Final Report for OS13-082
The pecan (Carya illinoinensis) is an economically important, native tree that is prevalent throughout the Southern USA. The USA produces >80% of the world’s pecans (worth $674 million in 2010) and the vast majority of pecans come from Southeastern states (Giesler, 2011). Although the pecan business has been extremely successful in recent years, the problems of alternate bearing trees, price fluctuations, and disease pressure remain problematic for growers (Anderson et al. 2012; Anderson & Cocker, 2012)
Pecan trees bear an exceptional nut crop in the canopy, but they also have an underutilized potential to yield a lucrative supplementary crop from their roots. The native pecan truffle (Tuber lyonii) is an ectomycorrhizal (ECM) fungus that forms a mutualistic symbiosis with pecan trees. Truffles are subterranean fungal fruiting bodies and T. lyonii is a native species that is related to the prized European truffles (Bonito et al., 2011). Symbiotic ECM fungi enhance plant health by increasing nutrient absorption, mitigating drought stress, and limiting root disease (Smith & Read, 2009).
Given their high culinary and economic value, truffles are the ultimate specialty crop. Valued at $160–320 per pound, pecan truffles are candidates for commercial production but have been neglected due to a lack of information about overall biology and production practices (Smith et al., 2012). However, pecan truffles are common and widespread in pecan-growing regions of the Southeast and some orchards can produce two or more pounds of truffles per acre per year (Smith & Brenneman, personal observation). This truffle fruits August to November on a variety of soil types. Unfortunately, like other symbiotic ECM fungi, pecan truffles cannot reproduce in the lab - they can only be cultivated with a living host tree.
Studies on the pecan truffle are timely because European truffle harvests have dwindled while global demand has grown. In the Pacific Northwest, interest in locally produced foods has driven a boom in the new truffle economy with >$300,000 of truffle income per year and additional revenues from associated businesses (Pilz et al., 2009). The pecan-truffle agro-ecosystem has the potential for even greater success due to the simultaneous harvest of pecans and truffles.
Like many other truffles, pecan truffles proliferate in habitats with soil disturbance, limited litter, and high soil pH so pecan orchards are an excellent truffle habitat (Bonito et al., 2011). Preliminary studies suggest that minor changes in nursery and orchard management could facilitate sustainable production of the pecan truffle with minimal costs to growers and positive impacts on trees. Production of this symbiotic, edible truffle will increase the sustainability of the pecan agro-ecosystem by providing important supplemental income. For nurseries, the production of truffle-inoculated pecan trees will ensure premium prices and allow the nurseries to sell to a wider audience that includes both pecan growers and homeowners. Lastly, the new truffle crop will generate regional economic benefits by spurring truffle-associated businesses such as truffle dog training, truffle marketing, and “food tourism” (Pilz et al., 2009).
The best approach to promote Tuber lyonii cultivation on pecans is to begin in the nursery. Data from European truffle production suggests that successful colonization of seedling roots with truffles is one of the most important factors in determining success of truffle orchards (Hall et al., 2007; Bonet et al., 2009). Accordingly, we are working with nurseries to:
1) identify the current distribution of the pecan truffle on seedlings in nurseries, and
2) develop protocols to inoculate pecan seedlings in nurseries with minimal changes in operating procedures.
Secondary objectives that developed over the course of the research project also included identify the main fungi that compete with Tuber lyonii in the nursery setting and determine whether Tuber lyonii is actually one species or actually part of a species complex.
To achieve our two main objectives we sampled ectomycorrhizal roots from pecan seedlings in typical pecan nurseries (mostly in Georgia and Florida but also from select nurseries in other Southeastern States as well) and also sampled ectomycorrhizal roots from experimental treatments in field nurseries that had been inoculated with Tuber lyonii spore slurries. Roots were isolated and washed, visualized under dissecting microscope, and then DNA was extracted using a Soil DNA kit after we homogenized roots in a tissue grinder. PCR was used to amplify fungal ITS ribosomal DNA and then PCR amplicons were sequenced using several methods (Sanger sequencing for individual roots or 454 pyrosequencing or Illumina MiSeq for pooled samples). To identify fungi on the roots the ITS rDNA was analyzed by DNA alignments and/or comparisons with known fungi on GenBank.
The project was successful in some ways but suffered some setbacks in other ways. Graduate student Maxwell Reitman received his Master's Thesis in Summer 2015 for his research on Tuber lyonii. Although this was a major milestone, he had been slated to complete a PhD in the Plant Pathology graduate program and some of the project difficulties arose due to his rapid and unexpected departure. On a more positive note, PI Smith was able to leverage support from the SARE grant to obtain additional funding from the University of Florida for an internal "Early Career Award" (which created additional possibilities to further study Tuber lyonii on pecans). The combination of these funds and preliminary data facilitated the arrival of a new PhD student, Arthur Grupe, in Fall 2015. Mr. Grupe is currently working on the project (with non-SARE funds) and is scheduled to complete his PhD dissertation in Summer 2019.
Objective 1: identify the current distribution of the pecan truffle on seedlings in nurseries.
This objective was partially achieved in that Sanger sequencing did identify Tuber lyonii on some roots from pecan nurseries. However, the distribution was spotty and the full dataset is incomplete. Mr. Grupe is currently working with ectomycorrhizal fungi data from Illumina MiSeq to more completely address the distribution of Tuber lyonii on seedlings. Additional data was obtained from mature pecan trees in the vicinity of nurseries. The data from mature trees (in multiple sites in FL and GA) indicates that mature trees host a wide variety of fungi (more than 50 species) and that the ECM fungi on nursery seedlings are mostly a small subset of disturbance-adapted specialists - including Tuber lyonii and other Tuber species. We identified several species of Tuber, Pachyphlodes, Pezizaceae, Scleroderma, and Tomentella as the main nursery-associated fungi, regardless of the site. A more detailed dataset that will also incorporate correlations with edaphic factors will be available soon. Mr. Grupe will present a poster on pecan-associated fungi from pecan nurseries at the Mycological Society of American meeting in August 2016.
Objective 2: Develop protocols to inoculate pecan seedlings in nurseries with minimal changes in operating procedures.
The results for year 1 were incomplete due to the rapid departure of Maxwell Reitman from the project. However, Mr. Grupe and Dr. Smith completed sampling of 2-year old pecan nursery seedlings in Fall 2015 and the MiSeq Illumina data should reveal more about those communities soon (see above). Preliminary morphological and Sanger sequencing, however, suggest that nursery seedling colonization by Tuber lyonii was not as successful as hoped. Many seedlings in the nursery after two years had large root systems with several different ectomycorrhizal types. Some of the Sanger sequencing from these produced non-Tuber lyonii sequences, indicating that other fungi are present. The extent of the contaminant fungi will be clear in the near future.
We also determined during the course of this project that Tuber lyonii is a species complex but that truffles in pecan orchards apparently belong to only one genetic lineage within this group. The species complex is comprised of ca. 11 different molecular species but many of these are restricted to particular regions of North America (e.g. northern Mexico, New England). In contrast, the main truffle lineage that is found in pecan orchards seems to be widespread in human-dominated and disturbed environments from Quebec to New Mexico to Florida.
Additional studies on mature trees by a postdoctoral researcher in the lab (Zaiwei Ge) indicate that Tuber lyonii is widespread but patchy on the root systems of adult trees. Sampling of adult trees using a haphazard method showed relatively poor correlation with truffle production in orchards, indicating that random or haphazard sampling of orchards is likely not the most effective approach. These data and other anecdotal observations suggest that the most effective way to identify Tuber lyonii production sites and to determine the presence of the truffles is to use dogs to assist searches.
Educational & Outreach Activities
Reitman, Maxwell. 2015. Further Explorations of the Tuber lyonii species complex. Master's Thesis in Plant Pathology. University of Florida
Healy RA, Bonito GM, Smith ME. (2016) A Brief Overview of the Systematics, Taxonomy, and Ecology of the Tuber rufum Clade. In: True Truffle (Tuber spp.) in the World.Eds. Alessandra Zambonelli et al. Springer. www.springer.com/in/book/9783319314341
Pecan Truffle Website (maintained by collaborator Dr. Tim Brenneman)
- The ectomycorrhizal fungi are highly variable in pecan nurseries and orchards, suggesting that many competing fungi are present in most environments. This suggests that future work may need to focus on specific sites for Tuber lyonii propagation (e.g. distant from other pecans, oaks, or other ectomycorrhizal host plants) or may need to use clean greenhouses. Complete results from studies by Mr. Grupe (currently underway) will help to give a more complete answer.
- Tuber lyonii can colonize seedlings but may not be a dominant competitor in the nursery environment. Preliminary results from second year seedlings suggest that it is not the lone colonizer of roots as we had hoped. However, the full dataset should be available by Summer 2016 and may provide more clues.
- Data on the diversity within the Tuber lyonii complex suggests that all of the truffles in orchards belong to the same genetic lineage and therefore are likely to produce truffles of a similar taste and smell and also to respond similarly during cultivation efforts.
- Pecan-associated ectomycorrhizal fungi in the pecan nurseries are mostly a subset of the larger ectomycorrhizal community that is found on mature pecans. These are likely the most disturbance-adapted taxa in the community and will be the taxa that are the main competitors of Tuber lyonii.
Thus far only four farmers are actively working with our research group and/or attempting to inoculate pecan seedlings with Tuber lyonii. However, depending on the results from this year's trials we expect that more farmers will be interested to adopt practices to enhance the production of Pecan Truffles. The results of "in-nursery" inoculation experiments are still not complete so we are not yet sure if this method will be highly successful or not and therefore cannot yet advise the farmers with great confidence. However, the number of contacts via email and phone have increased each year and we now know of two small farmers who are regularly selling Pecan Truffles. As interest continues to grow we expect more farmers will attempt to grow truffle-inoculated trees.
Areas needing additional study
Future research will obtain definitive answers to some of the questions above (later in 2016) - see above. Moving beyond 2016 it will be critical to test the effectiveness of nursery-grown vs. greenhouse-grown pecan seedlings inoculated with Tuber lyonii to determine whether greenhouse seedlings are more effective and therefore more cost effective too. Additional studies should focus on sustainable ways to reduce competition to favor the growth of Tuber lyonii over other species on the root systems of seedlings.