Progress 10/01/11 to 09/30/15
Outputs
Target Audience:The target audience are the many farmers engaged in reforestation of harvested trees, conversion of traditional crop fields to forests, and the production of pecans in North Carolina. Limited resources farmers who can utilize small acreage to grow such a high dollar crop are also the target recipient of the technology developed. North Carolina ranks 10th in pecan production in the US, and has an estimated 3.3 million acres of planted pine trees. On the other hand truffles are one of the most expensive coveted delicacies that have a potential to become highly profitable crop in the state of North Carolina. However, truffle farming which is still in its infancy across the US is fraught with many production problems and uncertainties, because of unavailable information on productions methods. The research conducted was focused one of the major steps to develop truffle production methods by co-farming truffles with loblolly pines and pecan trees in North Carolina. The research and methods developed so far does not represent a complete set of all the information a farmer needs to produce truffles. However, successful inoculation of seedlings with target truffles, their propagation in the greenhouse and out-planting in the field were achieved. Progress and method developed so far was on display to farmers during the Small Farms field day at North Carolina A&T State University in 2015. Changes/Problems: The inability to find truffles for use in inoculation at the beginning of the project resulted in the delay of seedlings production. Attempt to use mycelia based inoculation failed due to slow growth rate and the low number of individuals involved in the mycorrhization of root system. The lack of access to the proposed research plot in Rose Hill, NC led to the non-establishment and testing of the inoculated seedlings at a location outside the region of NC A&T School farm. What opportunities for training and professional development has the project provided?The PI and staff attended the following training opportunities: CUR-HBCU Grant Writing Workshop, Johnson Smith University, Charlotte. 2012 iPlant Tools and Services Workshop. NC State University, Raleigh, NC. May 31-June 1, 2012. Truffle hunting in the wild and collection training, by Prof Alessandra Zambonelli, Bologna University, Italy. March 4-8, 2013 Truffle collection, isolation, morphological characterization, Identification training by Prof Alessandra Zambonelli, Bologna University, Italy. March 25-28, 2014 Microbial Genomics & Metagenomics workshop, Joint Genome Institute, Walnut Creek, California. May 5-9, 2014 Genome Access Course. Cold Spring Habor. New York Genome Center. September 2-4, 2014. Droplet Digital PCR (ddPCR) 2014. CDC Biotechnology CORE Facility and Bio-Rad Laboratories, Emory Conference Center Hotel, Emory University, Atlanta. . September 17, 2014 How have the results been disseminated to communities of interest? Methods and protocols developed so far was on display to farmers during the Small Farms Field Day at North Carolina A&T State University in 2015 Knowledge from the project was used to teach participants how to prepare field plots for use as truffle orchards as well as the morphology and microscopic characteristics of truffle infected roots on loblolly pines. This was at the truffle orchard demonstration day organized by Mycorrhiza Biotech LLC., in Gibsonville, NC on April 29, 2014. A presentation on the potential for truffle farming utilizing loblolly pines was given to the Society of American Foresters, North Carolina Branch, in January 2014. Macro and micro morphology of truffle inoculated pine roots were demonstrated to participants at the meeting. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Major activities completed: Protocols for inoculating Tuber borchii onto loblolly pines and T. lyonii onto pecan seedlings were developed. Marginal shift in microbial communities was detected pre/post liming activities in the field plots. The Presence of T. borchii and T. lyonii on the roots systems of loblolly pine and pecan seedlings out-planted to field plots were confirmed. Specific objectives met Propagation and inoculation of loblolly pine seedlings with truffle (Tuber borchii) and pecan with native truffle (T. lyonii); Completed. Establishing a test plot at A&T SU Research Farm and an offsite plot located in Rose Hill Duplin County, NC. Only filed plots at NC A&T SU were established. Screening of microbial community for the presence of organisms capable of competing against the truffle; completed. Evaluation of quality (percent of root system colonization) of seedlings to be out-planted into plots; Completed Monitoring for the presence and abundance of truffle mycorrhizae throughout out-planted seedlings; completed. Significant results achieved The project focused on understanding the initial stages of starting a truffle farm. Note that truffle farming takes a minimum of six years after out-planting to yield truffles; and it takes one a half to two years to produce truffles. Therefore the significant results in this project lays the foundation for further research to improve seedling inoculation an d propagation practices, as well as the research into growth and development through truffle production in out-planted seedlings. The major results achieved are: Stratification of Pine and pecan seeds to initiate seed germination were successful flowing the existing protocols in the nursery industry. Inoculation of seedlings in both plants should not start until the seedlings are at least 90 days old, when secondary root systems starts to develop. Watering inoculated seedlings in the greenhouse should not be more than once per week. Nutrient management in seedlings during the long stay in the greenhouse is critical. The limitation that fertilizer application can have on seedling/mycorrhization led to a new grant proposal and project that is ongoing. Soils in field plots does have very diverse fungi, including some that could compete against the target truffles and heaving liming did not result in significant change in the microbial diversity within the test period. All out-planted seedlings of (pine inoculated with T. borchii and pecan inoculated with T. lyonii) were positive in DNA testing for their presence in the root systems six months post planting. One plot each (about an acre in size) for T. borchi/pine and T. lyonii/pecan have been established at NCA&T farm. This is the first in a University in the entire southeastern US. It will be useful for further and training of farmers and extension agents. NCA&T has become the largest holder of T. borchii and T. lyonii culture strains in the whole to the US. The overall knowledge gained from this project has raised many questions upon which our current research is being driving. Now were are asking the following questions: What is the effect of fertilizer application on root mycorrhization vis-à-vis inoculated plant growth and development? Two major research proposal have been submitted: USDA-NIFA Specialty crop grant:Advancing truffle Farming in the U.S. through Precision Agricultural Technologies ($5,000,000) NSF-DEB grant: Preliminary Proposal RUI "Studies on the ecology of native trees in the Mid-Atlantic Forests and tree orchards that are in symbiosis with hypogenous, truffle-producing fungi" A grant proposal submitted to the Capacity Building Grant opportunity of USDA-NIFA in 2014. The proposal title is: Nutrient supplementation and identification of helper microbes for enhanced seedling growth and mycorrhization of truffle inoculated pine and pecan seedlings (A resubmission is in the works).
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Osejie O, Anike F. N., Isikhuemhen O. S. Requirements for in vitro mycelia growth of Tuber borchii Vittad. Mid Atlantic States Mycology Conference (MASMC), April 26-28 2013, Beltsville MD.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
F. N. Anike, O. S. Isikhuemhen. Growth Requirement for in vitro Cultivation of Tuber lyonii biomass. IMMC7 2013, Beijing, China.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
O. Oriaifo, F. N. Anike, O. S. Isikhuemhen. Requirements for in vitro mycelia growth of Tuber borchii. IMMC7 2013, Beijing, China.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2015
Citation:
Osejie Oriaifo. In vitro Mycelial Growth and Root Infection of Loblolly Pine Seedlings by Bianchetto Truffle (Tuber borchii)
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Co-farming truffles with loblolly pines and pecan trees will benefit the many farmers engaged in reforestation of harvested forests, conversion of traditional crop fields to forests, and the production of pecans in North Carolina. The development of truffle production methods, which is the expected result from the proposed research, will ensure a high return on investment in pine forests and pecan farms, and meet the forest and soil conservation needs of USDA. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Training activities: Five students and two research associates had the opportunities to be trained in the field of truffle research. Professional development 1. The PI and a research associate attended a training course on Microbial Genomics & Metagenomics at The Joint Genome Institute to learn modern tools for microbial diversity assessment in environmental samples. 2. The PI and a research associate attended a training course (Genome Access) at the New York Genome Center, to learn bioinformatics and analysis of sequence information. How have the results been disseminated to communities of interest? Results to date have been communicated as follows: 1. Presentation: Society of the American Foresters, North Carolina Branch. 2. Field day demonstration, organized by Mycorrhiza Biotech, LLC. 3. A master's degree thesis has been published. What do you plan to do during the next reporting period to accomplish the goals? Further evaluation of quality (percent of root system colonization) of seedlings to be out-planted into plots: The recommendation to re-inoculate seedlings has been performed. The morphological and DNA based evaluation of seedlings to be out-planted into field plots will be conducted in October - December 2014, before out-planting to field plots. Monitoring for the presence and abundance of truffle mycorrhizae throughout out-planted seedlings: Twenty-five percent of out-planted seedlings will be randomly selected for root sampling. The screening will be carried out in June/July 2015. This work will provide data about the rate of mycorrhization of the seedlings after out-planting, and will allow tracking changes in microbiota in truffle plots after introduction of mycorrhized seedlings. Workshop for farmers and extension agents. A one-day workshop for farmers and extension agents will be conducted within the Small Farmers Week activity organized by the Cooperative Extension at N.C. A&T. The workshop will cover the basics of truffle cultivation (site selection, soil prepping, inoculated seedlings quality, etc.). Farmers and Extension agents will visit the demonstration plot. The workshop will be an important step toward establishing a network of farmers and Extension agents for truffle cultivation. The PI and Co-PI will communicate results and achievements of the project during the workshop.
Impacts
What was accomplished under these goals?
Evaluation of seedlings to be out-planted into demonstration field plots at the N.C. A&T University Farm were conducted. In loblolly pines and pecan seedlings, the average level of mycorrhization was less than 20%. We sought the advice of Drs. A. Zambonelli and M. Iotti from Bologna University, Italy, and were advised to re-inoculate the seedlings to increase levels of mycorrhization prior to out-planting. Re-inoculation has been completed and the plants will be ready for out-planting by October-December 2014.
Publications
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Co-farming truffles with loblolly pines and pecan trees will benefit the many farmers engaged in reforestation of harvested trees, conversion of traditional crop fields to forests, and the production of pecans in North Carolina. The development of truffle production methods, which is the expected result from the proposed research will ensure a high return on investment in pine forests and pecan farms and meet the forest and soil conservation needs of USDA. Changes/Problems: As indicated in the previous year’s report, the time to complete this project has been pushed forward at least one year. We have serious challenges in nutrient management in seedling propagation. Fertilizers cannot be applied to the seedlings as it is and at the rate used in the nursery today. This is because such levels of fertilizer application could kill or significantly impact the growth of the target truffle fungi and their ability to colonize the roots of the plants. This problem is so significant that it has formed the basis for our writing another grant to look into the interactions between nutrient application, seedling and target fungi growth and development in the greenhouse. What opportunities for training and professional development has the project provided? A master’s degree student involved in this project will write a thesis and graduate in May, 2014. Two undergraduate students have been added to the project. The learning opportunities are: The undergraduate students are learning truffle biomass generation under optimized conditions in vitro. The extraction of DNA from soil samples and PCR amplification of soil fungi micro-biota. Students prepared two abstracts that have been presented at an International Conference (www.IMMC7.org), in China in September 2013. All students in the project will attend the upcoming MASMC in Appalachian State University in April, 2014. How have the results been disseminated to communities of interest? Two presentations at an international conference (IMMC 7, Beijing 2013) A major presentation on truffle farming at the Society for American Foresters meeting, Charleston South Carolina, October 23-27, 2013. The title was: Diversifying Timber Crops with Gourmet Truffles. What do you plan to do during the next reporting period to accomplish the goals? Continue to work on the project as previously outlined in the proposed objectives not completed so far, as follows: Evaluation of quality of seedlings to be out planted to plots. Screening of inoculated seedlings for successful mycorrhization, Out planting to field plots and screening of out-planted seedlings for survival of truffle fungi. Comparative analysis of microbial diversity pre- and post-soil remediation prior to out planting and during inoculated plant growth in test plots.
Impacts
What was accomplished under these goals?
a). Propagation and inoculation of loblolly pine seedlings with truffle (Tuber borchii ) and pecan with native truffle (T. lyonii) – Tuber lyonii truffles were available in October 2013. Fresh collections were used for pure culture isolation. This is a slow process. However, using the preferred media composition already developed and reported earlier, we have 12 new pure culture isolates of T. lyonii. The truffle fruit body inoculation just began and we have successfully inoculated some of the seedlings. Further studies on nutritional requirement for mycelia growth were conducted on Tuber borchii. The study determined the optimum carbon and nitrogen source concentrations for best mycelia growth in pure culture. Results indicated that a medium containing 5 g/L soluble starch and 15 g/L yeast extract best support mycelia growth. Truffles of T. borchii were used to inoculate germinated pine seedlings in October 2013. b). The PI is establishing atest plots at the N.C. A&T University Farm and at an offsite plot located in Rose Hill, in Duplin County, NC; The second round of lime application was performed, and results of analysis at a test laboratory are pending. c). Screening of a microbial community for the presence of organisms capable of competing against the truffle was conducted. Test plots were sampled again. Amplification of all DNA samples using ITSI and ITSII primers were successful. Amplified PCR products were cleaned, followed by a sequencing reaction and these were later sent to MWG-Operon in Alabama for sequence elucidation. Further analysis and assemblage of sequences obtained is ongoing. d). Evaluation of quality (percent of root system colonization) of seedlings to be out-planted into plots: No activity yet. e). Monitoring for the presence and abundance of truffle mycorrhizae throughout outplanted seedlings: No activity yet
Publications
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The goals and objectives of this project were: a) Propagation and inoculation of pecan with native truffle (T. lyonii and loblolly pine seedlings with truffle (Tuber borchii ): T. lyonii: The growth rate of 4 strains (MBFBL 1289, 1290, 1291, and 1293) was conducted. The pH range was from 4.0 to 9.0, the temperatures tested were 15C to 35C, and the carbon sources used in this experiment were glucose, sucrose, fructose, soluble starch, mannose, xylose, sorbitol and mannitol. For each of the parameters, the strains were observed for four weeks. Seeds of pecan were acquired and are being stratified for germination. This process takes 9 month and it is ongoing. T. borchii: Six isolates (MBFBL 1320, 1321, 1322, 1323, 1324, and 1325) were studied just as for T. lyonii above. pH ranges tested were from 4.0 to 9.0, with 0.5 increments and the addition of 6.6. Temperatures tested were 15C to 30C only and glycerol was tested, in addition to sugars used above. Loblolly pine seeds were acquired, stratified and germinated. This process takes about three months for stratification, which was followed by test germination and seedlings development. b) Establishing a test plot at A&T SU Research Farm and an offsite plot located in Rose Hill Duplin County, NC: Three plots at the North Carolina A&T State University School farm have been identified. A one acre plot was also located at the NCCFRF facility in Rose Hill, NC. The plots have been sampled for analysis of pH and soil characteristics. The plots have been tilled and adequate amounts of lime applied to raise the pH to 8. c) Screening of microbial community for the presence of organisms capable of competing against the truffle: Soil samples were obtained from the identified plots in b. above and used for DNA extraction. DNA extraction was done on 27 samples from Plot 66, 31 from plot 67, 24 from plot 69 and 20 from the Rose Hill plot. Four pooled DNA samples were made per plot, except Rose Hill Plot, where 6 were made; the extra being from outliers. These pooled samples (psDNA) were used for PCR amplification with ITS 1 and 4 primers. Resulting PCR products were cloned into competent E. coli cells. Twenty three clones per psDNA sample were selected and used for colony PCR using same primers (ITS 1&4). Resulting PCR products (1,692 in total for all psDNA) were exo-sap cleaned and sent for sequencing at MWG-Operon laboratories. Results from the sequencing facility are currently being analyzed. PARTICIPANTS: The PI, Dr. O. S. Isikhuemhen worked on this project during the period covered by this report. Dr. F. N. Anike and Mr. Lynn Luffman assisted with this project. Three graduate assistants also worked on this project and two of them will write their thesis from part of the studies in this project TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: To our surprise, a county agent brought to our lab one truffle. Microscopic examination proved it to have truffle-like spores. It was found growing on pen oak in Danbury, Stokes County. DNA-PCR analysis showed it to amplify with T. lyonii specific oligonucleotide primers. Four culture isolates were made from fruit bodies collected under one pen oak tree, and are now stored in our culture bank. This find was a surprise and it being genetically similar to T. lyonii and found here in North Carolina has led us to include it in our T. lyonii studies. Initial characterization for temperature, pH, carbon and nitrogen source requirements are ongoing.
Impacts
a) Propagation and inoculation of pecan with native truffle (T. lyonii and loblolly pine seedlings with truffle (Tuber borchii ): T. lyonii: This is the first time growth studies have been conducted on T. lyonii. There are variations in the temperature and pH requirements, as well as the preferred carbon source for best growth in the tested strains. In the strain MBFBL 1289 the optimum pH was 8.5; optimum temperature was 2C,; best carbon source was sucrose and the second best carbon source was starch. For the strain MBFBL 129, optimum pH was 6.5, optimum temperature was 25C, best carbon source was sucrose and the second best carbon source was starch. For MBFBL 1291, optimum pH was 8, optimum temperature was 25C, best carbon source was sucrose and the second best carbon source was starch. Finally for strain MBFBL 1293, optimum pH was 8, optimum temperature was 25C, best carbon source was mannose and the second best carbon source was glucose. T. borchii: Simialr to T. lyonii, there are variations in the temperature, and pH requirements, as well as the preferred carbon source for best growth in the tested strains. In strain MBFBL 1320, optimum pH was 7.0, optimum temperature was 20C, best carbon source was glycerol and the second best carbon source was starch. For strain MBFBL 1321, optimum pH was 7.0, optimum temperature was 25C, best carbon source was glycerol and the second best carbon source was sorbitol and starch. For MBFBL 1322, optimum pH was 8.0, optimum temperature was 25C, best carbon source was sucrose and the second best carbon source was glucose. For MBFBL 1323, optimum pH was 6.6, optimum temperature was 20C, best carbon source was manitol and the second best carbon source was sucrose. For MBFBL 1324, optimum pH was 9.0, optimum temperature was 25C, and best carbon source was glycerol. The knowledge about each strain requirement could be used to conduct further studies to obtain optimized conditions for biomass generation in selected strains. b) Establishing a test plot at A&T SU Research Farm and an offsite plot located in Rose Hill Duplin County, NCL: Plots 66, 67 and 69 at the NC A&T School farm and about one acre of land at the Rose Hill facility are now designated as truffle research plots. Soil types for each plots are: Plot 66 -MhB2 (55.2%), MhC2 (42.7%) and WhA (2.1%), Plot 67 - EnC (41.8%) and MhB2 (58.2%). Plot 69 - MhB2 (16.7%), MhC2 (78.0%), WhA (5.3%) and Rose Hill plot - AuB (100%). Plots 66, 67, 69 and Rose Hill Plot have average pH of 5.83, 5.73, 6.07 and 4.81 respectively. Adequate amount of fast acting lime was applied to each plot to bring their pH to 8. The methods of pooling DNA samples followed by PCR amplification and the subsequent cloning into E. coli, clones selection and colony PCR gave amplicons that were successfully processed and used for DNA sequencing. The expected sequences will enable us to identify the abundance of potential competing fungi against the target truffle in the test plots. It will also serve as baseline data on diversity and abundance of fungi in test plots, against which we will measure the effect of the pretreatment of soil plots prior to out-planting of inoculated seedlings.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Sites for the out-planting of the inoculated trees both at the A&T School Farm and off campus location were identified and demarcated. Two master's students to work on the project were admitted. DNA extraction and microbial diversity assessment protocols were optimized. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This project has just begun. Protocols are in place for DNA extraction from soil and PCR application from soil samples.
Publications
- No publications reported this period
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