennifer Andrew, University of Florida, to identify the best material to be used for building the spore traps. In particular, we discussed potential tests to assess the hydrophobicity and electrostatic properties of F. circinatum spores, to be able to select a substrate for spore trapping that has the best adhesiveness toward the spores, but that could also be selective for the spores of the target species. One of the objectives of this project is to minimize the number of steps for DNA extraction for on-site testing. In this regard, we tested adhesive paper labels (Avery ® mailing labels) and a synthetic membrane (Breathe Easy ®, Sigma-Aldrich) commonly used for sealing culture plates. Three circles were punched out of each adhesive materialand a spore solution was added on the adhesive side of each circle and allowed to air-dry at room temperature. Each circle was placed into a centrifuge tube and a rapid DNA extraction (X-Tract-N-Amp, Sigma-Aldrich) was directly performed as per the manufacturer's instructions. The advantage of using this extraction protocol is that it requires less time and fewer steps compared to other standard DNA extraction protocols. The samples were then amplified by PCR using F. circinatum specific primers. Results showed product amplification of only the samples extracted from the Breathe Easy® membrane, indicating this as a potentially suitable material for direct DNA extraction and amplification. These membranes are also transparent, which would allow visualization through light or electron microscopy.Amplification was unsuccessful with the Avery® labels. Given the good results obtained with the Breathe-Easy ® membrane, we tested the minimum amount of spores on the membrane that could be successfully extracted and amplified using PCR, and results showed that as low as 10 spores could be detected with this method, which would be a satisfactory threshold for field applications. After successful selection of the synthetic adhesive membrane, we also developed 3D printed "cassettes" to replace the glass microscope slides, as these are fragile and may shatter, resulting in sample loss and potential injuries. We tested these cassettes in the field using the synthetic membranes by placing four traps on two different sites: Austin Cary Forest and the Millhopper sites, both located in Alachua County, Florida. Each trap held two 3D-printed cassette prototypes and an adhesive Breathe-Easy® strip. Cassettes were replaced weekly for the duration of the test, which was 12 weeks, between June and September 2020. DNA was extracted using the new protocol and half of the volume of each sample was used for real-time PCR whereas the other half was sent to the Villari lab for testing using the LAMP assay. For the conventional PCR tests, we used three different primer sets: one totest whether fungal DNA was present on the membranes, one totest whether Fusarium sp. DNA was present, and one to test whether F. circinatum DNA was present. Results showed that we were able to detect fungal DNA from the spore trap membranes with the fungal generic primers, meaning that fungal material from the environment was being retained by the spore traps. Increased specificity using Fusarium-specific primers, as well as F. circinatum specific primersalso showed that the membranes were able to capture Fusarium species in some traps and F. circinatum in one of the traps tested. This suggests that while F. circinatum was detected in at least one trap, there were possibly low amounts of F. circinatum in the environment to be retained in the membranes and detected in sufficient quantities by the conventional PCR test. Testing of the DNA with the LAMP assay has not been completed yet.We applied for an invention disclosure with the Office of Technology Licensing (OTL) at UF for the 3D printed cassette in the spore traps but OTL recommended focusing on innovating the adhesive membrane to make it more selective as the spore trap itself was not sufficient for patenting. We have discussed with Dr. Jennifer Andrew about further researching on the membrane but at the moment we have focused on the other objectives. We plan to revisit this component of the project in the next quarter. •Objective 3- Provide training and hands-on opportunities to forest health practitioners of commercial pine stakeholders and diagnostic clinics for the implementation of the integrated detection system.We have planned the first hands-on training workshop, which will take place in October 2021 in Gainesville, FL. The workshop has already been advertised to regional forestry industry stakeholders. We have also drafted the first extension publication on pitch canker disease, and have started drafting the script for the video tutorial on the use of the LAMP assay.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Villari C. Diseases of southern pine plantations: new tools for diagnosis and risk assessment. Seminar at the Department of Forestry and Environmental Resources, North Carolina State University. October 2020, Raleigh, North Carolina, USA
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Meinecke C. & Villari C. Results in real time: a field-capable molecular test for Fusarium circinatum. Oral presentation at the Southern Appalachian Forest Entomology & Pathology Seminar, March 2021, virtual meeting.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Meinecke C., Niyas A.M. & Villari C. Molecular approaches to identify emerging needle diseases in managed loblolly pine. Oral presentation at the 2021 Warnell Graduate Student Symposium, February 2020, virtual meeting.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Meinecke C. & Villari C. Defining and diagnosing emerging diseases in commercially managed southern pines. Invited oral presentation at the Society of American Foresters (SAF) National Convention, October 2020, virtual meeting.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Smith J.A. Using spore traps to detect and manage tree pathogens. Invited presentation at Trees Florida Conference, Florida Chapter International Society of Arboriculture, June 2021, Palm Coast, Florida.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Quesada T., Benitez B., Villari C., Smith K., Andrew J., Dominguez-Gutierrez P.R., Smith J. Improving environmental sampling of Fusarium circinatum . Oral presentation at the Forest Biology Research Cooperative Annual Meeting, October 2020, virtual meeting.
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Progress 07/01/19 to 06/30/20
Outputs
Target Audience:We presented our preliminary results to the members of theUGA Southern Pine Health Research Cooperative, which comprisesforestry industry stakeholders and the members of theRegion 8 FS-Forest Health and Protection Program.Preliminary results have also been presented to the scientific community of the University of Georgia. Changes/Problems:Straing March 23rd 2020, we had to temporarily suspend the research activities because of the COVID-19 pandemic, but at this time, provided research at both the University of Georgia and University of Florida will be restored within the next couple of months, we do not anticipate a significant impact on the work timeline. What opportunities for training and professional development has the project provided?To date, this project has allowed for the training of oneundergraduate student and one graduate student. The undergraduate student,Brianna Benitez, hasbeen trained on workingwithfungal cultures, spore count estimation, DNA extraction and PCR techniques. This student is currently applying for a master's program in Plant Pathology, and these skills will benefit her professional development during her graduate career. The graduate student, Colton Meinecke, is pursuing a MS in Forest Pathology, and in additiona to his MS class work, he has been trained on working with the pathogen Fusarium circinatum, and in the use of LAMP, including the design of LAMP primers and probes. The student had also the opportunity to travel to South Africa to perform project-related experiments, with the great benefit of exploring different work environments and expanding his professional network. How have the results been disseminated to communities of interest?The results are only preliminary and have not been extensively disseminated yet to communities of interest. Some of the preliminary results on the LAMP primers development have been shared in the format of poster presentations tothe members of theUGA Southern Pine Health Research Cooperative. Preliminary results regarding the molecular component of the project have also been disseminated by the graduate student in the form of poster and oral presentations to the scientific community of the University of Georgia. What do you plan to do during the next reporting period to accomplish the goals? LAMP probes validation. Finalize the validation of the developed LAMP primers and probes for the species-specific detection of F. circinatum. This will include testing of the sensitivity of the assay using both DNA dilutions and a spore standard curve; testing the robustness of the assay when using naturally infected samples and crude DNA extracts; testing of field portable instruments. Collaboration with material science engineer team. Perform hydrophobicity and electrostatic tests on F. circinatum spores to evaluate potential materials that would be selective for F. circinatum spores. Develop and test a suitable frame for the synthetic membranes to be used for direct DNA extraction. This would be used in the spore traps for non-selective trapping and DNA extraction followed by selective PCR or LAMP amplification using specific primers for F. circinatum. Field testing of spore traps with new frames and synthetic membranes. These tests would complete the series of experiments to include in a scientific publication. Workshop for stakeholders and forestry professionals. The first workshops on the use and implementation of this detection system is scheduled for the second year of the project. Production of the YouTube tutorial. During the second year of the project we will start the filming and post-production of the YouTube tutorial for the use of the LAMP technology. Publication of a Fact Sheet. During the second year of the project we will produce the first outreach publication on pitch canker, the disease caused by F. circinatum.
Impacts
What was accomplished under these goals?
Pitch canker, caused by Fusarium circinatum, is an aggressive disease affecting most Pinus species and control of this pathogen is one of the top forest health priorities of the forestry industry in the southern US region, as well as in other countries. The goal of this project is to develop an affordable system and portable tool for the early detection of airborne F. circinatum spores and infected tissues, and subsequently ensure thetechnology transfer of the protocol to forest health practitioners and forestry industry stakeholders. The implementation of a low-cost LAMP technology can be a game-changer in the early detection of F. circinatum and will allow for successful management of pitch canker. Moreover, this assay would be crucial to monitor the presence of this quarantine pathogen during international trades of seeds and seedlings, preventing its further spread worldwide. To date, our accomplishments have been the following: Objective 1- Design and validate a LAMP assay to specifically detectF. circinatum. We designed six LAMP primer sets in a 12,000 base pair insertion encoding for five genes putatively involved with pathogenicity, and which has been identified as species-specific for F. circinatum in previous genome comparison studies. After preliminary tests performed with both target and non-target species, we selected one of the six primer sets as the most promising, and discarded the other five. Preliminary tests were performed with F. circinatum isolates and other closely related species from the collection of Co-PI Dr. Quesada. To finalize the specificity testing of the selected primer set, the graduate student working on the project was hosted by the Forestry and Agricultural Biotechnology Institute at the University of Pretoria, South Africa, in order to have access to the large Fusarium fungal collection of Dr. Brenda Wingfield. The specificity of the primer set was successfully validated with 45 F. circinatum isolates and other 14 non target Fusarium species. The specificity of the primer set was also successfully tested both on infected and healthy pine tissues, to make sure the assay is neither inhibited by nor cross-reacts with the host plant. While the validation of the LAMP assay still needs to be finalized, the preliminary results are very promising, and we do not foresee encountering any specific problem in the further validation steps. Objective 2- Identify the best low-cost spore trap, sampling strategy and in-field DNA extraction method to be coupled with the LAMP assay. We established a collaboration with a material science engineer, Dr Jennifer Andrew, University of Florida, to identify the best material to be used for building the spore traps. In particular, we discussed potential tests to assess the hydrophobicity and electrostatic properties of F. circinatum spores, to be able to select a substrate for spore trapping that has the best adhesiveness toward the spores, but that could also be selective for the spores of the target species. We also performed laboratory tests to evaluate the effects of different solvents (which will be used to dissolve the adhesive substrate in the spore traps) on the integrity of F. circinatum microconidia. We evaluated water (control), ethanol, isopropanol, and acetone, and results shows that none of the solvents were affecting the integrity of the spores, which means that all of them can be potentially used in the sampling strategy. The actual solvent to be used will be selected depending on which adhesive substrate will be used in the traps, but the fact that the solvent is not a liming factor will allow to choose from a broader set of options.One of the objectives of this project is to minimize the number of steps for DNA extraction for on-site testing. At this regard, we tested adhesive paper labels (Avery ®, 1" x 3" mailing labels) and a synthetic membrane (Breathe Easy ®, Sigma-Aldrich) commonly used for sealing culture plates. Three circles were punched out of each adhesive material using a standard paper punch, and a spore solution was added on the adhesive side of each circle and allowed to air-dry at room temperature. Each circle was placed into a centrifuge tube and a rapid DNA extraction (X-Tract-N-Amp, Sigma-Aldrich) was directly performed as per manufacturer's instructions. The samples were then amplified by PCR using F. circinatum specific primers. Results showed product amplification of only the samples extracted from the Breathe Easy® membrane, indicating this as a potential suitable material for direct DNA extraction and amplification. These membranes are also transparent, which would allow visualization through light or electron microscopy. One disadvantage of the membranes is that they are very thin, which makes them difficult to manipulate. This could be solved by using a rigid support frame. Amplification was unsuccessful with the Avery® labels. Given the good results obtained with the Breathe-Easy ® membrane, we tested the minimum amount of spores on the membrane that could be successfully extracted and amplified using PCR, and results showed that as low as 10 spores could be detected with this method, which would be a satisfactory threshold for field applications. Objective 3- Provide training and hands-on opportunities to forest health practitioners of commercial pine stakeholders and diagnostic clinics for the implementation of the integrated detection system. This objective has not been approached yet, and accomplishment will begin only during the second year of the project. Accomplishments during this first ten months are on time according to our predicted project timetable, and the data collected so far are instrumental for the following stages of the project. We expect to have a finalized field-tested early detection system for F. circinatum by the end of the third year of project. The products of this project will bring a substantial advancement in the strategy for the early detection of pitch canker, allowing for timely planning and response, and for effective screening of potentially infected plant material, which are all key components for the proper management and containment of this critical disease.
Publications
- Type:
Other
Status:
Other
Year Published:
2019
Citation:
Meinecke, C. & Villari, C. Molecular methods for detecting and identifying fungal pathogens of southern pines. Poster presented at Southern Pine Health Research Cooperative Semi-annual Meeting. September 26, 2019. Athens, Georgia.
- Type:
Other
Status:
Other
Year Published:
2019
Citation:
Meinecke, C. & Villari, C. Molecular methods for detecting and identifying fungal pathogens of southern pines. Poster presented at the Plant Center at the University of Georgia 2019 Fall Retreat. October 24, 2019. Helen, Georgia.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Meinecke, C. & Villari, C. Development of a rapid, field-capable molecular detection assay for pitch canker. Presentation at the Warnell Graduate Student Symposium. February 14, 2020. Athens, Georgia.
|
Progress 05/01/19 to 04/30/20
Outputs
Target Audience:We presented our preliminary results to the members of theUGA Southern Pine Health Research Cooperative, which comprisesforestry industry stakeholders and the members of theRegion 8 FS-Forest Health and Protection Program.Preliminary results have also been presented to the scientific community of the University of Georgia. Changes/Problems:Straing March 23rd 2020, we had to temporarily suspend the research activities because of the COVID-19 pandemic, but at this time, provided research at both the University of Georgia and University of Florida will be restored within the next couple of months, we do not anticipate a significant impact on the work timeline. What opportunities for training and professional development has the project provided?To date, this project has allowed for the training of oneundergraduate student and one graduate student. The undergraduate student,Brianna Benitez, hasbeen trained on workingwithfungal cultures, spore count estimation, DNA extraction and PCR techniques. This student is currently applying for a master's program in Plant Pathology, and these skills will benefit her professional development during her graduate career. The graduate student, Colton Meinecke, is pursuing a MS in Forest Pathology, and in additiona to his MS class work, he has been trained on working with the pathogen Fusarium circinatum, and in the use of LAMP, including the design of LAMP primers and probes. The student had also the opportunity to travel to South Africa to perform project-related experiments, with the great benefit of exploring different work environments and expanding his professional network. How have the results been disseminated to communities of interest?The results are only preliminary and have not been extensively disseminated yet to communities of interest. Some of the preliminary results on the LAMP primers development have been shared in the format of poster presentations tothe members of theUGA Southern Pine Health Research Cooperative. Preliminary results regarding the molecular component of the project have also been disseminated by the graduate student in the form of poster and oral presentations to the scientific community of the University of Georgia. What do you plan to do during the next reporting period to accomplish the goals? LAMP probes validation. Finalize the validation of the developed LAMP primers and probes for the species-specific detection of F. circinatum. This will include testing of the sensitivity of the assay using both DNA dilutions and a spore standard curve; testing the robustness of the assay when using naturally infected samples and crude DNA extracts; testing of field portable instruments. Collaboration with material science engineer team. Perform hydrophobicity and electrostatic tests on F. circinatum spores to evaluate potential materials that would be selective for F. circinatum spores. Develop and test a suitable frame for the synthetic membranes to be used for direct DNA extraction. This would be used in the spore traps for non-selective trapping and DNA extraction followed by selective PCR or LAMP amplification using specific primers for F. circinatum. Field testing of spore traps with new frames and synthetic membranes. These tests would complete the series of experiments to include in a scientific publication. Workshop for stakeholders and forestry professionals. The first workshops on the use and implementation of this detection system is scheduled for the second year of the project. Production of the YouTube tutorial. During the second year of the project we will start the filming and post-production of the YouTube tutorial for the use of the LAMP technology. Publication of a Fact Sheet. During the second year of the project we will produce the first outreach publication on pitch canker, the disease caused by F. circinatum.
Impacts
What was accomplished under these goals?
Pitch canker, caused by Fusarium circinatum, is an aggressive disease affecting most Pinus species and control of this pathogen is one of the top forest health priorities of the forestry industry in the southern US region, as well as in other countries. The goal of this project is to develop an affordable system and portable tool for the early detection of airborne F. circinatum spores and infected tissues, and subsequently ensure thetechnology transfer of the protocol to forest health practitioners and forestry industry stakeholders. The implementation of a low-cost LAMP technology can be a game-changer in the early detection of F. circinatum and will allow for successful management of pitch canker. Moreover, this assay would be crucial to monitor the presence of this quarantine pathogen during international trades of seeds and seedlings, preventing its further spread worldwide. To date, our accomplishments have been the following: Objective 1- Design and validate a LAMP assay to specifically detectF. circinatum. We designed six LAMP primer sets in a 12,000 base pair insertion encoding for five genes putatively involved with pathogenicity, and which has been identified as species-specific for F. circinatum in previous genome comparison studies. After preliminary tests performed with both target and non-target species, we selected one of the six primer sets as the most promising, and discarded the other five. Preliminary tests were performed with F. circinatum isolates and other closely related species from the collection of Co-PI Dr. Quesada. To finalize the specificity testing of the selected primer set, the graduate student working on the project was hosted by the Forestry and Agricultural Biotechnology Institute at the University of Pretoria, South Africa, in order to have access to the large Fusarium fungal collection of Dr. Brenda Wingfield. The specificity of the primer set was successfully validated with 45 F. circinatum isolates and other 14 non target Fusarium species. The specificity of the primer set was also successfully tested both on infected and healthy pine tissues, to make sure the assay is neither inhibited by nor cross-reacts with the host plant. While the validation of the LAMP assay still needs to be finalized, the preliminary results are very promising, and we do not foresee encountering any specific problem in the further validation steps. Objective 2- Identify the best low-cost spore trap, sampling strategy and in-field DNA extraction method to be coupled with the LAMP assay. We established a collaboration with a material science engineer, Dr Jennifer Andrew, University of Florida, to identify the best material to be used for building the spore traps. In particular, we discussed potential tests to assess the hydrophobicity and electrostatic properties of F. circinatum spores, to be able to select a substrate for spore trapping that has the best adhesiveness toward the spores, but that could also be selective for the spores of the target species. We also performed laboratory tests to evaluate the effects of different solvents (which will be used to dissolve the adhesive substrate in the spore traps) on the integrity of F. circinatum microconidia. We evaluated water (control), ethanol, isopropanol, and acetone, and results shows that none of the solvents were affecting the integrity of the spores, which means that all of them can be potentially used in the sampling strategy. The actual solvent to be used will be selected depending on which adhesive substrate will be used in the traps, but the fact that the solvent is not a liming factor will allow to choose from a broader set of options.One of the objectives of this project is to minimize the number of steps for DNA extraction for on-site testing. At this regard, we tested adhesive paper labels (Avery ®, 1" x 3" mailing labels) and a synthetic membrane (Breathe Easy ®, Sigma-Aldrich) commonly used for sealing culture plates. Three circles were punched out of each adhesive material using a standard paper punch, and a spore solution was added on the adhesive side of each circle and allowed to air-dry at room temperature. Each circle was placed into a centrifuge tube and a rapid DNA extraction (X-Tract-N-Amp, Sigma-Aldrich) was directly performed as per manufacturer's instructions. The samples were then amplified by PCR using F. circinatum specific primers. Results showed product amplification of only the samples extracted from the Breathe Easy® membrane, indicating this as a potential suitable material for direct DNA extraction and amplification. These membranes are also transparent, which would allow visualization through light or electron microscopy. One disadvantage of the membranes is that they are very thin, which makes them difficult to manipulate. This could be solved by using a rigid support frame. Amplification was unsuccessful with the Avery® labels. Given the good results obtained with the Breathe-Easy ® membrane, we tested the minimum amount of spores on the membrane that could be successfully extracted and amplified using PCR, and results showed that as low as 10 spores could be detected with this method, which would be a satisfactory threshold for field applications. Objective 3- Provide training and hands-on opportunities to forest health practitioners of commercial pine stakeholders and diagnostic clinics for the implementation of the integrated detection system. This objective has not been approached yet, and accomplishment will begin only during the second year of the project. Accomplishments during this first ten months are on time according to our predicted project timetable, and the data collected so far are instrumental for the following stages of the project. We expect to have a finalized field-tested early detection system for F. circinatum by the end of the third year of project. The products of this project will bring a substantial advancement in the strategy for the early detection of pitch canker, allowing for timely planning and response, and for effective screening of potentially infected plant material, which are all key components for the proper management and containment of this critical disease.
Publications
- Type:
Other
Status:
Other
Year Published:
2019
Citation:
Meinecke, C. & Villari, C. Molecular methods for detecting and identifying fungal pathogens of southern pines. Poster presented at Southern Pine Health Research Cooperative Semi-annual Meeting. September 26, 2019. Athens, Georgia.
- Type:
Other
Status:
Other
Year Published:
2019
Citation:
Meinecke, C. & Villari, C. Molecular methods for detecting and identifying fungal pathogens of southern pines. Poster presented at the Plant Center at the University of Georgia 2019 Fall Retreat. October 24, 2019. Helen, Georgia.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Meinecke, C. & Villari, C. Development of a rapid, field-capable molecular detection assay for pitch canker. Presentation at the Warnell Graduate Student Symposium. February 14, 2020. Athens, Georgia.
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