Propagation of edible Pecan Truffle (Tuber lyonii) in pecan nurseries

View the project final report

• 2014 annual report

Commodities

• Nuts: pecans

Practices

• Crop Production: biological inoculants, double cropping, multiple cropping
• Production Systems: general crop production
• Soil Management: soil microbiology

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 pecanscome 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 forgrowers (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.

This project seeks to enhance the sustainability of pecan production in the southeast by promoting the commercialization and widespread production of the edible pecan truffle with pecans. Based on preliminary studies (Bonito et al., 2011? 2012? Brenneman et al., 2013) and data on the truffle production systems of Europe (Hall et al.,2007? Bonet et al., 2009) we know that pecan truffles have a widespread but patchy distribution and that nursery production of truffle inoculated trees is integral to successful production of truffles on a large scale. In order to further develop the truffle cropping system, we will work on site with pecan nurseries in Florida and Georgia to identify the current distribution of the pecan truffle in pecan nurseries and to develop protocols to inoculate seedlings using high throughput approaches in nurseries.

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 achieve two goals: 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.

Identify current distribution of the pecan truffle –

Observations on T. lyonii distribution within orchards suggest a patchy distribution with no obvious explanatory variables. There are two main routes whereby T. lyonii could establishin orchards: 1) T. lyonii may establish in the nursery and arrive on the roots of live seedlings, or 2) T. lyonii may enter pecan orchards from nearby woodlands via natural dispersal. Based on data from European truffles, we hypothesize that seedlings are the main route of dispersal into orchards. Pecans trees are propagated in nurseries by grafting productive cultivars onto rootstocks germinated from seed. Seedlings are grown 2–3 years in the nursery before being transplanted to orchards. Nursery practices are variable, dependent on the size and location of thenursery. We know that T. lyonii is capable of establishing symbiotic associations with pecan seedlings (Bonito et al., 2012) but we have not sampled roots in nurseries to confirm that T. lyonii is common. If pecan truffles have a patchy distribution in nurseries, this will help explain the distribution in orchards.

We hypothesize that some nurseries are highly colonized by T. lyonii whereas the truffle is rare in others. To determine pecan truffle prevalence in nurseries, we will sample 20 seedlings per nursery from five FL and GA nurseries (n=100 seedlings). We will sample 10 ECM roots per seedling and identify fungal communities via pyrosequencing of the ITS rDNA barcode region followed by phylogenetic and ordination analyses (Talbot et al., 2013). These molecular methods have proven effective in documenting ECM fungal communities on pecan roots and in determining T. lyonii frequency belowground (Bonito et al., 2011). Soils will be subjected to standard analyses for nutrients, organic matter, pH, and minerals. All sampled roots will be studied to document the diversity of fungi in nurseries and identify the most common 'competitor' fungi. We will use data on site history, distance to the nearest ECM tree, and soil variables to see how these factors influence T. lyonii presence and frequency of competitor fungi.

Inoculating pecan seedlings with truffles in the nursery – 

European truffles are commonly inoculated onto oaks in the greenhouse and then planted in orchards. However, pecan nurseries are usually established directly in field soils. Although greenhouse approaches produce reliable truffle trees, these methods require additional infrastructure, economic inputs, and increased labor costs. Preliminary studies of pecan truffle seedling interactions (Bonito et al.,2012) suggest that it may be possible to inoculate pecan truffles onto seedlings in the nursery once appropriate techniques have been developed.

Another complication is that many nurseries prepare the soil by fumigation. This reduces weeds, nematodes, and soilfungi and therefore may enhance the success of truffle inoculations due to reduced competition from other fungi. Alternatively, truffles may benefit from a rich biota of other soil microbes. Thus, it is unclear whether soil preparations are beneficial for establishment or if T. lyonii can initiate the symbiosis in natural nursery soil. In addition to determining the success of inoculations from fumigated vs. non-fumigated soils, it is important to determine optimum inoculum levels so that truffle spores can be used efficiently.

We hypothesize that spores of T. lyonii will readily colonize pecan seedlings in all nursery soils. Although fumigation may enhance T. lyonii colonization and reduce 'competitor' fungi, we hypothesize that it will be possible to directly inoculate T. lyonii in unaltered soils and achieve good colonization. We will conduct inoculation experiments on pecan seedlings at 2 sites: the UGA Ponder Orchard (Ty Ty, GA) andShiloh Farms nursery (Ray City, GA). Each orchard will contain 200 experimental seedlings. 100 seedlings per site willbe divided into four treatments in randomized blocks: truffles & fumigation, truffles & no fumigation, no truffles &fumigation, no truffles & no fumigation (control). After 1 year, ECM roots will be sampled and morphologically assessed to determine the percent colonization by T. lyonii as well as the diversity and percent colonization of otherECM fungi. We will sample 10 ECM roots from each seedling and identify fungal communities by ITS rDNA barcode pyrosequencing (see above). The combination of above treatments will determine: 1) whether pecan seedlings canbe readily inoculated with T. lyonii in the field, 2) whether fumigation enhances or hinders inoculation success, and 3) what other common ECM fungi can readily compete with T. lyonii in the nursery habitat.

Bonito et al. (2011, 2012) showed that pecans were readily colonized by T. lyonii when 1 gram of ground truffles was used to inoculate seedlings in greenhouse pots. However, inoculation may not be as successful in a nonsterile,, field environment. Empirical tests are therefore needed to determine the minimum amount of truffle spores required for successful inoculation. Accordingly, 100 seedlings per site will be treated with 4 different levels of T. lyonii spores (2,1, 0.5, and 0.1 grams of truffle per seedling). After 1 year, roots of the seedlings will be examined to determine the percent colonization by T. lyonii and also to determine whether any other ECM fungi are present. To verify colonization by T. lyonii, select roots will be subjected to PCR with T. lyonii-specific primers (Bonito et al., 2011). If any roots are colonized by 'competitor' ECM fungi, the ITS region will be sequenced using standard protocols to determine their identity (Smith et al., 2011).