INCREASING SUCCESS OF PITCH PINE RESTORATION THROUGH SOIL MICROBE MANAGEMENT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: MCINTIRE-STENNIS
• Reporting Frequency: Annual
• Accession No.: 1010281
• Project Start Date: Aug 15, 2016
• Project End Date: Sep 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
STATE UNIV OF NEW YORK
(N/A)
SYRACUSE,NY 13210

Performing Department
Environmental & Forest Biology

Non Technical Summary
The proposed project will investigate if managing ectomycorrhizal fungi associated with pitch pine will improve pitch pine restoration at the Albany Pine Bush Preserve. A number of studies have shown that fungi in two closely related genera, Suillus and Rhizopogon, are unique among other ectomycorrhizal fungi in that they facilitate conifer invasions in the southern hemisphere. Examples of these fungi are present at the Albany Pine Bush Preserve and can be exploited to facilitate better restoration of Pitch Pine within its native range. This project will contribute to the maintenance of one of only a few inland pine - scrub oak barrens in the world. It is an urban forest that is extensively visited by the public annually. It will be one of the first restoration projects that will use locally adapted mycorrhizal fungi to facilitate the restoration of a native plant community, an approach that can help reduce the introduction of invasive microbes while restoring plant communities.

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
124	0599	1070	100%

Knowledge Area
124 - Urban Forestry;

Subject Of Investigation
0599 - Recreational resources, general/other;

Field Of Science
1070 - Ecology;

Keywords

suillus

rhizopogon

pine restoration

mycorrhizae

molecular techniques

Goals / Objectives
This project will use local and native fungus species related to those that factiliate pine invasions in the southern hemisphere to facilitate restoration of pine in their home range. Managers at the Albany (NY) Pine Bush Preserve are removing invasive locust and restoring pitch pine. Establishment of the pines has failed in some locations and we believe we can increase establishment success with local mycorrhizal fungi. We will:Investigate fungal diversity associated with naturally established pine seedlings along ten 50m transects starting within mature pine stands and ending well away from the original or any other pine stand.Evaluate establishment success of pine seedlings in areas where restoration of pine has been successful and in areas where restoration has not been successful.Evaluate establishment success of pine seedlings inoculated with live suilloid spore slurries added to the planting holes versus those with autoclaved spores slurries. Evaluate establishment success of pine seedlings planted with live suilloid mushrooms added to the planting holes versus those with autoclaved sulloid mushrooms.We will also evaluate edaphic factors to rule the role of N, P, pH, moisture in the establishment pattern.

Project Methods
Objective 1 - Descriptive study: Investigate fungal diversity associated with naturally established pine seedlings along ten 50m transects starting within mature pine stands and ending well away from the original or any other pine stand, collecting the nearest seedling to the transect every 10 meters. Seedlings will be collected in the fall, mycorrhizal infection recorded, and ectomycorrhizal species identified with molecular techniques (Ashkannejhad and Horton 2006; Horton and Bruns 2001).Hypothesis 1: EM infection, species richness and Shannon Weiner diversity will decline along the transects using seedlings as the sample unit. Approach for evaluating hypothesis 1: I will use regression analyses with distance from existing pine stands as the independent variable and species richness and diversity as dependent variables. Expected results 1: I expect a species rich group of fungi will be observed on seedlings in and near existing pine stands with no clear dominants and a reduction in richness along the transects to a species poor group of fungi dominated by suilloids at increasing distances from pine stands.Objective 2 - Manipulative study 1 - Facilitated pine establishment with suilloids in sites with a previously low success rate of pine establishment: Inoculate pine seedlings in lab using suilloid spore slurries following a modified version of a protocol we have used previously (Ashkannejhad and Horton 2006). The fungi were collected at the Preserve this fall and are now stored in the refrigerator as spore slurries (good for at least 6 months). We will grow pitch pines from seed in a growth chamber with two inoculation treatments in otherwise sterile soil: live suilloid spore slurry and autoclaved suilloid spore slurry (to control for a fertilizer effect). Pitch pine seed has been collected from the Preserve and more are available for our use. After 8 10 months, 10 seedlings will be randomly selected from each inoculation treatment and used to verify the presence or absence of mycorrhizal root tips. The remaining seedlings will be planted at the Preserve in five replicate areas where locust has been removed. In each of the replicate areas we have locations where pines are and are not establishing, which we will use as treatments. Ten field seedlings will be planted in each plots (10 seedlings x 5 areas x 2 establishment zones) in the fall and harvested the following fall after one year of growth (the fall season is when mycorrhizal root tips and networks are most active). The fungi from the subset of laboratory bioassay seedlings and from the field bioassay seedlings will be identified following our standard molecular protocols.Hypothesis 2a: Establishment success of pine seedlings will be greater in areas where restoration of pine has been successful than in areas where restoration has not been successful. Hypothesis 2b: Establishment success of pine seedlings will be greater for seedlings inoculated with live suilloid spore slurries versus those with autoclaved spores slurries.Approach for evaluating Hypothesis 2a and 2b: This is an ANOVA experimental design with inoculation type (with/without) and previous establishment success (with/without) as treatments.Expected Results 2: I expect that a greater number of seedlings inoculated with suilloid fungi will become established in all sites. There will likely be an interaction between site and inoculation treatments. The critical result will be that sites with a previously low success rate of pine establishment will show greater success with seedlings inoculated with suilloid fungi.Objective 3 - Manipulative study 2 - Low tech facilitated pine establishment in difficult areas using suilloid fungi added to planting holes. Ten seedlings will be planted at the preserve in each of three replicate areas with poor success rates for pine establishment. At each area, we will add sporocarp tissue of suilloid fungi to the planting holes of five of the seedlings following (Nuñez et al. 2009), adding the equivalent of one mushroom and being sure the seedling roots are in contact with the fungal tissue. We will add autoclaved sporocarp tissue to planting holes of ten additional seedlings (to control for a fertilizer effect), again being sure that the seedling roots are in contact with the fungal tissue. Seedlings will be obtained from the Saratoga Tree Nursery, the source of seedlings used in the restoration effort at the Preserve. Seedlings will be planted in the fall and harvested the following fall after one year of growth. Ectomycorrhizal infection will be measured and the ectomycorrhizal fungi will be identified with molecular techniques as described below.Hypothesis 3: Establishment success of pine seedlings will be greater for seedlings planted with live suilloid mushrooms versus those with autoclaved sulloid mushrooms. Approach for evaluating Hypothesis 3: Parametric or non parametric t tests will be used to test for differences in establishment success for seedlings exposed to live or autoclave sporocarp tissue. Expected Results 3: I expect a statistically significant difference in seedling establishment between the two treatments, with live inoculant leading to greater establishment. Seedlings exposed to suilloid inoculant are expected to be dominated by suilloid fungi on their roots at harvest. If seedlings exposed to autoclaved fungal tissue survive, it is expected they will be colonized by a diverse group of ectomycorrhizal fungi existing as soil inoculant at the sites.Objective 4 - Manipulative study 3 - Low tech facilitated pine establishment in difficult areas using soil from local pine stands added to planting holes. Ten seedlings will be planted at the Preserve in five replicate areas with low rates of success for pine establishment. In this case, we will add soil from local pine stands to the planting holes following (Nuñez et al. 2009). The soil will either be alive or autoclaved (to control for a fertilizer effect). Seedlings will be obtained from the Saratoga Tree Nursery. Mycorrhizal infection and species will be identified on the seedlings.Hypothesis 4: Establishment success of pine seedlings will be greater for seedlings planted with live soil from nearby forest stands added to the planting hole than those with autoclaved soil added to the planting hole. Approach for evaluating Hypothesis 4: Depending on the normality of the data, a parametric or non parametric t test will be used to test for differences in seedling establishment success exposed to live or autoclaved inoculant.Expected Results 4: I expect a statistically significant difference in seedling establishment between the two treatments, with live inoculant leading to greater establishment.Citations:Ashkannejhad S, Horton TR (2006) Ectomycorrhizal ecology under primary succession on the coastal sand dunes: interactions involving Pinus contorta, suilloid fungi and deer. New Phytologist 169:345-354.Horton TR, Bruns TD (2001) The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Molecular Ecology 10:1855-1871.Nuñez MA, Horton TR, Simberloff D (2009) Lack of belowground mutualisms hinders Pinaceae invasions. Ecology 90:2352-2359.

Progress 08/15/16 to 09/30/19

Outputs
Target Audience:Target audiences: Interested public at the Albany Pine Bush. We gave several talks and posters to the public highlighting our restoration of pine barren habitat. Natural History enthusiasts: I organized a symposium on mycorrhizal fungi at th NENHC (North East Natural History Conference) in April 2019. My lab gave three talks supported by this funding at this symposium. My lab gave 2 lectures as part of the Albany Pine Bush Preserve Science Lecture series (one by Horton and one by Hudon). We also contributed 2 posters for display during a public overview of the science going on at the preserve (one by Patterson and one by Hudon). Colleagues: My students gave one poster and one talk at the 11th International Mycological Congress in San Juan Puerto Rico, July 2018. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The following two students completed their masters thesis: Taylor Patterson and Aimee Hudon. Both were trained in mycorrhizal ecology and the use of molecular techniques for identifying fungi. How have the results been disseminated to communities of interest?We presented two 60 minutes talks at the Albany Pine Bush Preserve, Albany NY 2017, 2018. We presented one talk at the MassMyco Conference in Cambridge, MA October 2018. We presented one poster and one talk at the International Mycological Congress in San Juan, Puerto Rico July 2018. We presented two talks at the Northeastern Natural History Conference in Springfield, MA April 2019. Dissertations by Taylor Patterson and Aimee Hudon were given to the Albany Pine Bush Preserve Discovery Center. What do you plan to do during the next reporting period to accomplish the goals?Aimee Hudon is preparing her collection of fungi for the Albany Pine Bush Preserve Discovery center as voucher specimens.

Impacts
What was accomplished under these goals? 1. Taylor Patterson demonstrated that planting pitch pine seedlings inoculated with ectomycorrhizal fungus spores increased seedling establishment on the Albany Pine Bush Preserve. We used spores collected from Suillus and Rhizopogon, both collecte from the Albany Pine Bush Preserve. We used locally collected fungi to avoid introducing novel genotypes. 2. Aimee Hudon conducted the first formal survey of fungi and lichen on the Albany Pine Bush Preserve. She collected 142 species in 65 genera (including two slime molds). This is nowhere a complete inventory, but serves as a baseline for future work. 3. Aimee Hudon characterize the mycorrhizal fungal assemblage of Quercus ilicifolia (scrub oak) in the Albany Pine Bush Preserve. Fungi on root tips were identified using molecular techniques. A different assemblage of ectomycorrhizal fungi was observed on oak roots collected from the field site than on greenhouse seedlings grown in soils collected from the site. In addition, a greater diversity of ectomycorrhizal fungi was observed in the field collected samples than the laboratory grown seedlings. These results suggest some fungal species occur in soils as a resistant spore bank, a feature observe in fire adapted pine communities. These results help elucidate how forests recover from disturbance and inform restoration efforts in the area.

Publications

• Type: Theses/Dissertations Status: Accepted Year Published: 2019 Citation: Patterson, Taylor, Legacy of Robinia pseudoacacia invasion and use of ectomycorrhizal fungi to restore Pinus rigida in the Albany Pine Bush Preserve, NY. 2019. Dissertation. 66.
• Type: Theses/Dissertations Status: Accepted Year Published: 2019 Citation: Hudon, Aimee, Fungi of the Albany Pine Bush Preserve, Albany, New York and Assemblage structure of ectomycorrhizal fungi on Quercus ilicifolia (scrub Oak) roots in fire-adapted pine barrens. 2019. 106.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience: Nothing Reported Changes/Problems:I added a second master's student on this project. Aimee Hudon conducted work on the mycorrhizl ecology of Quercus Illicifolia, another plants species involved in the restoration effort at the Albany Pine Bush Preserve What opportunities for training and professional development has the project provided?Graduate student Taylor Patterson is finishing his MS focused on this project. Taylor also presented his work in an oral presentation at an international conference (IMC11, Puerto Rico, July 2018). How have the results been disseminated to communities of interest?In addition to items listed previously, we presented this work at the 11th International Mycological Congress in Puerto Rico as an invited talk. What do you plan to do during the next reporting period to accomplish the goals?Faclitate Taylor Patterson's completion of his masters degree. Facilitate publication of Patterson's work.

Impacts
What was accomplished under these goals? MS student Taylor Patterson took care of these goals. His thesis is under review by the by his graduate committee. He will defend in April. Details of his work will be submitted in the final report.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Hudon AT, Horton TR. Assemblage structure of ectomycorrhizal fungi on scrub oak (Quercus ilicifolia) roots in fire adapted pine barrens. Poster. 11th International Mycological Congress. July 2018, San Juan, Puerto Rico.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Patterson TR, Horton TR. Increasing success of pitch pine restoration in the Albany Pine Bush Preserve using suilloid fungi. Invited Talk. 11th International Mycological Congress. July 2018, San Juan, Puerto Rico.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The data from these projects will inform efforts in pine and oak restoration. Planting of pine at the Albany Pine Bush Preserve has failed in some areas where invasive locust was removed. By inoculating pine seedlings with mycorrhizal fungi we expect to see better establishment success. We have quite a bit of data to suggest the inoculation methodology will work on the pines. The work has shown that pine specific mycorrhizal fungi produce resistant spore banks analogous to resistant seed banks, both of which support establishment of plants following disturbance (locust invasion and removal in this case) and we are using this knowledge in our restoration effort. There are no studies investigating this phenomenon with oak. We do know that the fungi supporting pine establishment are pine specific and cannot be used for oak establishment. We are therefore generating baseline data to see if resistant propagules occur in soils that will support oak establishment following disturbance. Beyond the local and regional practioners interested in maintaining pitch pine and scrub oak plant communities (and their associated species including the Karner Blue butterfly), Conservation and Restoration Ecologists will be generally interested in this work. Changes/Problems:Because there were few mushroom produced and harvested in the first year of this project, we had to adjust the project. This resulted in a delay of about one year in getting the experiments in place. The project start date was August 15, 2016. We were able to collect mycorrhizal roots from the field and grow inoculated seedlings in the growth chamber fall 2016 - Spring 2017. But the planting of those seedlings into the field had to wait until spring 2017, and the harvest of those seedlings had to wait until the fall of 2017. This was all accomplished and all the fieldwork is now done. However, the PCR-based work to identify the fungi from the seedlings was delayed by about one year. What opportunities for training and professional development has the project provided? Taylor Patterson, MS student. Finishing summer 2018. Submitted abstract for the 11th International Mycological Congress in Puerto Rico to present his pine restoration data (accepted - as an oral symposium presentation). Aimee Hudon, MS student. Finishing Fall 2018. Submitted abstract at the 11th International Mycological Congress in Puerto Rico to present her oak restoration data (accepted as poster). Both Taylor and Aimee have been successful TAs, gaining valuable teaching experience. How have the results been disseminated to communities of interest?One book chapter: 1. Horton TR (2017) Spore Dispersal in Ectomycorrhizal Fungi at Fine and Regional Scales. In: Tedersoo L ed. Biogeography of Mycorrhizal Symbiosis: Ecological Studies Vol. 230. Springer. pp. 61-78. Two talks: Horton TR. Ectomycorrhizal fungi from resistant spore banks support post-fire pines. Northeastern Natural History Conference. Cromwell, Connecticut. April 21 2018. Horton TR. Ectomycorrhizal fungi from resistant spore banks support post-fire pines. Invited talk, SUNY-Delphi. October 26, 2018. One Poster: Taylor P, Horton TR. Improving pitch pine restoration in the Albany Pine Bush Preserve using ectomycorrhizal fungi. Research Symposium, Albany Pine Bush Preserve. What do you plan to do during the next reporting period to accomplish the goals?Pine Restoration: Mycorrhizal fungi from pine seedlings grown in the lab will be identified with PCR-based methods. This will allow us to know what fungi were on the seedlings when out planted and learn whether those fungi survived the field conditions or replaced by fungi in the field soils. Oak Restoration: Mycorrhizal fungi from oak roots collected in the field will be identified with PCR-based methods. This will allow us to know what fungi were on the oaks in the field and compare to this to the list of species found on seedlings grown in the field soils under growth chamber conditions. The second list will show which fungi are in the soils as resistant propagules and might be exploited for restoration efforts of oak at the Albany Pine Bush Preserve. Data from these projects will be presented in local meetings and conferences.

Impacts
What was accomplished under these goals? We attempted to collect enough suilloids mushrooms to add to pine seedlings planting holes but the 2017 fruiting season was not very productive Considering the poor of mushroom production in the 2017 season, the objective was modified to address the question whether seedlings inoculated with mycorrhizal spore slurries would survive better than non-mycorrhizal seedlings. We used the mushrooms we did find to make spore slurries, which offered enough inoculum for the new objective (adding spores to germinating seedlings in the growth chamber) but not enough for the old objective (adding spores to each planting hole in the field). The seedlings were grown in a growth chamber for one year and then out planted in mature pitch pine stands or in areas where invasive locust trees had recently been removed. We met with Dr. Jeff Corbin from Union College who is characterizing soil nutrient dynamics including rates of nitrogen cycling to share data between the two labs. We discussed plans to submit a manuscript that investigates restoration success at the Albany Pine Bush Preserve that combines data from the two labs. Although the Albany Pine Bush Preserve Commission labels the restored habitat for the Karner Blue butterfly as 'Pine Barrens', it is actually a mixed plant community with pitch pine and scrub oak as co-dominant species. I brought on an excellent second MS student to investigate the mycorrhizal condition of scrub oak to better inform the restoration effort. Both graduate students designed their field and laboratory experiments and met with Dr. Stehman to check that the experimental plans were statistical sound. Pine Restoration: Taylor Patterson (MS) planted his pine seedlings at the Albany Pine Bush Preserve. The seedlings were grown in a growth chamber non-mycorrhizal or mycorrhizal with inoculum from mushrooms harvested at the Preserve. The seedlings were planted in two forest treatments. One treatment was areas where invasive locust had been removed within the past year. The other treatment was mature pine stands that had never been invaded by locust. Oak Restoration: Aimee Hudon (MS) harvested oak mycorrhizae from the Albany Pine Bush Preserve. Roots were cleaned of soil and stored in CTAB buffer until they can be further processed with PCR-based methods to identify the mycorrhizal fungi. At the same time, soils were harvested from these samples and used to set up a growth chamber bioassay to learn what mycorrhizal fungi are in the soils as resistant propagules capable of supporting oak establishment following disturbance.

Publications

• Type: Other Status: Published Year Published: 2017 Citation: Taylor P, Horton TR. Improving pitch pine restoration in the Albany Pine Bush Preserve using ectomycorrhizal fungi. Research Symposium, Albany Pine Bush Preserve.
• Type: Book Chapters Status: Published Year Published: 2017 Citation: Horton TR (2017) Spore Dispersal in Ectomycorrhizal Fungi at Fine and Regional Scales. In: Tedersoo L ed. Biogeography of Mycorrhizal Symbiosis: Ecological Studies Vol. 230. Springer. pp. 61-78.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 1. Horton TR. Ectomycorrhizal fungi from resistant spore banks support post-fire pines. Northeastern Natural History Conference. Cromwell, Connecticut. April 21, 2017.

Progress 08/15/16 to 09/30/16

Outputs
Target Audience:I gave a lecture on our restoration efforts at the Albany Pine Bush Preserve. Target: Public visiting the Albany Pine Bush Preserve. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?I am now training my new masters student Taylor Patterson on mycorrhizal ecology. In addition, I recruited Stephen Garney, an undergraduate student, to help us on this project. How have the results been disseminated to communities of interest?Public lecture What do you plan to do during the next reporting period to accomplish the goals?Taylor Patterson (MS student) will continue his effort to understand how mycorrhizal fungi at the site can best be used to increase restoration success.

Impacts
What was accomplished under these goals? We collected sporocarps of two ectomycorrhizal genera that are important for pine establishment at the preserve: Suillus and Rhizopogon. We made spore slurries from these fungi and inoculated pitch pine seedlings with the slurries. A check of the roots on a random selection of seedlings 4 months later showed they were colonized by the fungi. These seedlings will be planted at the preserve as part of Taylor Patterson's MS project.

Publications