Source: STATE UNIV OF NEW YORK submitted to NRP
ENVIRONMENTAL IMPACTS OF GE AND CONVENTIONALLY PRODUCED AMERICAN CHESTNUT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1017038
Grant No.
2018-33522-28708
Cumulative Award Amt.
$500,000.00
Proposal No.
2018-02688
Multistate No.
(N/A)
Project Start Date
Sep 1, 2018
Project End Date
Jun 30, 2022
Grant Year
2018
Program Code
[HX]- Biotechnology Risk Assessment
Recipient Organization
STATE UNIV OF NEW YORK
(N/A)
SYRACUSE,NY 13210
Performing Department
(N/A)
Non Technical Summary
The American chestnut was once a common, economically and environmentally important tree in the eastern U.S. forests until an invasive fungal disease killed over three billion trees and brought the species to near extinction. Using the tools of biotechnology, a blight tolerant American chestnut tree has been developed that could rescue this species and benefit the environment. But although federal regulators have experience evaluating genetically engineered (GE) agricultural crops, using biotechnology to rescue a wild species sets up a novel paradigm requiring new information. The GE American chestnut is an ideal case study to help not only with its own restoration, but with understanding the process for other threatened trees such as ash, hemlock, American elm, and many others.This research will directly compare environmental impacts of GE, conventionally bred, and wild-type trees. We will establish plantings of GE and wild-type American chestnuts, advanced backcross and hybrid chestnuts, and red oak controls at three sites spanning the American chestnut's historic range. Core objectives will 1) address baseline soil and atmospheric conditions at the experimental sites prior and subsequent to site preparation and planting, 2) evaluation of health, form, growth, and physiological performance of GE and conventional genotypes, 3) comparison of species richness and diversity of soil fungi associated with the different trees, and 4) examine insect communities and feeding responses across tree types and sites.These forests will last far beyond this specific proposaland thus were designed to facilitate decades of future research while continuously providing feedback to regulators. There is also a critical public outreach and education component to this project so that educators and the public can follow these trees from seedlings to canopy trees. Most importantly, this project serves as a critical template for informing future tree restoration projectsProgram area addressed:4a) Assessment of the influence of GEas compared tonon-GE organisms on agricultural or forest ecosystems.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1360620107050%
2010699108050%
Knowledge Area
136 - Conservation of Biological Diversity; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0620 - Broadleaf forests of the North; 0699 - Trees, forests, and forest products, general;

Field Of Science
1070 - Ecology; 1080 - Genetics;
Goals / Objectives
Federal regulators have experience evaluating genetically engineered (GE) agricultural crops, but using biotechnology to rescue a wild species is novel and requires new information. American chestnut is an exemplar case study. This heritage tree was once a keystone species providing significant agricultural, ecological, and social benefits until chestnut blight, an invasive fungal pathogen, removed more than 4 billion trees from the landscape. To restore American chestnut, blight-tolerance was developed by adding a gene that detoxifies the pathogen's primary weapon, oxalate. This research will directly compare environmental impacts of GE and conventionally bred trees. We will establish plantings of GE and wild-type American chestnuts, advanced backcross and hybrid chestnuts, and red oak controls at three sites spanning chestnuts historic range. Core objectives will address (1) baseline soil and atmosphere/microclimate conditions at the experimental sites prior and subsequent to site preparation and planting, 2) evaluation of health, phenology, growth, and physiological performance of GE and conventional genotypes, 3) comparison of species richness and diversity of soil fungi associated with the trees, and 4) enumerate insect herbivore communities and feeding responses of select herbivore species across tree genotypes and sites. These forests will last far beyond this specific proposal, and thus were designed to facilitate decades of future research while continuously providing feedback to regulators. Most importantly, this project serves as a critical template for informing future tree restoration projects. The specific objects of this project are:Objective 1: Establish long-term, research forests plots at three locations, comparing genetically engineered American chestnut to chestnut produced by traditional breeding and wild-type. (year 1) The goal of objective one is to establish three long-term field trials designed to make direct comparisons between the bioengineered American chestnut trees (genetically engineered), conventionally bred chestnut trees (hybrids), wild-type American chestnut trees, and an unrelated tree typically found in chestnut/oak forests (red oak). These plantings will support the research objectives 2 thru 5 of this research over the next three years and beyond. The plantings will be available for additional research for decades into the future. The land used for the orchard-type and shelterwood-type plantings are being provided by SUNY-ESF in central New York State, Penn State in central Pennsylvania, and Virginia Tech University in southwestern Virginia.Objective 2: Establish long-term monitoring protocols for soils, all-taxa biological diversity, and atmosphere/microclimate conditions at experimental sites, to be used for data collection prior to, and at regular intervals following, site preparation and experimental plantings. (years 1-3)Long-term monitoring (LTM) is a widely-employed technique for evaluating experimental outcomes in complex systems such as plant communities, which are shaped over long time periods by interacting controls and feedbacks among soils, plants, and atmospheric conditions, as well as human influences. In this study, LTM protocols are focused on variables that allow us to investigate short- and long-term effects of experimental plantings (Objective 1) on key ecosystem controls in situ: soils, biodiversity, and microclimate. Some LTM data will be used to evaluate observed variation in seedling performance across experimental plots and over time; for example, site-level weather data will be used to interpret phenological observations as well as impacts of heat or frost injury.Objective 3: Perform a comparative assessment of health, phenology, growth, and physiological performance of contrasting taxa of C. dentata and red oak seedlings. (years 1-3)Regular monitoring of seedling survivorship, health, and phenology during the first few growing seasons after planting provides a basis to assess seedling performance and its many causal factors. Newly established seedlings are vulnerable to many stressors, including damping off (pathogens), frost injury, herbivory, desiccation, and mechanical injury. A quantitative census provides data on the relative contributions of such factors, which are thought to be unrelated to blight resistance, to overall seedling health and survivorship.The relative growth analysis provides a method of statistically comparing the performance of these taxa. Small differences in growth rates of young seedlings can strongly influence the subsequent growth trajectories of developing trees, as small initial differences compound over time.The measurements of photosynthetic performance allow for a new assessment of the potential effect of transgene insertion on the basic and fundamental photosynthetic processes of these trees. This is directly relevant to the question posed in the RFA: "Is there an effect above and beyond what might occur with an organism that has similar traits, but was developed using other technologies?". Furthermore the photosynthetic parameters generated by these measurements provide potential physiological mechanisms underpinning differences in growth across taxa.Objective 4: Compare the species richness and diversity of soil fungi associated with Darling T1s GE-AC, B3F2 backcross AC, and wild-type American chestnut controls in established research forest plots. (years 1-3)The GE-AC will depress fungi growing on above-ground parts of the trees, and we will use this experiment to investigate if the genetic resistance also depresses soil fungi. The goal of objective 2 is to assess if there are detectable differences in the communities of soil fungi associated with Darling T1s GE-AC, B3F2 backcross AC, and wild-type AC controls. Our previous work suggests resistance to chestnut blight does not affect colonization of chestnut roots by ectomycorrhizal fungi. Here we will sample DNA from in-growth sand bags installed in the experimental plots.Objective 5: Evaluate the effects of GE, hybrid, and wild-type American chestnut on terrestrial insect herbivore communities and the feeding responses of select species representing important functional feeding guilds. (years 1-3)Evaluating unintended phenotypic change associated with GE trees on processes important to ecosystem functioning is a critical component of evaluating their release in natural environments. Insects profoundly influence forest ecosystems, impacting nutrient cycling, inter and intraspecific competition among trees, stand structure, health, as well as comprising the base of complex food webs. Although assessments of transgenic expression products on non-target insects are common, especially in agriculture, few studies have involved plants transformed for purposes other than insect resistance. In this study insect herbivory of traditionally bred, GE, and wild-type American chestnut trees will be compared.
Project Methods
Objective 1: Establish long-term, research forests plots at three locations, comparing genetically engineered American chestnut to chestnut produced by traditional breeding methods and to wild-type trees. (year 1) The goal of objective one is to establish long-term field trials of GE American chestnut, B3F2 [3rd generation backcross 1st generation intercross chestnut (American (A) x Chinese (C), x A, x A, x A, then B3 xB3)], commonly planted chestnut hybrids (A x C, and complex hybrids of A, C, Japanese, and European chestnut), and wild-type American chestnut. The primary plantings will consist of a randomized complete-block experiment with trees in orchard-style plantings at each site. This experimental design provides the ability to robustly compare the performance of these genotypes across a wide range of environmental conditions. A secondary planting design will also be established at all three sites in which American chestnut trees will be established in shelterwood cuts in forested areas, representing a more ecologically realistic restoration possibility for this iconic species. The land used for the orchard and shelterwood plantings are being provided by SUNY-ESF in central New York State, Penn State in central Pennsylvania, and Virginia Tech University in southwestern Virginia.Objective 2: Establish long-term monitoring (LTM) protocols for soils and atmosphere/microclimate conditions at experimental sites for data collection prior to and following site preparation and planting.(years 1-3)Soils: We will measure the physical and chemical characteristics of soils within each orchard plot. Bulk density, organic matter content, soil C:N and cations (Na, Ca, Mg, K, Mn, Al) will be evaluated.Atmosphere/microclimate: We will install a data-logging weather station to continuously record temperature, humidity, photosynthetic photon flux density (PPFD), wind speed and direction, and precipitation at all three sites. Objective 3: Perform a comparative assessment of health, phenology, growth, and physiological performance of contrasting taxa of C. dentata and red oak seedlings. (years 1-3)The health and phenology of C. dentata seedlings will be assessed with a monthly census. Every seedling will be observed and scored with a visual assessment of blight and other potential contributing factors of seedling mortality, including frost damage, herbivory, or desiccation. During the Spring and Autumn, this assessment will be done weekly to document phenological transitions of leaf on/off - and in future years, flowering times. We will also explore using an automated web camera for site-level phenological observations. We will perform a rigorous comparative assessment of growth and physiological performance across the C. dentata taxa.Objective 4: Compare the species richness and diversity of soil fungi (from in-growth sand bags) associated with Darling T1s GE-AC, B3F2 back-cross AC, and wild-type American chestnut controls in established research forest plots. (years 1-3)We willinvestigate the communities of soil fungi associated with GE-AC and TACF B3F2 backcross and wild type American chestnut controls. We will insert one in-growth sand bag(0.44 µm nylon mesh)within the drip line of each of the four treesto allow fungal hyphae but not roots to grow into the bags during the experiment. We will generate community profiles from these extracts based on the fungal ITS1 region, using the Illumina MiSeq platform. We have over 300 modified primers with unique barcodes to track individual samples in the MiSeq run. Sequence data will be processed using the BBMap package, VSearch 2.5.1 and Qiime 1.9, while FUNGuild will be used to identify EMF taxa.Objective 5: Evaluate the effects of GE, hybrid, and wild-type American chestnut on terrestrial insect herbivore communities and the feeding responses of select species representing important functional feeding guilds. (years 1-3)Survey of herbivorous insect community and relative herbivory level: This objective holistically captures any general effects of chestnut genotype on herbivorous insect diversity and abundance. Insects and their damage will be quantified within each of the planting blocks at each site during three seasonal time intervals in each year of the project. Standard census techniques for leaf-feeding insect herbivoreswill be used. A subset of leaves on each tree will be carefully examined and any herbivorous insects present enumerated and partitioned into Lepidoptera, Diptera, Hemiptera, Coleoptera, and Hymenoptera, similar to the classification used in a study of insect communities on transgenic pine. In each year of the study, a randomly selected subset of 25 trees of each chestnut genotype will be assessed in early June, mid-July, and mid-September) to capture the effects of different seasonal feeding guilds. On each tree, all leaves on two representative branches will be examined for feeding damage. Each leaf will be assigned to one of six damage classes ranging from no damage to complete consumption.Direct Effects on Insect Herbivores: These experiments are not intended as a complete ecological risk assessment but rather, by using a few exemplar insect herbivores, as a guide to more detailed and comprehensive studies if results warrant. We will utilize both laboratory and field bioassays to evaluate potential non-target effects of transgenic trees on insect herbivores, specifically gypsy moth, forest tent caterpillar, Polyphemus, and orange-striped oakworm. These represent a diverse array of life-histories: native and invasive (gypsy moth), colonial (forest tent caterpillar and oakworm) and solitary, and a phenological gradient from spring to early fall. All assays of free-feeding larvae will be conducted in the laboratory. Effects on Natural Enemies: To test whether transgenic chestnut changes the probability of mortality from predators and parasitoids, we will use two common ecologically and economically important defoliators, the invasive gypsy moth and the native forest tent caterpillar. We will couple an on-tree field experimental approach using sentinel larvae with specific laboratory assays.

Progress 09/01/18 to 06/30/22

Outputs
Target Audience:The target audience still includes USDA-APHIS-BRS and project collaborators, as described previously. Since regulatory documents have now been submitted to all three biotechnology regulatory agencies (USDA, EPA, and FDA), potential applications of this project for both public and private groups are becoming more tangible. We anticipate that regulatory review will be completed by all three agencies by the fall of 2023, which will dramatically expand the potential for GE trees to be used by public and private groups in various types of plantings. Studies on environmental impacts of GE trees are increasingly important to environmental conservation groups and forestry agencies as American chestnut restoration with transgenic trees becomes feasible in the near future, so groups like these are becoming more important target audiences as well. Multiple scientific journal articles and graduate theses describing research results have been submitted or are in preparation, which will reach additional scientific professionals and researchers. The dentataBase chestnut planting database (described in previous reports) is currently being revised and expanded to facilitate use by small-scale growers and private individuals, which also expands the target audience beyond researchers to general-public chestnut enthusiasts. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project continues to provide numerous opportunities for students (both undergraduate and graduate) to learn and apply research techniques. Recent training and method development have included soil collection and analysis, insect herbivory with multiple species of insects, mycorrhizal root collection and fungal identification, leaf physiological measurements, plant pathology (chestnut blight canker observations), biological experimental design, and several types of statistical analysis. The project has provided training opportunities to at least six undergraduate students, six M.S. degree candidates, two PhD candidates, research staff, field staff, and volunteers. How have the results been disseminated to communities of interest?As described in the "Target Audience" and "Outputs" section, results have been disseminated in several different ways. Publications in scientific journals and graduate theses are most direclty applicable to academic communities. Results have been described in great detail to federal regulatory agencies (USDA-APHIS, EPA, and FDA) for their review of Darling 58 chestnuts for potential distribution in the near future. Only one of these regulatory documents is currently available to the public ( https://www.aphis.usda.gov/brs/aphisdocs/19-309-01p.pdf ), but we intend that others (or key summaries) will eventually be shared depending on relevant agency guidelines. There have also been multiple popular-press news articles, blogs, and podcasts about this project that reach much broader public audiences. When appropriate, results are also shared on social media, resulting in conversations and interactions with interested communities that would not otherwise be reached by more traditional scientific reporting. 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. Plots (including both open field and shelterwood in NY, PA, and VA) were established in year 1 and have been maintained since then. Overall tree mortality was highest at the VA shelterwood plot, but there were not strong differences in survival among tree types. Objective 2. Detailed baseline data for several soil characteristics were collected and analyzed in 2021-2022. This included at least 9 spatially distinct soil samples from each plot in each state, to compare soil characteristics both among and within plots. Analyses included pH, phosphorus (P), potassium (K), calcium (Ca), carbon:nitrogen ratio (C:N), and organic matter (OM). The most obvious differences were between different plots: for example, Ca was higher in the VA open field plot and the NY shelterwood plot, but generally low and less variable in the remaining plots. pH and P were quite consistent within plots, while K varied more within some plots. Shelterwood plots consistently had higher C:N and more OM than the nearby open field plots in each state. These baseline analyses will allow future observers to determine whether chestnut tree types affect soil chemistry over time. Objective 3. Several growth and physiological traits were analyzed over the past year: To increase our silvicultural understanding of the environmental conditions most favorable to the growth and survival of American chestnut relative to species with which it historically co-existed, we examined effects of shelterwoods with variable residual densities on the growth and physiology of blight-tolerant chestnut seedlings relative to sugar maple, and several oak and hickory species across a broad range of light availabilities (gap-light index; GLI; of 4-50%). To assess the hypothesis that American chestnuts are more physiologically plastic and responsive to light than oaks and hickories, we measured seedling relative diameter and height growth rates and photosynthetic light-response curves across a GLI gradient. The diameter and height growth of chestnut increased with GLI, while oaks and hickories did not. All species varied aspects of their photosynthetic physiology across the GLI gradient in an approximately equivalent manner: The dark respiration rate and light compensation point increased equivalently across species in response to increases in light availability. A multivariate analysis identified three major axes of variation across ten measured traits, largely reflecting differences in species averages and phenotypic plasticity across the GLI gradient. American chestnut seedlings were spread across these multivariate dimensions more than other species, consistent with the hypothesis of higher phenotypic plasticity in this focal species. Therefore, relatively low residual shelterwood densities may better promote the establishment of underplanted chestnut seedlings relative to associated tree species provided it does not result in detrimental increases in the productivity of competing understory species. Mature, fully-expanded leaf samples were collected from five taxa (non-transgenic American chestnut; NT; transgenic American chestnut, T2+; Red oaks, RO; backcross hybrids, B3F3; and hybrids) in both growing conditions (open field and shelterwoods) at all three states in the summer of 2020. A single leaf was removed from one individual tree of each taxa within each of the blocks at each location (N = 173 leaves). Leaves were processed the next day for measurements of leaf mass per unit area, leaf dry matter content, and leaf N concentration. The resulting data were analyzed with Analysis of Variance (ANOVA) with the full interactions between state (NY, PA, VA), condition (open field vs. shelterwood), and taxa. When "taxa" effects were significant, we did post-hoc Tukey tests to specifically address whether NT and T2+ taxa differed in their responses. In general, these results show expected difference in leaf traits across states and growing conditions that were largely equivalent across taxa. All taxa seem to be adjusting leaf traits to their growing conditions in a similar or equivalent manner. When differences were identified, they were most strongly associated with condition (open field vs. shelterwood) or species background (RO and hybrid chestnuts vs. American chestnuts); these analyses did not reveal significant differences between transgenic and non-transgenic American chestnuts. Tree height was measured and compared for all trees at all sites. There were strong differences between states (NY, PA, VA) in height trends. In NY, open field trees were consistently taller than shelterwood trees, but heights between plots were similar in PA. At most sites, hybrid chestnuts were taller than all other taxa, but this was not the case in VA (where American chestnuts were tallest). Transgenic chestnuts were generally slightly shorter on average than their non-transgenic counterparts, especially in the NY open field plot, but this difference was not statistically significant at all sites. Expression of the OxO transgene was measured at the NY open field site to assess its potential impact on tree height, but no correlation was found between height and OxO expression. Chestnuts in this study are still younger than those typically infected by chestnut blight, but we did observe some natural blight infections. As expected, natural infections on non-transgenic American chestnuts were more severe than those on other types of chestnuts (transgenic, backcross, and hybrid). However, hybrid chestnuts had more individual cankers than any other tree type on average. Controlled inoculations are in progress on transgenic & non-transgenic chestnuts in nearby plots (NY open field), and preliminary results show that cankers are far smaller on transgenic trees than on their non-transgenic siblings. Objective 4. Analyses of mycorrhizal fungi associated with each tree type are in progress and will be featured in an upcoming M.S. thesis. Preliminary results (based on fungal morphology rather than DNA) indicate that fungal diversity is broadly similar among all tree types. Hybrid chestnuts may have slightly less fungal diversity than American chestnuts, but this difference is not statistically significant, and there is no difference between related transgenic and non-transgenic American chestnuts. PA plots showed slightly more fungal diversity NY and VA plots, and trees in shelterwood plots have significantly more fungal diversity associated with their roots than open field plots. More detailed DNA identification of fungal associates is underway on all tree types from all states. Objective 5. Insect herbivory has been analyzed previously (see publications and theses), and additional studies are in progress as of summer 2022. Feeding studies with spongy moth (Lymantria dispar) are being repeated with additional tree types this year, and new studies with Japanese beetle (Popillia japonica) are being added for the first time. Preliminary results on Lymantria dispar growth rates show that there is significant variability among unrelated genotypes of American chestnut, but not between related transgenic & non-transgenic American chestnut. Hybrid chestnuts may also result in slower Lymantria growth rates than some American chestnut types, but red oak is similar to most other chestnuts, and growth rate differences between all leaf types were relatively small (range ~0.33 - 0.40 mg / day). Anecdotal observations show that Japanese beetles are widespread on all tree types in this study; none of these taxa apparently deter beetle activity.

Publications

  • Type: Theses/Dissertations Status: Accepted Year Published: 2022 Citation: M.S. Thesis: Measuring Transgenic American Chestnut Growth and Photosynthesis Across a Light-Availability Gradient to Ascertain Potential Reintroduction Strategies. May 2022. SUNY-ESF, Syracuse, NY.
  • Type: Other Status: Accepted Year Published: 2022 Citation: "Supplemental Information to USDA-APHIS-BRS in support of Petition 19-309-01p for Determination of Nonregulated Status for Blight-Tolerant Darling 58 American Chestnut (Castanea dentata)" Submitted to USDA-APHIS, May 2022.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Anuli Onwumelu, William A. Powell, Andrew E. Newhouse, Garrett Evans, Gwen Hilles, Dakota F. Matthews, Vernon Coffey & John E. Drake. 2022. "Oxalate oxidase transgene expression in American chestnut leaves has little effect on photosynthetic or respiratory physiology." New Forests. https://link.springer.com/article/10.1007/s11056-022-09909-x (Note: this article was described in an earlier report as "submitted", but was later re-submitted and accepted in a different journal. This is the final citation.)
  • Type: Other Status: Accepted Year Published: 2021 Citation: Biotechnology Notification File No. BNF000185, safety and nutritional assessment of Darling 58 American Chestnut. For evaluation by FDA Center for Food Safety & Applied Nutrition. Accepted November 17, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: "Environmental Impacts of GE and Conventionally Produced American Chestnut" Presentation at USDA BRAG 2022 Annual Project Director's Meeting, by Andrew Newhouse, April 26, 2022.


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:The target audience still includes USDA-APHIS-BRS and project collaborators, as described last year. With regulatory documents submitted to both EPA (FIFRA registration) and FDA (Biotechnology Consultation), potential applications of this project for both public and private groups are becoming more tangible. Multiple scientific journal articles describing research results have been submitted or are in preparation, which will reach additional scientific professionals and researchers. One recently published article (Newhouse & Powell 2021, see "Products" below) also focuses on more logical and philosophical perspectives of using biotechnology for conservation, which further expands target audiences to conservation biologists who might not otherwise be actively involved in biotechnology research. The dentataBase chestnut planting database (described in previous reports) is currently being revised and expanded to facilitate use by small-scale growers and private individuals, which also expands the target audience beyond researchers to general-public chestnut enthusiasts. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has continued to provide training and experience to both graduate and undergraduate students, including both field and laboratory techniques. Specifically during this growing season (summer 2021), faculty trained new graduate students in physiological measurements and subsequent statistical analysis, whiile technicians and graduate students trained undergraduates on mycorrhizal collections and soil analysis. Entomology faculty also trained a new graduate student on several aspects of leaf herbivory experiments. The plots are also also frequent foci for field tours to researchers, reporters, students, and interested public: this not only trains the tour groups, but also provides development for project staff and students in effective science communication. How have the results been disseminated to communities of interest?Scientific results have been disseminated in the form of publications and a dissertation (see "Products"), with another publication submitted and expected soon, and multiple graduate theses in progress. Results are also included in regulatory documents submitted to federal agencies (both EPA and FDA) this year. General project descriptions and updates have been disseminated to both academic and public audiences via Zoom conferences and in-person field tours. What do you plan to do during the next reporting period to accomplish the goals?Analysis should conclude within the next few months for Objectives 2, 3, and 4, so we should be able to provide result summaries in our upcoming Final Report. Objective 5 (insect herbivory) involves two experiments that we hope to repeat next summer (2022). We intend that detailed results from all of these studies will be included in both graduate theses and peer-reviewed publications. We expect that all plots should be maintained for the forseeable future, so long-term experimentation should continue pending funding and research priorities of overlapping projects.

Impacts
What was accomplished under these goals? Objective 1: Research plots were successfully established previously and have been continually maintained this year. Objective 2: Climate monitoring systems have been installed and weather data are being collected at all sites. Detailed soil testing is in progress: several replicate soil samples were collected from each site in summer 2021, and detailed physical and chemical analyses are currently in progress. Objective 3: Detailed physiological measurements including photosynthetic efficiency were collected at all sites in 2020 and 2021. These were incorporated into the Onwumelu et al. article described previously, which was re-submitted in 2021 after being supplemented with the new data. Briefly, this work assessed potential transgene impacts on American chestnut physiology, with a focus on photosynthesis and respiration. We collected measurements of leaf respiration and photosynthetic capacity for transgenic (T) and non-transgenic (NT) sibling trees in two distinct experiments. Multiple measurements of photosynthesis (light and CO2 response curves) and foliar traits (leaf mass per unit area, foliar N concentration) were indicative of equally high rates of photosynthetic capacity across T and NT plants, with no significant differences between groups. Photosynthetic rates were equivalent between T and NT plants across two studies in two locations. We observed a modest stimulation of foliar dark respiration in T vs. NT plants (~5-15%) in across a range of temperatures, but no change in foliar respiration in the light. The modest stimulation of dark respiration did not seem to be associated with an alteration in growth rate, as stem diameter and length were equivalent between T and NT types. Our findings suggest that there may be a minor impact of transgene expression on the respiratory physiology in some situations, but this effect is not likely to strongly impact the physiological ecology of this historically important species. End-of-season height and diameter measurements have been collected at the NY site as of fall 2021, and measurements are in progress at the PA and VA sites. Generally, growth in open-field conditions is faster than growth in shelterwood conditions. Preliminary observations suggest that transgenic trees experience reduced growth rates compared to related non-transgenic controls, but the magnitude of this change varies somewhat by plot and is approximately similar to other genotypic differences among unrelated chestnut types. Detailed analyses should be complete in time to include in our upcoming final report. General health and survival of all tree types varied across sites, but did not strongly depend on transgene status. Objective 4: Both mycorrhizal experiments described last year (mesh ingrowth bags and direct soil sampling) were completed this year, samples were collected from all locations, and analyses are in progress. These are featured in two graduate student theses, and summaries should be ready to include in our upcoming final report. Objective 5: Two experiments on insect herbivory were conducted in 2021. These included herbivory by Japanese Beetles and Gypsy Moths, and after repetition next summer, are expected to be included in an upcoming graduate student thesis (planned fall 2022). Preliminary results indicate that Gypsy Moth performance was not significantly different on any of the transgenic vs. related non-transgenic tree types. There were two unrelated non-transgenic American chestnut controls and one hybrid chestnut between which Gypsy Moth growth was significantly different, again emphasizing the importance of including natural genotypic differences for experimental context. The initial (2021) Japanese Beetle experiment was confounded by collection difficulties with both insects and vegetation, and so will be repeated next summer (2022). Preliminary results do not indicate any significant differences on beetle growth between transegnic and non-transgenic tree types.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Newhouse, A.E. and Powell, W.A. (2021) Intentional introgression of a blight tolerance transgene to rescue the remnant population of American chestnut, Conservation Science and Practice, 3(4), p. e348. doi:https://doi.org/10.1111/csp2.348.
  • Type: Other Status: Under Review Year Published: 2021 Citation: Interregional Research Project No. 4, 2021. EPA Section 3 Registration for Darling 58 American Chestnut. CDX_2021_009326.
  • Type: Theses/Dissertations Status: Submitted Year Published: 2021 Citation: Newhouse, A.E. 2021. Regulatory, Scientific, and Social Considerations for the Use of Transgenic American Chestnut Trees in Conservation. PhD Dissertation, SUNY-ESF, Syracuse NY.


Progress 09/01/19 to 08/31/20

Outputs
Target Audience:The target audience still includes USDA-APHIS-BRS and project collaborators, as described last year. With the publication of SUNY-ESF's "Petition for Determination of Nonregulated Status for Blight-Resistant Darling 58 American Chestnut" on the Federal Register (Docket No. APHIS-2020-0030), importance of this project to the general public, APHIS, and other regulatory agencies has increased. Additionally, with experimental data being collected and analyzed, audiences are expanding to include researchers outside this specific project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has continued to provide both lab and field research training for undergraduate and graduate students. This has included a variety of techniques including molecular lab procedures such as PCR, advanced field measurements such as photosynthetic efficiency, statistical analysis, and crafting effective reports and presentations. Project researchers have gained experience in giving presentations and tours, while attendees (including students, other researchers, teachers, and public chestnut enthusiasts) have benefitted from these presentations. How have the results been disseminated to communities of interest?An important means of dissemination this year was the posting of our APHIS Petition for Nonregulated Status on the Federal Register (link above). The publication of this extensive and wide-ranging document has been of interest to chestnut researchers specifically, and more broadly to researchers and biotechnologists who are interested in potentially using biotechnology to address tree diseases, invasive species threats, or restoration. Additionally, there has been increased interest in the chestnut project from media outlets, other research institutions, and conservation groups. This has resulted in several public articles, interviews, podcasts, videos and virtual presentations to a variety of audiences. Selected examples are viewable at the following links: https://youtu.be/wG7Tirt-wyY , https://youtu.be/JbEUjupPPng , https://youtu.be/VHcDV2Mblo4 , https://www.indefenseofplants.com/podcast/2020/7/5/ep-272-restoring-the-american-chestnut , https://www.nytimes.com/2020/04/30/magazine/american-chestnut.html What do you plan to do during the next reporting period to accomplish the goals?We will continue with research efforts described above as in progress. Specifically, Objectives 2 (long-term monitoring) and 4 (soil fungi) will be addressed in 2021 by experiments that have started in 2020. As individual experiments conclude, they will be published in appropriate scientific outlets and incorporated into public presentations.

Impacts
What was accomplished under these goals? Objective 1: Research plots were established at all three locations last year, and have been maintained this year. Objective 2: Climate monitoring systems have been installed and weather data are being collected at all sites. Detailed soil testing at each site has been started, but analyses are not yet complete. Objective 3: Several studies have been conducted regarding relative growth, health, and physiology of the tree types. First, a subset of tree types (transgenic, non-transgenic, & hybrid) were measured during 2019 at the open field plots in NY & PA. This study is described in more detail in the "Petition for Nonregulated Status of Darling 58...", currently available for public comment and download on the Federal Register (https://www.federalregister.gov/documents/2020/08/19/2020-18135/state-university-of-new-york-college-of-environmental-science-and-forestry-petition-for). In summary, we found that the transgenic trees had similar photosynthetic and respiratory rates relative to non-transgenic control trees. We did find some correlations between transgene presence and an increased rate of respiratory release of CO2 during some timepoints on T1 (first generation offspring) trees, and correlations between transgene presence and reduced photosynthetic capacity on T1 trees, but it is possible that these differences are due to endogenously linked chestnut genes instead of the transgenes themselves. These results were not consistent with measurements on T2 (second generation) seedlings, which showed opposing trends at different planting locations. These correlations between photosynthetic activity and transgene presence in T1 trees, while not statistically significant in the current studies, could hypothetically reduce the growth rate of transgenic trees relative to non-transgenic control trees. This could be caused by changes in carbon uptake and release, which would also be expected when enhancing resistance by classical breeding. If these effects are biologically significant, they apparently have a small or negligible impact on tree growth rates, given that height and diameter of the tested T1 trees were not significantly different after two growing seasons. Mid-season height differences in certain families of T2 transgenic seedlings could be explained by this differential carbon uptake and release, but according to photosynthesis and respiration measurements performed on one family of T2 seedlings, photosynthetic activity does not differ according to transgene presence. And as with the T1 trees, T2 height differences were small and not consistent among all families. If these effects noted on T1 trees are biologically significant and persist or grow as Darling 58 offspring get older, a modestly lower photosynthetic capacity (and thus potentially slightly reduced growth rates) in transgenic plants would suggest that Darling 58 offspring could actually be less weedy or aggressive than otherwise-similar wildtype American chestnut. However, photosynthetic activity of T2 seedlings and hybrids at different sites suggests that planting location, environmental factors, or hybridization have larger impacts on photosynthesis than transgene presence. For comparison, a separate study on chestnut photosynthesis reported similar photosynthetic rates for American and backcross chestnuts, but significant differences between these types and Chinese chestnut (Knapp et al., 2014). Other experiments involving chestnut photosynthesis have shown differences among chestnuts grown under different thinning regimes (Joesting et al., 2007), and differences among chestnuts of different age (Joesting et al., 2009) Additionally, photosynthetic rates and gas exchange parameters were measured during 2020 on each tree type in the NY open field and shelterwood plots. The most obvious difference noted was that gas exchange results in the shelterwood reflect lower photosynthetic capacity relative to the open-field plants. Light saturated photosynthetic rates were variable across plants, but look similar across taxa. Stomatal conductance to water vapor was also variable, perhaps with some variation across genotypes. A fluorescence measurement, which is the quantum yield of photosystem II, reflects the fraction of absorbed photons that were used to drive photochemistry. The taxa measured were red oak, non-transgenic T2s, transgenic T2s, B3F3s, and sleeping giant hybrids (SG). The largest signal here is strong variation in photosynthetic capacity across growth environments, likely reflecting phenotypic plasticity (e.g., sun vs. shade leaves). A similar experiment (Onwumelu et al.) on photosynthesis of transgenic & non-transgenic chestnuts near the BRAG plots has been submitted for publication (see "Products"). Height growth measurements at both NY sites revealed that chestnuts grown in open conditions consistently grow faster than those grown in shelterwood conditions. All chestnut types (transgenic, non-transgenic, hybrid, and backcross) put on substantial new growth between 2019 and 2020, with the Sleeping Giant hybrid showing the most new growth. Relative growth rates varied more in the open field, with the red oak and one family of transgenic trees showing less growth than most other types, and the Sleeping Giant hybrid showing more. Other families of transgenic trees were statistically similar to their non-transgenic counterparts in terms of new growth. This matches data collected outside the BRAG plots in NY, which also suggests that transgenic plants from some backgrounds grow slightly slower than their non-transgenic relatives. Height measurements from VA and PA sites will be collected shortly, but not in time for this 2020 annual report. Objective 4: Two types of experiments to quantify soil fungi were initiated in summer 2020. These included mesh bags buried next to each tree type, which will be collected and analyzed in 2021, and a direct root sampling procedure, which should be completed by the end of 2020. Objective 5: The small size of the 2 yr old plantings precluded comprehensive evaluation of their effects on insect herbivores as excising foliage would have been detrimental to the survival and growth of the trees. Previous studies including larger transgenic chestnut trees, some of which originated in a previous BRAG project on transgenic chestnut, have recently been published (see "Products"). In 2020, we assessed performance of a generalist folivore, gypsy moth, on leaves collected from 4-5 trees of several of the genotypes in our planting array. After comparing mean relative growth rate (RGR) of 4th instar gypsy moth larvae, we found no difference among the transgenic event Darling 58 and the related wild-type American chestnut, Sleeping Giant hybrid chestnut, or red oak. We included Darling 4, an older, moderately tolerant transgenic line in the assay to facilitate comparison with previous assessments of transgenic chestnut on gypsy moth. RGR was higher on Darling 4 leaves, consistent with previous bioassays using this genotype, its control, and the wildtype progenitor of this line. Overall, there was no negative effect of transgenic American chestnut on the short term performance of gypsy moth.

Publications

  • Type: Other Status: Accepted Year Published: 2020 Citation: Newhouse et al., 2020. "State University of New York College of Environmental Science and Forestry; Petition for Determination of Nonregulated Status for Blight-Resistant Darling 58 American Chestnut." A Notice by the Animal and Plant Health Inspection Service on 08/19/2020. Document Citation: 85 FR 51008, Agency/Docket Number: Docket No. APHIS-2020-0030 Document Number: 2020-18135. https://www.federalregister.gov/d/2020-18135
  • Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Onwumelu, Anuli; Powell, William; Newhouse, Andrew; Matthews, Dakota; Coffey, Vernon; Drake, John. 2020. Physiological implications of oxalate oxidase transgene expression: A study of the American chestnut. Submitted to: Journal of Experimental Botany.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Brown, Aaron J., Andrew E. Newhouse, William A. Powell, and Dylan Parry. 2020. Comparative Efficacy of Gypsy Moth (Lepidoptera: Erebidae) Entomopathogens on Transgenic Blight-Tolerant and Wild-Type American, Chinese, and Hybrid Chestnuts (Fagales: Fagaceae). Insect Science 27 (5): 106778. https://doi.org/10.1111/1744-7917.12713. NOTE: This article was submitted and accepted earlier in 2019, but was just recently assigned a volume/issue number. It references a previous NIFA BRAG grant on similar chestnut trees.


Progress 09/01/18 to 08/31/19

Outputs
Target Audience:This first year was primarily for establishing USDA APHIS BRSfield permits, production of the trees to be tested, establishing the field plots, and collecting initial data. So the target audience this year was primarility theUSDA APHIS BRS and our research collaborators. In the following years the target audience will broaden to include researchers, policy makers, and the genral public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research did provided both field and lab research training opportunities for undergraduate and graduate students working on the project. This included chestnut breeding techniques and molecular lab techniques such as PCR and oxalate oxidase assays. It also provided training on database use. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will replace any trees that don't survive the winter. We have plantedholding plots near the research plots to maintain trees of the same age to be used for replacemenrts. We expect most of the trees to survive and therefore will follow-up on the studies mentioned in this report plus begin the other studies outlined in the objectives.

Impacts
What was accomplished under these goals? All of objective one, establishment long-term, research forests plots at three locations, comparing genetically engineered American chestnut to chestnut produced by traditional breeding and wild-type,was completed this year. This included the establishment of permits, hand pollinated outcrossing of transgenic American chestnut to produce a T2 generation, testing 1800 offspring for oxalate oxidase production to differentiate the transgenic and non-transgenic siblings, and completing a randomsurvey of offspring for blight resistance using leaf assays. SUNY ESFproduced, tested, and selected the transgenic and wild-type siblings, The American Chestnut Foundation (TACF) produced all the backcross trees at their Meadowview farm, VA, TACF Maryland chapter provided the offspring of the irradiated American chestnut trees, and the two hybrid varietiesand red oak were purchased. All trees were planted at the prepared open field and shelterwood sites in NY, PA, and VA, according to the proposed plan and databases established. In addition, initial data was collected on growth of the transgenic American chestnut compared to wild-type siblings. These initial measurements showedsome variation between the transgenic and non-transgenic, with only one lineof the transgenic having significantly less growthcompared to the non-transgenic siblings. All the other lines showed nosignificant differences in growth. Thedata also showed growth variation in offspring with respect to thier mother trees,and also variation in offspringwith respect to their father trees, irrespective of transgene presence. Measurements in future years will provide more information. Initial photosynthesis data was collected to compare timber-type hybrids, transgenic and non-transgenic American chestnut siblings and two field sites (open fields in NY and PA). There were no significant differences between the transgenic and non-transgenic American chestnut siblings at either sight, but there was one significant difference in mean light-saturated photosynthesisbetween the American chestnutand the hybrid at the NY site. From these initial measurements, it appears that the field site has the greatest effect on photosynthesis, with possibly the hybrid genotype being second, and the transgene having no effect. Future studies will help give further insight.

Publications