Mycorrhiza DOI 10.1007/s00572-014-0562-y
ORIGINAL PAPER
Cultivation of Mediterranean species of Tuber (Tuberaceae) in British Columbia, Canada Shannon M. Berch & Gregory Bonito
Received: 13 November 2013 / Accepted: 21 January 2014 # Her Majesty the Queen in Right of Canada 2014
Abstract Based on an assessment of soil and climatic conditions in British Columbia (BC), the Truffle Association of British Columbia (TABC) determined that the cultivation of Mediterranean Tuber melanosporum and Tuber aestivum might be possible in the warmer parts of the province. With the cooperation of independent truffle growers, TABC assessed the colonization of host tree roots collected from eight truffle orchards planted 2–7 years earlier using morphological and molecular criteria. Both Tuber species persisted on the roots of inoculated trees in six of the eight truffle orchards studied. The identity of Tuber ectomycorrhizas that had been characterized morphologically as differing from those of T. melanosporum and T. aestivum were determined using DNA sequence analysis to belong to three species of truffles native to the Pacific Northwest. One of those species, Tuber anniae, had been previously reported from BC, but the other two, Tuber menseri nom. prov. and Tuber beyerlei, are reported here from BC for the first time. Recently, production of three Périgord black truffles in one truffle orchard and one Burgundy truffle in another orchard demonstrates that these truffles are able to fruit in BC.
Keywords Truffle cultivation . Tuber melanosporum . Tuber aestivum . Native truffle species
Electronic supplementary material The online version of this article (doi:10.1007/s00572-014-0562-y) contains supplementary material, which is available to authorized users. S. M. Berch (*) British Columbia Ministry of Environment, Victoria, British Columbia V8W 9C4, Canada e-mail: [email protected] G. Bonito Royal Botanic Gardens, Melbourne 3141, Australia
Introduction Tuber melanosporum Vittad. (Périgord black truffle) and Tuber aestivum Vittad. (Burgundy truffle) are the two most valuable Mediterranean truffles to be cultivated well beyond their natural distribution, including New Zealand and Australia (Hall et al. 2007). A comparison of key climate attributes of selected regions of British Columbia with those described by Hall and Brown (2002) for known truffleproducing areas in Europe and the southern hemisphere (Table 1) suggested that it might be possible to grow these truffles in British Columbia (BC), Canada, although cooler summer temperatures in BC compared to truffle-producing regions were a concern. The Truffle Association of British Columbia (TABC) was formed in 2004 to provide support for the nascent truffle-growing industry in the province. Despite the many attempts to cultivate truffles in North America over the past two decades, commercial quantities of Périgord black truffles are being produced in only a couple truffières in the USA, while most truffières do not produce truffles at all, or at least not in large amounts, possibly due to issues such as poor site selection, orchard maintenance, and ectomycorrhiza formation (Lefevre 2012). Recent studies of inoculated seedlings and/or truffières on T. melanosporum in Australia (Linde and Selmes 2012), New Zealand (GuerinLaguette et al. 2013), and the USA (Bonito et al. 2010) have shown that lower quality black truffle species, such as Tuber brumale Vittad. and Tuber indicum Cooke & Massee, may be unwittingly used to inoculate seedlings and have contaminated some truffières. TABC received funding to study truffle ectomycorrhizas on host trees in BC truffières to provide information that would guide the development of the best management practices for Mediterranean truffle-growing in BC. The objective of this study is to assess the persistence of truffle ectomycorrhizas on hazelnuts (Corylus avellana L.) and
Mycorrhiza Table 1 Comparison of climate in northern and southern hemispheres’ truffle-producing areas and selected regions of British Columbia Select climate attributes
France// Italya
New Zealanda
Gotland, Swedena
South Vancouver Island
Lower Fraser Valley
Okanagan Valley
Annual rainfall (mm) Mean daily temperature, summer (°C) Mean daily temperature, winter (°C) Annual sunshine hours
628–948 17.0–24.6 1.6–5.4 1,762–2,837
537–1,754 13.5–19.7 1.6–19.7 1,704–2,447
514 15.9 −1.1 1,579
695–992 15.6–16.7 3.3–4.0 1,803– 1,985
1,573 14.7 4.5 1,865
332–380 14.75–15.9 0.7–2.4 1,955– 1,956
Summer sunshine hours (April–Sept) Approximate degree days (10 °C) Accumulated degree days (>10 °C)
1,191–1,814 – 852–2,009
1,034–1,377 – 337–2,154
1,148 – 570
1,352–1,454 957–1,158 –
1,326 934 –
1,462–1,470 978–1,180 –
a
Data collected and made available by Hall et al. 2008
English oaks (Quercus robur L.) growing in truffières in BC, with a focus on T. melanosporum and T. aestivum but including all Tuber species encountered, particularly the lower quality black truffles that have contaminated truffières in other truffle-growing areas.
Materials and methods The truffières (Table 2) that were included in this study have been established by independent farmers using host tree seedlings that were inoculated by truffle nursery companies. We did not assess the seedlings before they were planted. To the best of our knowledge, all seedlings had been inoculated using
truffles purchased from Europe. In accord with standard procedures (Hall et al. 2007), the host trees were C. avellana, hazelnut, and Q. robur, English oak. All eight of the truffières studied were growing trees inoculated with T. melanosporum but two also had trees inoculated with T. aestivum. Two of the studied truffières (L, P) are located on the southern Vancouver Island, four in the Lower Fraser Valley (G, N, Pr, S), and two in the Okanagan Valley (M, W) (Fig. S1). Soil and climate attributes for the truffières are provided in Table 2. All orchards had been limed to raise the soil pH to favor the Mediterranean truffle fungi and disadvantage the indigenous ectomycorrhizal fungi that are more adapted to the native acidic to neutral soil pH (Hall et al. 2007).
Table 2 Soil and climate conditions of the truffières in this study Location
Biogeoclimatic zonea
Vancouver Island
CDFmm L Warm, dry summers and mild, wet winters Growing seasons are very long with large water deficits. P
Lower Fraser Valley CWHxm1 Warm, dry summers and moist, mild winters with relatively little snowfall Growing seasons are long, and feature water deficits.
Truffière code
PPxh Hot, dry summers and cool winters with low snowfall Large growing season moisture deficits.
Moderately well to imperfectly drained, loam, fine marine veneer over gravelly, sandy moraine Moderately well to imperfectly drained, silt loam or silty clay loam, glaciomarine deposit
G
Potting medium
Pr
Well drained to rapidly drained, sandy loam (gravelly), gravelly glacial outwash
CWHdm N Warm, relatively dry summers and moist, mild winters with little snowfall Growing seasons are long, and feature only S minor water deficits. Okanagan Valley
Soil (moisture, texture, parent material)b
W M
Moderately well to well drained, sandy loam, littoral and glacial outwash Moderately well drained, silt loam, glaciomarine deposit
Well drained to rapidly drained, loam (gravelly), glaciofluvial deposits Imperfectly drained, clay, glaciolacustrine sediments
a
From Biogeoclimatic Ecosystem Classification (BEC) web portal http://www.for.gov.bc.ca/hre/becweb/
b
From Terrestrial Ecosystem Information (TEI) web portal http://www.env.gov.bc.ca/tei/access-soil.html
Mycorrhiza
Truffières P and M planted inoculated seedlings from a nursery located on coastal British Columbia and the other six orchards planted seedlings inoculated by a nursery in Oregon, USA that had worked in collaboration with a nursery in the Okanagan Valley, BC. Truffières were sampled in the fall after the rains had returned (October) except for truffières P and M which were sampled in the spring (April) during bud break. Depending on the number of trees in the truffle orchard, 5–15 trees were sampled per orchard. For each sampled tree, three subsamples of fine roots with ectomycorrhizas were excavated within 1 m of the stem at spots located equidistant apart around the tree and bulked per tree. The fine roots were collected from the top 10–15 cm of soil, placed in a labeled plastic bag with soil, kept cool, and examined within 1 month of collection. Roots were gently washed free of soil and 100–200 individual ectomycorrhizal root tips (depending on abundance of ectomycorrhizal root tips in the samples) per sample were sorted to morphotypes and counted under the dissecting microscope. Morphotypes were further characterized under the compound microscope with mantle and cystidium details (Agerer 1987–2002). Ectomycorrhizal tips representing each Tuber morphotype on each sampled tree in each orchard were frozen for subsequent DNA extraction and PCR amplification of the fungal internal transcribed spacer (ITS) region of nuclear rDNA. DNA was extracted from individual ectomycorrhizal root tips and truffles by using the Extract-N-Amp (Sigma) procedure. Briefly, single root tips or small truffle tissue samples were placed in wells (on ice) and 25 μl of the SIGMA extraction buffer (Sigma Aldrich) was immediately added to these wells. The samples were then covered with a transparent plate film, vortexed gently and centrifuged briefly, and incubated at 95 °C in a thermal cycler for 10 min. The samples were then removed from the thermal cycler, vortexed, and centrifuged again. Then 25 μl of the SIGMA Dilution Solution was added to each tube. Tubes were covered with a transparent plate film and were vortexed briefly and centrifuged. The samples were then stored in a freezer until used for PCR. Prior to performing PCR, the samples were diluted by pipetting 1 μl of extracted DNA into 99 μl of DNA-grade water (1 in 100 dilutions). This dilution was used as the DNA template in all PCRs. PCRs were carried out in 25-μl reactions that consisted of 6.0 μl DNA-grade water, 12.5 μl GoTaq Master Mix (Promega), 1.25 μl ITS 1 (10 μM) forward primer, 1.25 μl ITS 4 (10 μM) reverse primer, 1.50 μl MgCl2 (25 mM), 1.50 μl BSA (10 mg/ml), and 1.0 μl template DNA. Thermal cycler conditions for PCR included an initial incubation at 94 °C for 3 min to separate double-stranded DNA, which was followed by 35 cycles consisting of a 1 min at 94 °C, 1 min at 50 °C for primer annealing, and a 1-min elongation step at 72 °C. The final elongation step was extended for 10 min and then PCR products were cooled to 4 °C.
PCR products were visualized through gel electrophoresis and 20 μl of PCR product was cleaned using the Mag-Bind EZ Pure 96 Plate protocol (Omega Bio-Tek) according to the manufacturer’s recommendations. The DNA concentration (nanogram/microliter) of each PCR product was obtained using a NanoDrop ND-1000 spectrophotometer and ranged between 4.0 and 8.2 ng/μl. Then, 6.0 μl each of cleaned PCR product was placed into two separate wells (96 plate), and 1.0 μl of either the forward or reverse primer (5 μM) was added to the well for bidirectional sequencing. These were then submitted for sequencing using a 3130xL Genetic Analyzer (ABI). Sequences were identified through queries against GenBank with the BLASTN algorithm to verify their affiliation to Tuber. Sequences belonging to Tuber were then aligned with reference sequences using MUSCLE (Edgar 2004). Alignments were manually checked and ambiguous regions were excluded in Mesquite 2.5 (Maddison and Maddison 2009). Phylogenetic analyses (Fig. 1) were conducted with maximum parsimony (MP) in PAUP* (Swofford 2002) and Bayesian inference (BI) with MrBayes (Huelsenbeck and Ronquist 2001). The best-fit nucleotide substitution model was based on the Akaike information criterion and was implemented in PAUP* 4d106 (Swofford 2002). ML bootstrap support based on 1,000 replicates and BI analyses were run through the CIPRES Web portal (http://www.phylo.org/). BI was based on parallel runs of 20,000,000 generations with four chains, sampling every 1,000 generations. Sequences produced in this study are deposited in GenBank under accession numbers KF742719-KF742777.
Results and discussion Tuber melanosporum was detected on both hazelnut and oak host trees in six of the eight truffières studied (Table 3, Fig. S2). In two of the truffières (P, M), no T. melanosporum ectomycorrhizas were detected. Because only a subsample of trees and of fine roots in each orchard were examined, we cannot be certain that the fungus was absent from these two truffières. However, the fact that the plants in both these truffières had been supplied by the same nursery and those in the successful truffières by another nursery, suggests that there may have been differences in the quality of plants produced by the two nurseries. Similar results were found in Australia (Linde and Selmes 2012) with 25 % of inoculated seedlings lacking T. melanosporum ectomycorrhizas and the two major nursery samples showing as low as 35 % and as high as 95 % of seedlings with T. melanosporum ectomycorrhizas. No jurisdiction in North America currently has quality assurance standards in place for truffle-inoculated seedlings. In the Catalonia region of Spain, a quality assurance
Mycorrhiza Fig. 1 Most parsimonious tree (score 489) based on the analysis of ITS rDNA from ectomycorrhizas and reference taxa. Taxa recovered from field ectomycorrhizas are labeled in bold. The analysis consisted of 33 taxa and 387 included characters, 136 of which were parsimony informative. Nodes receiving significant bootstrap values (>70) based on parsimony and 1,000 sample replicates are indicated by thickened branches. Tuber gennadii was chosen as an outgroup based on previous analyses
protocol is in place, based on earlier work on the certification of truffle-inoculated seedlings (Fischer and Colinas 1996). Percent colonization by T. melanosporum varied among samples and among truffières and ranged from 0–80 % (data not provided). Because only a subsample of trees and roots were examined per truffière, we do not consider percent colonization to be as meaningful in this study as the presence/absence data by truffière. Morphotypes similar to T. aestivum were detected on plants in the two truffières growing this fungus (G, W), but molecular assessment of these mycorrhizas sometimes revealed the presence of Trichophaea species (Fig. S2). The Trichophaea woolhopeia species complex has been reported to form AD morphotype mycorrhizas in truffières in Europe, North America (Rubini et al. 2011), and New Zealand (Ian Hall, personal communication) and is considered a competitor of truffle species (Zambonelli et al. 2012).
One truffière (L) had some Tuber brumale contamination but Tuber indicum was not detected in any of the sampled truffières. Similarly, in New Zealand (Guerin-Laguette et al. 2013) and Australia (Australian Truffle Growers’ 2011), T. melanosporum orchards were found to contain some T. brumale. While New Zealand (New Zealand Ministry of Agriculture and Forestry 2003 amended 2011) and Australia (Commonwealth of Australia 2013) now have regulations aimed at stopping the importation of lower valued black truffles such as T. brumale and T. indicum, no such regulations currently exist in Canada. In one truffière (N), we detected the presence of Tuber borchii. This was a surprising find because bianchetto truffles (T. borchii) have a white peridium, are easily distinguished from T. melanosporum, and should not have accidently found their way into nursery inoculum. It turned out that the nursery had produced some T. borchii-inoculated oak seedlings and there had been a mix-up that resulted in some of these
4
3
3
6
4
7
5
2
L
P
G
N
Pr
S
W
M
NA not available
Years in ground
Truffière code
B
A
A
A
A
A
B
A
Nursery code
Corylus avellana
Corylus avellana Quercus robur
Corylus avellana
Corylus avellana
Quercus robur
Corylus avellana
Quercus robur
Quercus robur Corylus avellana
Corylus avellana
Corylus avellana
Tree species
Tuber melanosporum Tuber melanosporum Tuber aestivum Tuber melanosporum
Tuber melanosporum
Tuber melanosporum
Tuber melanosporum Tuber aestivum Tuber melanosporum Tuber aestivum Tuber melanosporum
Tuber melanosporum
Tuber melanosporum
Tuber species inoculated 5/5 2/5 0/15 7/15 2/15 0/5 3/3 0/1 3/3 1/1 4/6 2/6 3/5 2/5 5/9 2/9 2/10 4/10 1/10 3/3 3/3 1/1 0/5 1/5 2/5
Tuber-like with bristles Tuber melanosporum Tuber sp. Tuber-like boxy cells Tuber melanosporum Tuber melanosporum Tuber aestivum Tuber melanosporum Tuber-like with bristles Tuber-like with boxy cells
Number of trees colonized/sampled
Tuber melanosporum Tuber-like with bristles Tuber melanosporum Tuber-like with bristles Tuber-like with bristles Tuber melanosporum Tuber melanosporum Tuber aestivum Tuber melanosporum Tuber aestivum Tuber melanosporum Tuber-like with bristles Tuber melanosporum Tuber-like with bristles Tuber melanosporum
Tuber morphotype
Tuber beyerlei Tuber melanosporum Tuber beyerlei Trichophaea sp. Tuber melanosporum Tuber melanosporum Trichophaea sp. NA Tuber menseri Trichophaea sp.
Tuber melanosporum Tuber brumale NA Tuber menseri Tuber anniae NA Tuber melanosporum Trichophaea sp. Tuber melanosporum Tuber aestivum Tuber melanosporum Tuber menseri Tuber melanosporum Tuber borchii Tuber melanosporum
Tuber species from DNA sequencing
Table 3 Tuber species introduced on inoculated host seedlings into truffle orchards in southern British Columbia and Tuber species detected by morphological assessment and DNA sequencing
Mycorrhiza
Mycorrhiza
seedlings being shipped to and planted in the truffière. Because bianchetto truffles have been produced for a few years in a truffière in Idaho (Berch 2013), it seems likely that they could be produced in British Columbia; if so, this accidental introduction may prove to be fortuitous. A major surprise was the detection of numerous Tuber-like morphotypes with bristle-like cystidia (Fig. S2) that proved to be native species of Tuber. Most of these species are known from the USA but are first reports for British Columbia and Canada. Tuber anniae was detected in one truffière (P), Tuber beyerlei in two (Pr, S), and Tuber menseri nom. prov. (Bonito et al. 2010) in three (P, N, M) and all three species were found only on hazelnut (Table 1). Truffles of T. anniae (Puberulum clade, Bonito et al. 2010), or a species similar to it, have been collected near Williams Lake, BC (Bill Chapman and Jim Trappe, personal communications). Truffles of T. beyerlei (Maculatum clade, Guevara et al. 2013) and T. menseri nom. prov. (Puberulum clade, Bonito et al. 2010) have not yet been collected in BC, according to herbarium records. T. beyerlei was recently described from Oregon in association with Pseudotsuga menziesii (Douglas-fir), and the detection of this species in truffières in British Columbia expands both the range and host associations of this truffle species (Guevara et al. 2013). T. menseri nom. prov. has yet to be formally described but has also been reported from New Zealand and Europe (Bonito et al. 2010) although the natural distribution of this species is considered to be western North America. In March of 2013, three Périgord black truffles were discovered under hazelnut trees by trained truffle dogs in truffière S in the Lower Fraser Valley (KF742777, Berch 2013). While T. melanosporum ectomycorrhizas were found to be present in this truffière, their abundance appeared to be low, suggesting that for truffières the presence of truffle ectomycorrhizas may be as meaningful as an indicator for the likelihood of truffle production as percent root colonization. The other T. melanosporum truffières we studied are likely still too young to produce truffles as Hall et al. (2007) indicate that truffle production might be expected by about year 7. In August 2013, one Burgundy truffle was found under a hazelnut tree in a truffière on Vancouver Island that had not been part of this study (KF742776, unpublished data). This is the first independently confirmed report of the production of truffles of these two Mediterranean Tuber species in Canada. Acknowledgments We are grateful to the Agroforestry Development Initiative Fund for funding this research, to all of the truffle growers who participated in this study, and to Charles Lefevre of New World Truffières for supporting this work with advice on truffle cultivation and root sampling. Special thanks go to Wayne Haddow, BC Ministry of Agriculture, for being one of the first people in BC to see the possibility of a Mediterranean truffle industry in British Columbia. The Fragment Analysis and DNA Sequencing Services (FADSS) at the University of British Columbia-Okanagan carried out the PCR amplification and sequencing of ectomycorrhizas. Dr. Mary Berbee, University of British Columbia, sequenced the Tuber melanosporum and Tuber aestivum truffles produced
in BC. Gregory Bonito was supported through the US Department of Energy, Office of Biological and Environmental Research, Genome Science Program.
References Agerer R ed (1987–2002) Colour atlas of ectomycorrhizae, 1st–12th del., Einhorn-Verlag, Schwäbisch Gmünd ISBN: 3-921703-77-8 Australian Truffle Growers’ Association (2011) Science helping Australia produce some of the world’s best quality truffles. http:// www.trufflegrowers.com.au/wp-content/uploads/2011/06/WinterTruffle-Press-Release-170611.pdf Berch, SM (2013) Truffle cultivation and commercially harvested native truffles. In: Proceedings of international symposium on forest mushroom. Hosted by Korea Forest Research Institute and Korean Forest Mushroom Society. August 6, 2013, Seoul, Republic of Korea. Pp. 85–97. http://www.for.gov.bc.ca/hfd/pubs/docs/scv/SCV872.pdf Bonito GM, Gryganskyi AP, Trappe JM, Vilgalys R (2010) A global meta-analysis of Tuber ITS rDNA sequences: species diversity, host associations and long-distance dispersal. Mol Ecol 19:4994–5008. doi:10.111/j.1365-294.2010.04855.x Commonwealth of Australia (2013) ICON database, Department of Agriculture, Fisheries, Forests, accessed September 12, 2013 Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinforma 5:1–19. doi: 10.1186/1471-2105-5-113 Fischer C, Colinas C (1996) Methodology for certification of Quercus ilex seedlings inoculated with Tuber melanosporum for commercial application. In: Proceedings of the 1st international conference on mycorrhizae. Berkeley, CA, USA Guerin-Laguette A, Cummings N, Hesom-Williams N, Butler R, Wang Y (2013) Mycorrhiza analyses in New Zealand truffières reveal frequent but variable persistence of Tuber melanosporum in coexistence with other truffle species. Mycorrhiza 23:87–98. doi:10. 1007/s00572-012-0450-2 Guevara G, Bonito G, Trappe JM, Cazares E, Williams G, Healy RA, Schadt C, Vilgalys R (2013) New North American truffles (Tuber spp.) and their ectomycorrhizal associations. Mycologia 105(1): 194–209. doi:10.3852/12-087 Hall IR, Brown G (2002) The black truffle: its history, uses and cultivation. Reprint of second edition on CD ROM plus booklet. New Zealand Institute for Crop & Food Research Limited, Christchurch Hall IR, Brown GT, Zambonelli A (2007) Taming the truffle. The history, lore, and science of the ultimate mushroom. Timber, Portland, p 305 Hall IR, Frith A, Haslam W (2008) A rough climatic comparison of various centres adjacent to or with similar climates to Périgord black, Italian white, Burgundy and bianchetto truffle producing areas. Dunedin, Truffles & Mushrooms (Consulting) Ltd. (updated 19 October 2013). www.trufflesandmushrooms.co.nz/free% 20downloads.html, accessed January 2014 Huelsenbeck JP, Ronquist F (2001) MrBayes: Bayesian inference of phylogeneric trees. Bioinforma 17:754–755. doi:10.1093/ bioinformatics/17.8.754 Lefevre C (2012) Native and cultivated truffles of North America. Chapter 12 in Edible Ectomycorrhizal Mushrooms, edited by Zambonelli A and Bonito G. Soil Biol 34:209–226 Linde CC, Selmes H (2012) Genetic diversity and mating type distribution of Tuber melanosporum and their significance to truffle cultivation in artificially planted truffières in Australia. Appl Environ Microbiol 78(18):6534–6539
Mycorrhiza Maddison WP, Maddison DR (2009) Mesquite 2.6: a modular system for evolutionary analysis. Periodical mesquite: a modular system for evolutionary analysis. http://mesquiteproject. org New Zealand Ministry of Agriculture and Forestry (2003) (amended 2011) Importation into New Zealand of specified fresh and frozen Tuber species (truffles). Biosecurity New Zealand Standard BNZIMP-TUBER. http://www.biosecurity.govt.nz/files/ihs/bnz-imptuber.pdf Rubini A, Belfiori B, Passeri V, Baciarelli Falini L, Arcioni S, Riccioni C, Paolocci F (2011) The AD-type ectomycorrhizas, one of the most
common morphotypes present in truffle fields, result from fungi belonging to the Trichophaea woolhopeia species complex. Mycorrhiza 21(1):17–25 Swofford DL (2002) PAUP* 4.0: phylogenetic analysis using parsimony (*and other methods). Sunderland, Massachusetts: Sinauer Associates Zambonelli A, Iotti M, Boutahir S, Lancellotti E, Perini C, Pacioni G (2012) Ectomycorrhizal fungal communities of edible ectomycorrhizal mushrooms. Chapter 7 in edible ectomycorrhizal mushrooms, edited by Zambonelli A and Bonito G. Soil Biol 34: 105–124
ORIGINAL PAPER
Cultivation of Mediterranean species of Tuber (Tuberaceae) in British Columbia, Canada Shannon M. Berch & Gregory Bonito
Received: 13 November 2013 / Accepted: 21 January 2014 # Her Majesty the Queen in Right of Canada 2014
Abstract Based on an assessment of soil and climatic conditions in British Columbia (BC), the Truffle Association of British Columbia (TABC) determined that the cultivation of Mediterranean Tuber melanosporum and Tuber aestivum might be possible in the warmer parts of the province. With the cooperation of independent truffle growers, TABC assessed the colonization of host tree roots collected from eight truffle orchards planted 2–7 years earlier using morphological and molecular criteria. Both Tuber species persisted on the roots of inoculated trees in six of the eight truffle orchards studied. The identity of Tuber ectomycorrhizas that had been characterized morphologically as differing from those of T. melanosporum and T. aestivum were determined using DNA sequence analysis to belong to three species of truffles native to the Pacific Northwest. One of those species, Tuber anniae, had been previously reported from BC, but the other two, Tuber menseri nom. prov. and Tuber beyerlei, are reported here from BC for the first time. Recently, production of three Périgord black truffles in one truffle orchard and one Burgundy truffle in another orchard demonstrates that these truffles are able to fruit in BC.
Keywords Truffle cultivation . Tuber melanosporum . Tuber aestivum . Native truffle species
Electronic supplementary material The online version of this article (doi:10.1007/s00572-014-0562-y) contains supplementary material, which is available to authorized users. S. M. Berch (*) British Columbia Ministry of Environment, Victoria, British Columbia V8W 9C4, Canada e-mail: [email protected] G. Bonito Royal Botanic Gardens, Melbourne 3141, Australia
Introduction Tuber melanosporum Vittad. (Périgord black truffle) and Tuber aestivum Vittad. (Burgundy truffle) are the two most valuable Mediterranean truffles to be cultivated well beyond their natural distribution, including New Zealand and Australia (Hall et al. 2007). A comparison of key climate attributes of selected regions of British Columbia with those described by Hall and Brown (2002) for known truffleproducing areas in Europe and the southern hemisphere (Table 1) suggested that it might be possible to grow these truffles in British Columbia (BC), Canada, although cooler summer temperatures in BC compared to truffle-producing regions were a concern. The Truffle Association of British Columbia (TABC) was formed in 2004 to provide support for the nascent truffle-growing industry in the province. Despite the many attempts to cultivate truffles in North America over the past two decades, commercial quantities of Périgord black truffles are being produced in only a couple truffières in the USA, while most truffières do not produce truffles at all, or at least not in large amounts, possibly due to issues such as poor site selection, orchard maintenance, and ectomycorrhiza formation (Lefevre 2012). Recent studies of inoculated seedlings and/or truffières on T. melanosporum in Australia (Linde and Selmes 2012), New Zealand (GuerinLaguette et al. 2013), and the USA (Bonito et al. 2010) have shown that lower quality black truffle species, such as Tuber brumale Vittad. and Tuber indicum Cooke & Massee, may be unwittingly used to inoculate seedlings and have contaminated some truffières. TABC received funding to study truffle ectomycorrhizas on host trees in BC truffières to provide information that would guide the development of the best management practices for Mediterranean truffle-growing in BC. The objective of this study is to assess the persistence of truffle ectomycorrhizas on hazelnuts (Corylus avellana L.) and
Mycorrhiza Table 1 Comparison of climate in northern and southern hemispheres’ truffle-producing areas and selected regions of British Columbia Select climate attributes
France// Italya
New Zealanda
Gotland, Swedena
South Vancouver Island
Lower Fraser Valley
Okanagan Valley
Annual rainfall (mm) Mean daily temperature, summer (°C) Mean daily temperature, winter (°C) Annual sunshine hours
628–948 17.0–24.6 1.6–5.4 1,762–2,837
537–1,754 13.5–19.7 1.6–19.7 1,704–2,447
514 15.9 −1.1 1,579
695–992 15.6–16.7 3.3–4.0 1,803– 1,985
1,573 14.7 4.5 1,865
332–380 14.75–15.9 0.7–2.4 1,955– 1,956
Summer sunshine hours (April–Sept) Approximate degree days (10 °C) Accumulated degree days (>10 °C)
1,191–1,814 – 852–2,009
1,034–1,377 – 337–2,154
1,148 – 570
1,352–1,454 957–1,158 –
1,326 934 –
1,462–1,470 978–1,180 –
a
Data collected and made available by Hall et al. 2008
English oaks (Quercus robur L.) growing in truffières in BC, with a focus on T. melanosporum and T. aestivum but including all Tuber species encountered, particularly the lower quality black truffles that have contaminated truffières in other truffle-growing areas.
Materials and methods The truffières (Table 2) that were included in this study have been established by independent farmers using host tree seedlings that were inoculated by truffle nursery companies. We did not assess the seedlings before they were planted. To the best of our knowledge, all seedlings had been inoculated using
truffles purchased from Europe. In accord with standard procedures (Hall et al. 2007), the host trees were C. avellana, hazelnut, and Q. robur, English oak. All eight of the truffières studied were growing trees inoculated with T. melanosporum but two also had trees inoculated with T. aestivum. Two of the studied truffières (L, P) are located on the southern Vancouver Island, four in the Lower Fraser Valley (G, N, Pr, S), and two in the Okanagan Valley (M, W) (Fig. S1). Soil and climate attributes for the truffières are provided in Table 2. All orchards had been limed to raise the soil pH to favor the Mediterranean truffle fungi and disadvantage the indigenous ectomycorrhizal fungi that are more adapted to the native acidic to neutral soil pH (Hall et al. 2007).
Table 2 Soil and climate conditions of the truffières in this study Location
Biogeoclimatic zonea
Vancouver Island
CDFmm L Warm, dry summers and mild, wet winters Growing seasons are very long with large water deficits. P
Lower Fraser Valley CWHxm1 Warm, dry summers and moist, mild winters with relatively little snowfall Growing seasons are long, and feature water deficits.
Truffière code
PPxh Hot, dry summers and cool winters with low snowfall Large growing season moisture deficits.
Moderately well to imperfectly drained, loam, fine marine veneer over gravelly, sandy moraine Moderately well to imperfectly drained, silt loam or silty clay loam, glaciomarine deposit
G
Potting medium
Pr
Well drained to rapidly drained, sandy loam (gravelly), gravelly glacial outwash
CWHdm N Warm, relatively dry summers and moist, mild winters with little snowfall Growing seasons are long, and feature only S minor water deficits. Okanagan Valley
Soil (moisture, texture, parent material)b
W M
Moderately well to well drained, sandy loam, littoral and glacial outwash Moderately well drained, silt loam, glaciomarine deposit
Well drained to rapidly drained, loam (gravelly), glaciofluvial deposits Imperfectly drained, clay, glaciolacustrine sediments
a
From Biogeoclimatic Ecosystem Classification (BEC) web portal http://www.for.gov.bc.ca/hre/becweb/
b
From Terrestrial Ecosystem Information (TEI) web portal http://www.env.gov.bc.ca/tei/access-soil.html
Mycorrhiza
Truffières P and M planted inoculated seedlings from a nursery located on coastal British Columbia and the other six orchards planted seedlings inoculated by a nursery in Oregon, USA that had worked in collaboration with a nursery in the Okanagan Valley, BC. Truffières were sampled in the fall after the rains had returned (October) except for truffières P and M which were sampled in the spring (April) during bud break. Depending on the number of trees in the truffle orchard, 5–15 trees were sampled per orchard. For each sampled tree, three subsamples of fine roots with ectomycorrhizas were excavated within 1 m of the stem at spots located equidistant apart around the tree and bulked per tree. The fine roots were collected from the top 10–15 cm of soil, placed in a labeled plastic bag with soil, kept cool, and examined within 1 month of collection. Roots were gently washed free of soil and 100–200 individual ectomycorrhizal root tips (depending on abundance of ectomycorrhizal root tips in the samples) per sample were sorted to morphotypes and counted under the dissecting microscope. Morphotypes were further characterized under the compound microscope with mantle and cystidium details (Agerer 1987–2002). Ectomycorrhizal tips representing each Tuber morphotype on each sampled tree in each orchard were frozen for subsequent DNA extraction and PCR amplification of the fungal internal transcribed spacer (ITS) region of nuclear rDNA. DNA was extracted from individual ectomycorrhizal root tips and truffles by using the Extract-N-Amp (Sigma) procedure. Briefly, single root tips or small truffle tissue samples were placed in wells (on ice) and 25 μl of the SIGMA extraction buffer (Sigma Aldrich) was immediately added to these wells. The samples were then covered with a transparent plate film, vortexed gently and centrifuged briefly, and incubated at 95 °C in a thermal cycler for 10 min. The samples were then removed from the thermal cycler, vortexed, and centrifuged again. Then 25 μl of the SIGMA Dilution Solution was added to each tube. Tubes were covered with a transparent plate film and were vortexed briefly and centrifuged. The samples were then stored in a freezer until used for PCR. Prior to performing PCR, the samples were diluted by pipetting 1 μl of extracted DNA into 99 μl of DNA-grade water (1 in 100 dilutions). This dilution was used as the DNA template in all PCRs. PCRs were carried out in 25-μl reactions that consisted of 6.0 μl DNA-grade water, 12.5 μl GoTaq Master Mix (Promega), 1.25 μl ITS 1 (10 μM) forward primer, 1.25 μl ITS 4 (10 μM) reverse primer, 1.50 μl MgCl2 (25 mM), 1.50 μl BSA (10 mg/ml), and 1.0 μl template DNA. Thermal cycler conditions for PCR included an initial incubation at 94 °C for 3 min to separate double-stranded DNA, which was followed by 35 cycles consisting of a 1 min at 94 °C, 1 min at 50 °C for primer annealing, and a 1-min elongation step at 72 °C. The final elongation step was extended for 10 min and then PCR products were cooled to 4 °C.
PCR products were visualized through gel electrophoresis and 20 μl of PCR product was cleaned using the Mag-Bind EZ Pure 96 Plate protocol (Omega Bio-Tek) according to the manufacturer’s recommendations. The DNA concentration (nanogram/microliter) of each PCR product was obtained using a NanoDrop ND-1000 spectrophotometer and ranged between 4.0 and 8.2 ng/μl. Then, 6.0 μl each of cleaned PCR product was placed into two separate wells (96 plate), and 1.0 μl of either the forward or reverse primer (5 μM) was added to the well for bidirectional sequencing. These were then submitted for sequencing using a 3130xL Genetic Analyzer (ABI). Sequences were identified through queries against GenBank with the BLASTN algorithm to verify their affiliation to Tuber. Sequences belonging to Tuber were then aligned with reference sequences using MUSCLE (Edgar 2004). Alignments were manually checked and ambiguous regions were excluded in Mesquite 2.5 (Maddison and Maddison 2009). Phylogenetic analyses (Fig. 1) were conducted with maximum parsimony (MP) in PAUP* (Swofford 2002) and Bayesian inference (BI) with MrBayes (Huelsenbeck and Ronquist 2001). The best-fit nucleotide substitution model was based on the Akaike information criterion and was implemented in PAUP* 4d106 (Swofford 2002). ML bootstrap support based on 1,000 replicates and BI analyses were run through the CIPRES Web portal (http://www.phylo.org/). BI was based on parallel runs of 20,000,000 generations with four chains, sampling every 1,000 generations. Sequences produced in this study are deposited in GenBank under accession numbers KF742719-KF742777.
Results and discussion Tuber melanosporum was detected on both hazelnut and oak host trees in six of the eight truffières studied (Table 3, Fig. S2). In two of the truffières (P, M), no T. melanosporum ectomycorrhizas were detected. Because only a subsample of trees and of fine roots in each orchard were examined, we cannot be certain that the fungus was absent from these two truffières. However, the fact that the plants in both these truffières had been supplied by the same nursery and those in the successful truffières by another nursery, suggests that there may have been differences in the quality of plants produced by the two nurseries. Similar results were found in Australia (Linde and Selmes 2012) with 25 % of inoculated seedlings lacking T. melanosporum ectomycorrhizas and the two major nursery samples showing as low as 35 % and as high as 95 % of seedlings with T. melanosporum ectomycorrhizas. No jurisdiction in North America currently has quality assurance standards in place for truffle-inoculated seedlings. In the Catalonia region of Spain, a quality assurance
Mycorrhiza Fig. 1 Most parsimonious tree (score 489) based on the analysis of ITS rDNA from ectomycorrhizas and reference taxa. Taxa recovered from field ectomycorrhizas are labeled in bold. The analysis consisted of 33 taxa and 387 included characters, 136 of which were parsimony informative. Nodes receiving significant bootstrap values (>70) based on parsimony and 1,000 sample replicates are indicated by thickened branches. Tuber gennadii was chosen as an outgroup based on previous analyses
protocol is in place, based on earlier work on the certification of truffle-inoculated seedlings (Fischer and Colinas 1996). Percent colonization by T. melanosporum varied among samples and among truffières and ranged from 0–80 % (data not provided). Because only a subsample of trees and roots were examined per truffière, we do not consider percent colonization to be as meaningful in this study as the presence/absence data by truffière. Morphotypes similar to T. aestivum were detected on plants in the two truffières growing this fungus (G, W), but molecular assessment of these mycorrhizas sometimes revealed the presence of Trichophaea species (Fig. S2). The Trichophaea woolhopeia species complex has been reported to form AD morphotype mycorrhizas in truffières in Europe, North America (Rubini et al. 2011), and New Zealand (Ian Hall, personal communication) and is considered a competitor of truffle species (Zambonelli et al. 2012).
One truffière (L) had some Tuber brumale contamination but Tuber indicum was not detected in any of the sampled truffières. Similarly, in New Zealand (Guerin-Laguette et al. 2013) and Australia (Australian Truffle Growers’ 2011), T. melanosporum orchards were found to contain some T. brumale. While New Zealand (New Zealand Ministry of Agriculture and Forestry 2003 amended 2011) and Australia (Commonwealth of Australia 2013) now have regulations aimed at stopping the importation of lower valued black truffles such as T. brumale and T. indicum, no such regulations currently exist in Canada. In one truffière (N), we detected the presence of Tuber borchii. This was a surprising find because bianchetto truffles (T. borchii) have a white peridium, are easily distinguished from T. melanosporum, and should not have accidently found their way into nursery inoculum. It turned out that the nursery had produced some T. borchii-inoculated oak seedlings and there had been a mix-up that resulted in some of these
4
3
3
6
4
7
5
2
L
P
G
N
Pr
S
W
M
NA not available
Years in ground
Truffière code
B
A
A
A
A
A
B
A
Nursery code
Corylus avellana
Corylus avellana Quercus robur
Corylus avellana
Corylus avellana
Quercus robur
Corylus avellana
Quercus robur
Quercus robur Corylus avellana
Corylus avellana
Corylus avellana
Tree species
Tuber melanosporum Tuber melanosporum Tuber aestivum Tuber melanosporum
Tuber melanosporum
Tuber melanosporum
Tuber melanosporum Tuber aestivum Tuber melanosporum Tuber aestivum Tuber melanosporum
Tuber melanosporum
Tuber melanosporum
Tuber species inoculated 5/5 2/5 0/15 7/15 2/15 0/5 3/3 0/1 3/3 1/1 4/6 2/6 3/5 2/5 5/9 2/9 2/10 4/10 1/10 3/3 3/3 1/1 0/5 1/5 2/5
Tuber-like with bristles Tuber melanosporum Tuber sp. Tuber-like boxy cells Tuber melanosporum Tuber melanosporum Tuber aestivum Tuber melanosporum Tuber-like with bristles Tuber-like with boxy cells
Number of trees colonized/sampled
Tuber melanosporum Tuber-like with bristles Tuber melanosporum Tuber-like with bristles Tuber-like with bristles Tuber melanosporum Tuber melanosporum Tuber aestivum Tuber melanosporum Tuber aestivum Tuber melanosporum Tuber-like with bristles Tuber melanosporum Tuber-like with bristles Tuber melanosporum
Tuber morphotype
Tuber beyerlei Tuber melanosporum Tuber beyerlei Trichophaea sp. Tuber melanosporum Tuber melanosporum Trichophaea sp. NA Tuber menseri Trichophaea sp.
Tuber melanosporum Tuber brumale NA Tuber menseri Tuber anniae NA Tuber melanosporum Trichophaea sp. Tuber melanosporum Tuber aestivum Tuber melanosporum Tuber menseri Tuber melanosporum Tuber borchii Tuber melanosporum
Tuber species from DNA sequencing
Table 3 Tuber species introduced on inoculated host seedlings into truffle orchards in southern British Columbia and Tuber species detected by morphological assessment and DNA sequencing
Mycorrhiza
Mycorrhiza
seedlings being shipped to and planted in the truffière. Because bianchetto truffles have been produced for a few years in a truffière in Idaho (Berch 2013), it seems likely that they could be produced in British Columbia; if so, this accidental introduction may prove to be fortuitous. A major surprise was the detection of numerous Tuber-like morphotypes with bristle-like cystidia (Fig. S2) that proved to be native species of Tuber. Most of these species are known from the USA but are first reports for British Columbia and Canada. Tuber anniae was detected in one truffière (P), Tuber beyerlei in two (Pr, S), and Tuber menseri nom. prov. (Bonito et al. 2010) in three (P, N, M) and all three species were found only on hazelnut (Table 1). Truffles of T. anniae (Puberulum clade, Bonito et al. 2010), or a species similar to it, have been collected near Williams Lake, BC (Bill Chapman and Jim Trappe, personal communications). Truffles of T. beyerlei (Maculatum clade, Guevara et al. 2013) and T. menseri nom. prov. (Puberulum clade, Bonito et al. 2010) have not yet been collected in BC, according to herbarium records. T. beyerlei was recently described from Oregon in association with Pseudotsuga menziesii (Douglas-fir), and the detection of this species in truffières in British Columbia expands both the range and host associations of this truffle species (Guevara et al. 2013). T. menseri nom. prov. has yet to be formally described but has also been reported from New Zealand and Europe (Bonito et al. 2010) although the natural distribution of this species is considered to be western North America. In March of 2013, three Périgord black truffles were discovered under hazelnut trees by trained truffle dogs in truffière S in the Lower Fraser Valley (KF742777, Berch 2013). While T. melanosporum ectomycorrhizas were found to be present in this truffière, their abundance appeared to be low, suggesting that for truffières the presence of truffle ectomycorrhizas may be as meaningful as an indicator for the likelihood of truffle production as percent root colonization. The other T. melanosporum truffières we studied are likely still too young to produce truffles as Hall et al. (2007) indicate that truffle production might be expected by about year 7. In August 2013, one Burgundy truffle was found under a hazelnut tree in a truffière on Vancouver Island that had not been part of this study (KF742776, unpublished data). This is the first independently confirmed report of the production of truffles of these two Mediterranean Tuber species in Canada. Acknowledgments We are grateful to the Agroforestry Development Initiative Fund for funding this research, to all of the truffle growers who participated in this study, and to Charles Lefevre of New World Truffières for supporting this work with advice on truffle cultivation and root sampling. Special thanks go to Wayne Haddow, BC Ministry of Agriculture, for being one of the first people in BC to see the possibility of a Mediterranean truffle industry in British Columbia. The Fragment Analysis and DNA Sequencing Services (FADSS) at the University of British Columbia-Okanagan carried out the PCR amplification and sequencing of ectomycorrhizas. Dr. Mary Berbee, University of British Columbia, sequenced the Tuber melanosporum and Tuber aestivum truffles produced
in BC. Gregory Bonito was supported through the US Department of Energy, Office of Biological and Environmental Research, Genome Science Program.
References Agerer R ed (1987–2002) Colour atlas of ectomycorrhizae, 1st–12th del., Einhorn-Verlag, Schwäbisch Gmünd ISBN: 3-921703-77-8 Australian Truffle Growers’ Association (2011) Science helping Australia produce some of the world’s best quality truffles. http:// www.trufflegrowers.com.au/wp-content/uploads/2011/06/WinterTruffle-Press-Release-170611.pdf Berch, SM (2013) Truffle cultivation and commercially harvested native truffles. In: Proceedings of international symposium on forest mushroom. Hosted by Korea Forest Research Institute and Korean Forest Mushroom Society. August 6, 2013, Seoul, Republic of Korea. Pp. 85–97. http://www.for.gov.bc.ca/hfd/pubs/docs/scv/SCV872.pdf Bonito GM, Gryganskyi AP, Trappe JM, Vilgalys R (2010) A global meta-analysis of Tuber ITS rDNA sequences: species diversity, host associations and long-distance dispersal. Mol Ecol 19:4994–5008. doi:10.111/j.1365-294.2010.04855.x Commonwealth of Australia (2013) ICON database, Department of Agriculture, Fisheries, Forests, accessed September 12, 2013 Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinforma 5:1–19. doi: 10.1186/1471-2105-5-113 Fischer C, Colinas C (1996) Methodology for certification of Quercus ilex seedlings inoculated with Tuber melanosporum for commercial application. In: Proceedings of the 1st international conference on mycorrhizae. Berkeley, CA, USA Guerin-Laguette A, Cummings N, Hesom-Williams N, Butler R, Wang Y (2013) Mycorrhiza analyses in New Zealand truffières reveal frequent but variable persistence of Tuber melanosporum in coexistence with other truffle species. Mycorrhiza 23:87–98. doi:10. 1007/s00572-012-0450-2 Guevara G, Bonito G, Trappe JM, Cazares E, Williams G, Healy RA, Schadt C, Vilgalys R (2013) New North American truffles (Tuber spp.) and their ectomycorrhizal associations. Mycologia 105(1): 194–209. doi:10.3852/12-087 Hall IR, Brown G (2002) The black truffle: its history, uses and cultivation. Reprint of second edition on CD ROM plus booklet. New Zealand Institute for Crop & Food Research Limited, Christchurch Hall IR, Brown GT, Zambonelli A (2007) Taming the truffle. The history, lore, and science of the ultimate mushroom. Timber, Portland, p 305 Hall IR, Frith A, Haslam W (2008) A rough climatic comparison of various centres adjacent to or with similar climates to Périgord black, Italian white, Burgundy and bianchetto truffle producing areas. Dunedin, Truffles & Mushrooms (Consulting) Ltd. (updated 19 October 2013). www.trufflesandmushrooms.co.nz/free% 20downloads.html, accessed January 2014 Huelsenbeck JP, Ronquist F (2001) MrBayes: Bayesian inference of phylogeneric trees. Bioinforma 17:754–755. doi:10.1093/ bioinformatics/17.8.754 Lefevre C (2012) Native and cultivated truffles of North America. Chapter 12 in Edible Ectomycorrhizal Mushrooms, edited by Zambonelli A and Bonito G. Soil Biol 34:209–226 Linde CC, Selmes H (2012) Genetic diversity and mating type distribution of Tuber melanosporum and their significance to truffle cultivation in artificially planted truffières in Australia. Appl Environ Microbiol 78(18):6534–6539
Mycorrhiza Maddison WP, Maddison DR (2009) Mesquite 2.6: a modular system for evolutionary analysis. Periodical mesquite: a modular system for evolutionary analysis. http://mesquiteproject. org New Zealand Ministry of Agriculture and Forestry (2003) (amended 2011) Importation into New Zealand of specified fresh and frozen Tuber species (truffles). Biosecurity New Zealand Standard BNZIMP-TUBER. http://www.biosecurity.govt.nz/files/ihs/bnz-imptuber.pdf Rubini A, Belfiori B, Passeri V, Baciarelli Falini L, Arcioni S, Riccioni C, Paolocci F (2011) The AD-type ectomycorrhizas, one of the most
common morphotypes present in truffle fields, result from fungi belonging to the Trichophaea woolhopeia species complex. Mycorrhiza 21(1):17–25 Swofford DL (2002) PAUP* 4.0: phylogenetic analysis using parsimony (*and other methods). Sunderland, Massachusetts: Sinauer Associates Zambonelli A, Iotti M, Boutahir S, Lancellotti E, Perini C, Pacioni G (2012) Ectomycorrhizal fungal communities of edible ectomycorrhizal mushrooms. Chapter 7 in edible ectomycorrhizal mushrooms, edited by Zambonelli A and Bonito G. Soil Biol 34: 105–124
