Phase 2: Burgundy Truffle Orchard Establishment - The Burgundy Truffle as a new sustainable agro-forestry crop for Missouri

Final Report for FNC12-878

Project Type: Farmer/Rancher
Funds awarded in 2012: $7,500.00
Projected End Date: 12/31/2013
Region: North Central
State: Missouri
Project Coordinator:
Nicola Hellmuth
Ozark Forest Mushrooms, LLC
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Project Information


This phase of the Truffle Orchard establishment has focused on: final pH adjustment of the soil; incorporation of a large amount of added organic matter; formation of raised beds; installation of an electric fence; simultaneous inoculation and planting of the first 128 (8 rows of 16) oak trees in December – January 2013; and arrangement for further soil amendment with agricultural grade gypsum (CaSO4). 

Over the course of our project, new information led us to emphasize soil calcium (Ca) content over pH. This led to augmentation of soil organic matter level in order to raise soil CEC (cation exchange capacity), permitting our silty loam soil to retain more Ca while affording the benefits of organic matter for beneficial soil microfauna and microbial development.

Because our lime applications had already raised the soil pH to approximately 8.0, and because we would like to at least double the soil Ca level (to 3,000 ppm or more) over the course of the next two to three years, we will use agricultural gypsum (CaSO4) to accomplish this without further raising soil pH.

After having disked in the organic matter amendments, we borrowed a “rice levee plow” from the University of Missouri Horticulture and Agroforestry Research Center in New Franklin, MO, to construct 10 large East-West oriented raised beds spaced 5 meters apart on center.  The beds are approximately 2 m wide and are raised roughly 30 cm above the inter-bed alleys which are roughly 2.5 m wide. The shoulders on the beds are approximately 0.25 m wide. Thus rows of trees are spaced 5 m apart. Along each row, the trees are spaced 3 m apart. The resulting 3 m x 5 m spacing provides a density of 667 trees per ha (about 270 trees per ac). This phase culminated in planting the oak trees, inoculating them with the Burgundy Truffle spores during planting, mulching the trees and protecting them from deer browsing with an electric deer fence and individual cylindrical wire mesh cages. Further soil amendments will be made as necessary depending on soil test results.


The adjustment of the soil properties has continued in Phase II and the main goal of soil pH has been reached, however the targeted soil organic matter and calcium levels are still in the process of being adjusted.  The soil organic matter is still increasing as the various organic matter sources have been disked into the soil and break down over time.  The calcium levels will be adjusted with the addition of gypsum just to beds after the trees have already been planted.

Project Objectives:
1 - Soil pH

We hoped to achieve a minimum calcium (Ca) concentration of 3,000 ppm at a pH of 7.5 - 8.0 throughout the upper 30 cm (1 ft) of soil. Recent work by Dr. Gerard Chevalier (retired, INRA, France) suggests that the soil Ca level is more important for truffle formation than is soil pH per se (which may be merely a common indicator of adequate Ca). By late August 2013, however, our soil pH had reached 8.3 with about half the desired Ca concentration. In retrospect, we see that the high silt content (and low organic matter and clay contents) of our soil limited our soil’s uptake of Ca. In order to augment the soil’s cation exchange capacity (CEC), and to provide the truffle fungus with a source of organic matter in its saprophytic phase, we applied and incorporated a large amount of organic matter (see below). As the organic matter decomposes and becomes incorporated into the soil, we will add about 10 tons of agricultural gypsum (CaSO4, approx. 21 % Ca) to add another 1600 ppm Ca to the upper 30 cm of soil.

2 - Soil Organic Matter + 3 - Calcium

Reaching the target of at least 10% soil organic matter was a challenge. Finding a readily available source of organic matter is critical from both a logistical and cost perspective.

Initially, 16 1,000 lb rolls of hay (old and musty) were rolled out over the orchard as evenly as possible and were disked in.

Another locally available source of organic matter was our spent shiitake logs, which had rotted outdoors for over 10 years.  We rented a wood shredder and shredded two landscape dump truck loads of this material and disked it into the truffière soil as well. While it takes some time for this type of organic matter to decompose and contribute fully to soil CEC, the effect of these first two applications on soil organic matter content did not appear to be on track to become sufficient.

At this point, a large amount of organic material was needed to quickly boost the soil organic matter content (and to lower the soil pH a little), as the trees were scheduled to be planted in December 2013.  We were able to track down a source of double-ground white oak bark mulch produced in nearby Salem, Missouri (distributed by Missouri Mulch).  Fourteen loads of 14-16 CY of bark mulch were trucked in, spread evenly across the truffle orchard site and disked in thoroughly.  We continue to monitor soil organic matter content, which seems to be rising significantly now. The organic matter content in May 2014 had increased to approx. 9% (from about 2% in August 2013), and this value does not include the bark mulch that remains to decompose in the soil.

3 - Calcium

The May 2014 soil tests also showed that soil pH has dropped to approximately 8.0 (from 8.3 in August 2013). The calcium levels however are now almost 1600 ppm (just over half of our minimum target level of 3000 ppm. We are currently evaluating sources of agricultural gypsum (CaSO4, 21% Ca) to further supplement the soil around the he trees themselves. We estimate that 10 tons of gypsum should provide an additional 1600 ppm to the upper 30 cm of soil. Because gypsum is considerably more soluble in water than is lime (calcium carbonate, Ca CO3), we will need to add gypsum gradually as the oak bark decomposes and gradually contributes to soil CEC. If the bark supplement and gypsum cause the pH to drop below 7.5, we will consider further boosting soil Ca content with lime.


Click linked name(s) to expand/collapse or show everyone's info
  • Dr. Johann Bruhn
  • David Enloe
  • Daniel Hellmuth


Materials and methods:
Procurement + Inoculation + Root Preparation + Planting

Burgundy Truffle Acquisition

Dr. Bruhn purchased 1.28 kg of clean ripe fresh wild-harvested Burgundy truffles from suppliers on Gotland on 18 November 2013 while on a work trip to Sweden. We have settled on the Gotland source of Burgundy truffle for two reasons: because of the excellent organoleptic quality of Gotland’s truffles; and because Gotland represents the most northerly known productive Burgundy truffle habitat, with winters quite similar to those in Missouri. Dr. Bruhn escorted the Burgundy truffles back to the US by air, wrapped in paper towel and sealed loosely in glass jars in an insulated cooler.

Burgundy Truffle Inoculum Preparation

While Dr. Bruhn was in Sweden, inoculum preparation was initiated in his lab at the University of Missouri. A technician placed ½ liter of damp horticulture-grade vermiculite in each of 128 autoclavable plastic bags, which were then stapled shut and autoclave-sterilized on two consecutive days. The bags were then frozen until needed.

Upon arrival of the truffles, 10 g (fresh weight) of truffle were ground in a blender micro-cup with a minimum quantity of sterile distilled water, and the resulting truffle slurry was added to one of the bags of vermiculite, mixed by manipulation of the resealed bag, and frozen until needed. All 128 inoculum samples were transported to OFM and stored in one of their walk-in coolers until needed at planting.

RPM® Oak Seedling Preparation

RPM® oak seedlings were selected by Dr. Bruhn from among seedlings produced by the Forrest Keeling Nursery (Elsberry, MO) using acorns produced at HARC (the UM Horticulture and Agroforestry Research Center, New Franklin, MO). The acorns represented several sources: Quercus bicolor (swamp white oak); Q. macrocarpa (bur oak); and hybrids of Q. bicolor with Q. robur (English oak). All seedlings were smaller than typical RPM® seedlings, due to the withholding of fertilizer and standard nursery inoculation with commercial mycorrhiza preparations. The root systems of all seedlings were nevertheless typical of RPM® seedlings, demonstrating prolific lateral root development with very little evidence of taproot development. Fertilizer applications had been minimized and mycorrhiza inoculation avoided in the nursery in order to create root systems that would be most receptive to the Burgundy truffle inoculum that was provided in the planting process.

The criteria for final seedling selection were therefore caliper of the stem and evidence of inclination to produce an open branching pattern. As a result, most of the seedlings planted were either Q. bicolor or Q. macrocarpa. Seedlings were transported commercially from the nursery to Ozark Forest Mushrooms in a closed trailer, where they were laid on their sides and covered with tarps until planting. Planting was delayed for some time due to the early snow and freezing weather, waiting for a warm window long enough for the early snow to melt and the surface soil to thaw!

Tree Planting and Inoculation Procedure

We faced a dilemma in figuring out how to integrate the RPM® seedling production system’s establishment of an extraordinary lateral root system with a proven method of seedling inoculation. Research conducted by Dr. Bruhn with Drs. Grechen Pruett and Jeanne Mihail at MU had shown that undetermined factors associated with RPM® seedling production interfere with RPM® seedling infection by the Burgundy truffle in the greenhouse. Field observations at HARC (unpublished) indicated that RPM seedlings generate far more lateral roots presumptively capable of supporting truffle mycorrhizae and thus truffle fruiting than do traditionally produced seedlings. For this reason, we decided to test a protocol that relies on the RPM® method to generate the hypothetically desirable root architecture, while relying on Gerard Chevalier’s proven technique for inoculating seedlings at the time of planting (Dessolas et al., 2007). Of course, the success of Chevalier’s technique is predicated on the presence of soil conditions that favor spore germination and infection of seedling fine roots. But these same conditions would be required for the survival and proliferation of truffle infections established in the greenhouse. Bruhn hypothesizes that the combined objective may be met by removing the loose RPM® substrate from the seedling root systems at the time of planting and inoculation using Chevalier’s field inoculation technique.

RPM® seedling planting and inoculation proceeded as follows. Seedlings were delivered in plastic pots measuring 7.5 in tall by 8 to 10 in in diameter (total capacity 477 cu in), of which the seedling root system occupied from half to three-quarters. A larger plastic pot measuring 10.5 in tall and 10 to 11.5 in in diameter was also available. We decided to test both sizes of pot as appropriate frames for seedling inoculation at planting. To do this, we cut the bottoms out of pots of both sizes to serve as frames. At planting, an empty intact pot of the prescribed dimensions was filled with soil from the planting hole, and the soil was transferred to a large tub. A bag of inoculated vermiculite was thoroughly mixed into the soil in the tub. A bottomless pot was placed in the planting hole so that the top of the pot was even with the soil surface, and a root-pruned seedling was planted using the inoculum-containing soil. After back-filling around the pot with the remaining soil from the hole, the bottomless pot was carefully removed.

Prior to planting, seedlings were power-washed free of most RPM® substrate, and then severely root-pruned to approximately half their original depth and width. Top pruning was not employed. The purpose of the root-pruning was to require the seedling to generate new fine roots which would then grow a substantial distance through the inoculum-infested soil. The reason for considering the two sizes of frames was to test the effect of inoculum concentration and inoculated soil volume, given a constant 10 g of truffle as inoculum.

Success of our procedure will be tested later this summer prior to our open-house, by extracting fine roots from soil cores and examining them microscopically for the presence of characteristic Burgundy truffle mycorrhizae.

Research results and discussion:

Once we are able to test the seedlings for colonization by the truffle mycelium we will have a better idea on the outcome of this project and the impact that it can have on Missouri agroforestry.  If indeed we are successful, this promises to be an exciting new crop for the Ozark Region that combines local food production with agrotourism in the production of a very high value specialty crop.

Impact of Results/Outcomes

Despite one of the coldest winters on record and a late planting window, all of the oak seedlings have survived and are getting well established.

Participation Summary

Educational & Outreach Activities

Participation Summary:

Education/outreach description:

We are planning an Open House at the truffle orchard around September 21, 22 and will have the testing completed by then to know if the inoculation was successful.

A detailed presentation was given on Friday, November 2, 2012, to a group of 30 or more attendees of the National Small Farm Trade Show Conference in Columbia, Missouri at the NCR-SARE Farmers Forum. The presentation covered the state of the art understanding of burgundy truffle culture as well as the process and results to date of the Phase 1: Truffle Project. A video of this presentation can be viewed online through NCR-SARE's YouTube channel. Copy the following URL and paste it into your browser to view the video:

Project Outcomes


Potential Contributions

If this project is successful it will demonstrate how to cultivate the Burgundy Truffle in the Missouri Ozarks and demonstrate the first successful initiative of its kind in the state.  

If not successful, a careful analysis of the procedure can be completed as the knowledge related to the cultivation of the Burgundy Truffle evolves in order to see what steps could be taken to make it successful.

Future Recommendations

Based on the results of the tree colonization by the truffle mycelium we will be able to make recommendations for field preparation, soil amendment and root treatment and inoculation.

Information Products

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.