Mycorrhiza DOI 10.1007/s00572-013-0543-6
ORIGINAL PAPER
Effects of soil tillage on Tuber magnatum development in natural truffières E. Salerni & M. Iotti & P. Leonardi & L. Gardin & M. D’Aguanno & C. Perini & P. Pacioni & A. Zambonelli
Received: 30 August 2013 / Accepted: 12 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Tuber magnatum Pico, the Italian white truffle, commands the highest prices of any truffle. Despite its commercial value, it is the only edible European truffle which has not yet been successfully cultivated. Because of this, it is essential to safeguard natural truffières and to identify cultural practices to maximize their productivity. Soil tillage is successfully and extensively used in black truffle cultivation to enhance productivity, but its effects are not known on the development of T. magnatum . A recently developed realtime PCR assay was applied to evaluate the effects of tillage (10–15 cm depth) on T. magnatum mycelium in two different natural truffle grounds located in Tuscany and EmiliaRomagna. Tillage effects on bulk density, ectomycorrhizal fungal communities, and ascoma production were also assessed. Tilling significantly increased the quantity of T. magnatum mycelium which seemed to be related to an increase in soil porosity by up to 34 %, and the diversity of ectomycorrhizal fungal communities. On the contrary, no significant effects were found on ascoma production. The results highlight that real-time PCR is the most reliable method for evaluating the effects of cultural practices on the E. Salerni (*) : P. Leonardi : M. D’Aguanno : C. Perini BIOCONNET, BIOdiversity and CONservation NETwork, Department of Life Science, University of Siena, via Mattioli 4, 53100 Siena, Italy e-mail: [email protected] M. Iotti : A. Zambonelli Dipartimento di Scienze Agrarie, University of Bologna, via Fanin 46, 40127 Bologna, Italy L. Gardin Via Guerrazzi, 2rosso, 50132 Firenze, Italy P. Pacioni Department of Life, Health and Environmental Sciences, University of Aquila, Via Vetoio, Coppito8 1, 67100 L’Aquila, Italy
development of T. magnatum in soil avoiding long-term studies on fruiting body production. Keywords Tuber magnatum . Soil tillage . Mycelium . Real-time PCR . Ectomycorrhizal community
Introduction Truffles are hypogeous fruiting bodies of ectomycorrhizal (ECM) Ascomycetes belonging to the order Pezizales, which includes the Pezizaceae, Helvellaceae, Tuberaceae, Morchellaceae/Discinaceae, Pyronemataceae, Glaziellaceae, and Carbomycetaceae (Trappe et al. 2009; Læssøe and Hansen 2007). The majority of the truffles of commerce are housed in the Tuberaceae of which the genus Tuber, containing about 180 species, is the most important (Mello et al. 2006; Bonito et al. 2013). Some of these species have considerable culinary importance with the most highly prized species Tuber magnatum Pico, the Italian white truffle, Tuber melanosporum Vittad. (Périgord black truffle), Tuber aestivum Vittad. (Burgundy truffle), and Tuber borchii Vittad. (bianchetto) (Bonito et al. 2013; Hall et al. 2007). T. magnatum Pico is the most sought after of the truffles with its Italian wholesale price reaching €2,800–3.800 kg−1 in the autumn of 2012 (http://www.tuber.it/pagine/ita/la_borsa. lasso; http://acqualagna.com/fiere-tartufo/borsa-tartufo). As with any ECM fungus, truffle development in the soil requires specific environmental conditions which includes pH, moisture, fertility, temperature, aeration, texture, type of organic matter and content, and canopy cover (Hall et al. 2007). Cultural practices are able to modify these factors and may create conditions more favorable to truffle development. Liming, irrigation, pruning, soil tillage, fertilization, and mulching are extensively used in black truffle cultivation with generally positive results (Chevalier and Frochot 1997; Callot
Mycorrhiza
1999; Tibiletti and Zambonelli 1999; Hall et al. 2003). Among these, soil tillage is extensively used in Italy, France, and New Zealand, where it has been shown to have beneficial effects on black truffle production in cultivated truffières (Hall et al. 2007; Pargney et al. 2011). Despite its economic value, scientific knowledge concerning T. magnatum has not been sufficient to identify reliable methods for its cultivation (Hall et al. 2003; Murat et al. 2005; Iotti et al. 2012a; Leonardi et al. 2013). Some sporadic successes have been reported (Bencivenga et al. 2009), although, in general, attempts to cultivate this truffle species have met with failure (Hall et al. 1998, 2007; Gregori 2001). In the absence of reliable strategies for T. magnatum cultivation, it is important to safeguard natural truffières and to identify cultural practices to increase their productivity. Till now, there has been no research reporting the effects of cultural practices on the development of T. magnatum. This gap in knowledge is due to the difficulties in studying the development of the white truffle in the soil because its ectomycorrhizas are rare, even in the areas where its ascomata are found (Leonardi et al. 2013; Murat et al. 2005), and the production of ascomata is too scattered and variable to follow in short-term experiments. Recently, Zampieri et al. (2010) have shown that T. magnatum mycelium is widely distributed in the soil and its presence is not restricted to just those points where mycorrhizas or ascomata are found. Iotti et al. (2012a) have developed a specific real-time PCR assay using TaqMan chemistry to detect and quantify T. magnatum in soil and concluded that its mycelium is a better indicator than mycorrhiza for assessing T. magnatum in the soil and predicting ascoma production. The aim of the work reported here was to determine the effect of soil tillage on the development of T. magnatum mycelium in the soil by using a real-time PCR assay. The effects of soil tillage on the ECM fungal communities and T. magnatum ascoma formation were also studied.
Materials and methods Experimental truffières The research was conducted in two different natural T. magnatum production areas, located in northern and central Italy in the following Italian regions: Emilia-Romagna [Parco del Museo della Bonifica, Argenta, FE, lat 44° 37′ 10″ N, long 11° 48′ 55″ E, altitude 5 m above sea level (ASL)] and Tuscany (Barbialla Nuova, Montaione, FI, lat 43° 35′ 30″ N, long 10° 50′ 55″ E, altitude 135 m ASL). All these truffières are closed to the public so the scientific data on production collected are more meaningful. The experimental areas, covering between 2 and 5 ha, are characterized by different habitats: a mixed broad-leaved forest in Tuscany and a man-
made park surrounding some buildings in Emilia-Romagna. This latter truffière is representative of the natural T. magnatum production areas in the Po valley and the putative host plants are poplar (Populus nigra L.) and linden (Tilia vulgaris Hayne). The soil is calcareous (13–19 % of total CaCO3) with a pH ranging from 8.0 to 8.3 in the different plots. The putative T. magnatum host plants in the Tuscan truffière are hornbeam (Ostrya carpinifolia Scop.), poplar (Populus alba L.), and oaks (Quercus cerris L., Q. petraea (Mattuschka) Liebl., Q. ilex L.). The soil has a CaCO3 content ranging from 4.1 to 9.3 % and a pH of 7.7–8.3 (Table 1).
Experimental design A Before–After–Control–Impact (BACI) design (Downes et al. 2002) to investigate the effects of soil tillage on T. magnatum development was applied in the studied truffières. The strength of a BACI design lies in the assumption that changes over time in the impact area, unrelated to the impact, are controlled for by changes over time in the control area because the time (before or after)×area (impact or control) interaction is analyzed (Downes et al. 2002). In order to apply BACI analysis, nine plots of about 500 m2 each, homogeneous for vegetation and soil parameters, were delimited in each truffière. The most important soil characteristics affecting the growth and fruiting of truffles were described for each Table 1 Main soil characteristics of the two truffières in L1 (tilled 1 year), L2 (tilled 2 years), and control (C) plot (CEC cationic exchange capacity, OM organic matter) Sand Silt (%) (%)
Argenta
Clay (%)
pH
Total CaCO3 (%)
CEC meq/ 100 g
OM (%)
C
54.30 31.90 58.80 L1 59.50 33.10 42.40 L2 60.00 31.60 36.70 Barbialla C 61.20 62.30
31.70 49.20 31.60 28.60 48.80 42.30 29.70 49.80 42.30 28.70 26.00
14.00 18.90 9.60 11.90 18.10 15.30 10.30 18.60 21.00 10.10 11.60
8.20 8.30 8.00 8.10 8.20 8.10 8.20 8.20 8.10 8.20 8.30
16.05 19.15 13.00 15.85 17.45 15.80 17.05 17.05 15.85 6.10 7.95
17.00 10.30 23.20 21.30 17.90 18.40 10.90 16.00 23.90 9.80 10.10
4.27 2.42 9.72 4.82 4.32 4.66 2.85 3.89 7.58 2.96 2.15
64.70 L1 67.60 63.70 62.70 L2 63.70 67.50 70.10
28.60 24.30 28.60 28.30 26.50 23.80 23.30
6.70 8.10 7.60 9.00 9.90 8.70 6.60
8.10 8.10 8.00 8.20 8.10 7.70 8.10
9.30 4.55 7.90 8.80 4.90 4.10 5.45
22.40 19.20 27.20 21.20 16.30 24.00 37.40
6.13 4.80 7.71 5.25 3.50 6.89 11.68
Mycorrhiza
plot using standard procedures (Soil Survey Staff 1993). Six plots (three per truffière) were tilled only once in 2009 (L1); six (three per truffiere) were tilled twice, one time each in 2009 and 2010 (L2); and six plots (three per truffière) were left as a control (C). Soil tillage was carried out at the end of March using a cutter operating up to a depth of 15 cm below ground, with respect to soil morphology (slope, rock outcrops, etc.). Assessment of bulk density and truffle production Soil sampling to assess bulk density was performed before and after soil tillage (in June 2008 and June 2011) by collecting two soil cores (0–5 cm depth) making three repetitions on diagonal of each plot (144 samples, 72 for each truffiére), taking care to remove litter and other coarse organic material from soil surface. Truffle production was assessed weekly from September to December for 3 years (2008–2010) (96 surveys in total, 48 in each truffière) by using trained dogs. All collected truffles were numbered, weighed, and recorded for each plot. Analysis of ECM fungal community ECM fungal communities of both experimental truffiéres before the treatments were analyzed in 2008 and reported in a recent study (Leonardi et al. 2013). In order to evaluate the effects of soil tillage on ECM communities also after the treatments, sampling was repeated in autumn 2010 in the same points of both experimental sites (40 samples in total, 20 for each truffière). After litter removal, soil cores (30 cm long and 6 cm in diameter) were taken both from the collection points of T. magnatum ascomata and from nonproductive patches as described by Leonardi et al. (2013). Samples were soaked overnight in tap water and then the roots were removed from the soil by careful washing over a 2-mm mesh sieve. Roots were examined under a dissecting stereomicroscope (×20) and ECM tips from each soil sample were sorted in morphotypes, counted, and washed from soil particles by three consecutive steps of vortexing (30 s) and centrifugation (1 min at 13,000 rpm). ECM morphotypes were stored at −20 °C in sterile water or at 4 °C in FAA (5 % of formaldehyde, 90 % of 70 %–ethanol, 5 % of acetic acid) until PCR amplification or morphological analysis, respectively. The relative abundance of ECM root tips was expressed in root tips density (no. of root tips per decimeter of soil). Each morphotype found in 2010 was morphologically compared to those described from the samples collected in 2008 and stored in FAA. Morpho-anatomical characteristics of the mantle, external elements (hyphae, rhizomorphs, and cystidia), as well as longitudinal and cross-sections of each morphotype, were examined under a microscope (Zeiss Imager Z1 Apotome, ×630) with differential interference
contrast. One ECM tip of each morphotype previously described and three representative ECM tips of each new morphotype were molecularly identified using a direct PCR approach as described by Iotti and Zambonelli (2006) with the primer pair ITS1F/ITS4 (White et al. 1990; Gardes and Bruns 1996). Identity of the previously characterized morphotypes was confirmed by treating their ITS1–ITS4 amplicons with the restriction enzymes AluI and HinfIII (Fermentas). They were then assigned to the operational taxonomic units (OTUs) identified by Leonardi et al. (2013). On the contrary, amplified fragments from the new morphotypes were sequenced using both the primers mentioned above and the obtained sequences were regarded as belonging to OTUs following the criteria reported by Landeweert et al. (2003) with the exceptions proposed by Leonardi et al. (2013). Analysis of T. magnatum soil mycelium Soil sampling to estimate T. magnatum mycelium was carried out in January 2009, 2010, and 2011, at the end of the annual white truffle season as reported by Iotti et al. (2012a). A set of nine equidistant soil cores (30 cm long and 16 mm in diameter) were taken from each plot along two diagonal lines, excluding a border 5 m wide all around. One sample per plot and year (54 samples in total, 27 for each truffiére) was made by pooling the nine soil cores after removal of all visible root fragments, stones, or litter compounds under a stereomicroscope. The soil was stored at −80 °C and then lyophilized for 3 days using the VirTis Benchtop 2 K freeze dryer (SP Industries). DNA extraction and T. magnatum quantification were carried out following the procedures and conditions set out by Iotti et al. (2012a). Briefly, isolation of total DNAs were performed with a CTAB-based protocol, followed by purification using a NucleoSpin Plant II kit (Macherey-Nagel, Düren, Germany), whereas TmgITS1for–TmgITS1rev primer pair and the TaqMan probe TmgITS1prob were employed for real-time PCR quantification. The thermal cycling conditions were: 10 min at 95 °C followed by 45 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The threshold fluorescence level was determined with the default adaptive baseline algorithm of the MxPro software (version 4.10) (Agilent Technologies) and the resulting Ct values were automatically converted to quantities of T. magnatum DNA using the standard curve method. A standard curve was generated for each run with a series of 10-fold dilutions of genomic DNA from T. magnatum (from 107 to 102 fg per reaction) as standards. Calibration curves were also generated to convert DNA concentration values resulting from real-time PCRs to absolute quantities of T. magnatum mycelium in soil. A total of seven serial dilutions of immature gleba tissue (from 2 to 2 * 10−6 mg) per gram of soil free from T. magnatum mycelium were processed in triplicate for each truffle ground. Gleba tissue and soil were lyophilized and grinded as described
Mycorrhiza
above to prepare the serial dilutions. An immature ascoma (without spores) was selected over mycelium because suitable pure cultures of T. magnatum are not available (Iotti et al. 2012b). Moreover, the presence of spores might be to the detriment of calibration curve reliability because DNA extraction from spores is difficult (Selosse et al. 2013). In turn, the soils used to generate the calibration curves were taken from unproductive truffle patches of each experimental site. Absence of T. magnatum mycelium within these soil samples was confirmed by species-specific PCRs (Amicucci et al. 1998) on soil DNAs previously isolated with the NucleoSpin Soil kit (Macherey-Nagel). Noninoculated soil samples were used as negative controls. DNA extractions and real-time PCRs were performed as previously described. Statistical analysis ECM fungal communities were described for each plot by the following: richness (total number of distinct OTUs detected), Pielou's index (describes how evenly the individuals are distributed among the OTUs) (E =0→1, where E =1 if all species occur at the same proportion), and Shannon–Wiener index (take into account the taxa richness and their relative abundance) (H ′=0→∞, increase of such value is due to additional unique species and/or a greater species evenness) (Magurran 2004). These indices were calculated using the software package Vegan version 1.17-9 (Oksanen et al. 2011), within the R system for statistical computing (version 2.12.2) (R Development Core Team 2011). Analysis of variance (ANOVA) and Tukey's HSD analysis were adopted to verify if the mean control– impact differences in bulk density, soil mycelium, truffle production (number of carpophores and total weight), and ECM fungal community in the “before” period were significantly different from the mean differences in the “after” period. Normality was checked using Shapiro–Wilk test and the homogeneity of variance was tested using Levene's test (Neter et al. 1996).
Table 2 ANOVA table for the effects of soil tillage on bulk density, ECM fungal communities, number of truffle, total weight, and soil mycelium
L1 and L2 soil tillage Bulk density Species richness ECM tip density (n./dm3) Number of truffle Total truffle weight Soil mycelium Before and after soil tillage Bulk density Species richness ECM tip density (n./dm3) Number of truffle Total truffle weight Soil mycelium Time (before or after)×treatment (L1/L2) Bulk density Species richness ECM tip density (n./dm3) Number of truffle Total truffle weight Soil mycelium
Type III, F
p level
7, 15,700 0, 28,919 0, 20,117
0.0029 0.7508 0.8189
0, 18,408 3, 77,645 2, 17,471
0.9068 0.0564 0.0996
21, 936 2, 54,961 1, 82,534 3, 69,231 0, 57,575 5, 36,787
0.0001 0.1202 0.1860 0.0591 0.6330 0.0237
3.365 0.35687 0.08289 1.247683 1.240824 318.333
0.0480 0.7028 0.9207 0.2999 0.3023 0.0297
10 %, respectively). Only slight differences were recorded in the control plots (Fig. 1). Moreover, Tukey's post hoc test revealed that the bulk density decreased significantly (p level0.05) (Tab. 2). 1.60
Results
Statistical differences were found between the bulk density values obtained before and after soil tillage and their interaction (Table 2). Soil tillage affected the soil bulk density in both the Argenta and Barbialla experimental truffières (Fig. 1). In the Argenta truffière (Emilia-Romagna), the bulk density measured before and after soil tillage decreased by approximately 34 % in the plots L2 and 15 % in those L1 (Fig. 1). Reductions in bulk density also occurred at Barbialla (Tuscany), although with lower percentage values (21 and
av. bulk density (g/cmc)
Effects of soil tillage on bulk density and truffle production
1.40 1.20 1.00 0.80
before soil tillage after soil tillage
0.60 0.40 0.20 0.00
C
L1 Argenta
L2
C
L1 Barbialla
L2
Fig. 1 Average bulk density and standard error (error bar) measured before and after soil tillage in the two truffières (Argenta and Barbialla)
Mycorrhiza
communities which remain dominated by Thelephoraceae and Sebacinaceae (Fig. 4).
Effects of soil tillage on ECM fungal communities Out of the 40 soil cores collected in autumn 2010, seven in the Argenta truffière and one in the Barbialla truffière, had no ectomycorrhizae, but only the roots of non-ECM trees (Aesculus hippocastanum , Acer campestre, Celtis australis, and Cornus sanguinea), as found in autumn 2008 (Leonardi et al. 2013). More than 7,000 colonized root tips were included in the analysis: 3,258 tips from the sampling performed in the autumn of 2008 and 4,141 tips from the 2010 sampling. In general, ECM root tip density increased in almost all plots through the experiment and, particularly, in the plots L2, even if no significant differences were found. In the Barbialla truffière, the ECM fungal community consisted of 45 species before soil tillage, whereas afterwards, only 27 were found. In Argenta truffière, the number of species decreased from 22 to 18 after the soil tillage. Seven new OTUs were molecularly identified with respect to the ECM fungal community characterized in 2008. Even if no statistical differences were found, the highest reduction in species richness was recorded in plots L1 and L2 of Barbialla and Argenta truffières, respectively (Table 3). Following soil tillage, the Shannon and Pielou's indices decreased (Table 3). In the L2 community of Barbialla, in particular, the Shannon index dropped from 3.39 to 3.21, confirming a loss of diversity. The Kruskal–Wallis test (α = 0.05) was performed between the Shannon and Pielou's indices to compare the different soil tillage treatments. Significant differences were only found with respect to the L2 soil tillage (p =0.049). The rank–abundance curves (Figs. 2 and 3) obtained from the control and the two differently tilled plots showed that the ECM fungal community structures differ between the truffières. After tillage treatments, ECM fungal communities tend to be dominated by a lower number of species. However, this reduction did not affect the composition of fungal Table 3 ECM fungal diversity in the total two truffières (Argenta and Barbialla), in L1, L2, and control C plot before and after soil tillage
In bold significant differences by the Kruskal–Wallis test (α =0.05)
Assessment of T. magnatum soil mycelium Real-time PCR data showed that T. magnatum mycelium markedly increased in the soil of all tilled plots in both truffières by the end of the last year of treatment (Fig. 5). ANOVA analysis demonstrated that the amount of T. magnatum mycelium was significantly higher (p
ORIGINAL PAPER
Effects of soil tillage on Tuber magnatum development in natural truffières E. Salerni & M. Iotti & P. Leonardi & L. Gardin & M. D’Aguanno & C. Perini & P. Pacioni & A. Zambonelli
Received: 30 August 2013 / Accepted: 12 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Tuber magnatum Pico, the Italian white truffle, commands the highest prices of any truffle. Despite its commercial value, it is the only edible European truffle which has not yet been successfully cultivated. Because of this, it is essential to safeguard natural truffières and to identify cultural practices to maximize their productivity. Soil tillage is successfully and extensively used in black truffle cultivation to enhance productivity, but its effects are not known on the development of T. magnatum . A recently developed realtime PCR assay was applied to evaluate the effects of tillage (10–15 cm depth) on T. magnatum mycelium in two different natural truffle grounds located in Tuscany and EmiliaRomagna. Tillage effects on bulk density, ectomycorrhizal fungal communities, and ascoma production were also assessed. Tilling significantly increased the quantity of T. magnatum mycelium which seemed to be related to an increase in soil porosity by up to 34 %, and the diversity of ectomycorrhizal fungal communities. On the contrary, no significant effects were found on ascoma production. The results highlight that real-time PCR is the most reliable method for evaluating the effects of cultural practices on the E. Salerni (*) : P. Leonardi : M. D’Aguanno : C. Perini BIOCONNET, BIOdiversity and CONservation NETwork, Department of Life Science, University of Siena, via Mattioli 4, 53100 Siena, Italy e-mail: [email protected] M. Iotti : A. Zambonelli Dipartimento di Scienze Agrarie, University of Bologna, via Fanin 46, 40127 Bologna, Italy L. Gardin Via Guerrazzi, 2rosso, 50132 Firenze, Italy P. Pacioni Department of Life, Health and Environmental Sciences, University of Aquila, Via Vetoio, Coppito8 1, 67100 L’Aquila, Italy
development of T. magnatum in soil avoiding long-term studies on fruiting body production. Keywords Tuber magnatum . Soil tillage . Mycelium . Real-time PCR . Ectomycorrhizal community
Introduction Truffles are hypogeous fruiting bodies of ectomycorrhizal (ECM) Ascomycetes belonging to the order Pezizales, which includes the Pezizaceae, Helvellaceae, Tuberaceae, Morchellaceae/Discinaceae, Pyronemataceae, Glaziellaceae, and Carbomycetaceae (Trappe et al. 2009; Læssøe and Hansen 2007). The majority of the truffles of commerce are housed in the Tuberaceae of which the genus Tuber, containing about 180 species, is the most important (Mello et al. 2006; Bonito et al. 2013). Some of these species have considerable culinary importance with the most highly prized species Tuber magnatum Pico, the Italian white truffle, Tuber melanosporum Vittad. (Périgord black truffle), Tuber aestivum Vittad. (Burgundy truffle), and Tuber borchii Vittad. (bianchetto) (Bonito et al. 2013; Hall et al. 2007). T. magnatum Pico is the most sought after of the truffles with its Italian wholesale price reaching €2,800–3.800 kg−1 in the autumn of 2012 (http://www.tuber.it/pagine/ita/la_borsa. lasso; http://acqualagna.com/fiere-tartufo/borsa-tartufo). As with any ECM fungus, truffle development in the soil requires specific environmental conditions which includes pH, moisture, fertility, temperature, aeration, texture, type of organic matter and content, and canopy cover (Hall et al. 2007). Cultural practices are able to modify these factors and may create conditions more favorable to truffle development. Liming, irrigation, pruning, soil tillage, fertilization, and mulching are extensively used in black truffle cultivation with generally positive results (Chevalier and Frochot 1997; Callot
Mycorrhiza
1999; Tibiletti and Zambonelli 1999; Hall et al. 2003). Among these, soil tillage is extensively used in Italy, France, and New Zealand, where it has been shown to have beneficial effects on black truffle production in cultivated truffières (Hall et al. 2007; Pargney et al. 2011). Despite its economic value, scientific knowledge concerning T. magnatum has not been sufficient to identify reliable methods for its cultivation (Hall et al. 2003; Murat et al. 2005; Iotti et al. 2012a; Leonardi et al. 2013). Some sporadic successes have been reported (Bencivenga et al. 2009), although, in general, attempts to cultivate this truffle species have met with failure (Hall et al. 1998, 2007; Gregori 2001). In the absence of reliable strategies for T. magnatum cultivation, it is important to safeguard natural truffières and to identify cultural practices to increase their productivity. Till now, there has been no research reporting the effects of cultural practices on the development of T. magnatum. This gap in knowledge is due to the difficulties in studying the development of the white truffle in the soil because its ectomycorrhizas are rare, even in the areas where its ascomata are found (Leonardi et al. 2013; Murat et al. 2005), and the production of ascomata is too scattered and variable to follow in short-term experiments. Recently, Zampieri et al. (2010) have shown that T. magnatum mycelium is widely distributed in the soil and its presence is not restricted to just those points where mycorrhizas or ascomata are found. Iotti et al. (2012a) have developed a specific real-time PCR assay using TaqMan chemistry to detect and quantify T. magnatum in soil and concluded that its mycelium is a better indicator than mycorrhiza for assessing T. magnatum in the soil and predicting ascoma production. The aim of the work reported here was to determine the effect of soil tillage on the development of T. magnatum mycelium in the soil by using a real-time PCR assay. The effects of soil tillage on the ECM fungal communities and T. magnatum ascoma formation were also studied.
Materials and methods Experimental truffières The research was conducted in two different natural T. magnatum production areas, located in northern and central Italy in the following Italian regions: Emilia-Romagna [Parco del Museo della Bonifica, Argenta, FE, lat 44° 37′ 10″ N, long 11° 48′ 55″ E, altitude 5 m above sea level (ASL)] and Tuscany (Barbialla Nuova, Montaione, FI, lat 43° 35′ 30″ N, long 10° 50′ 55″ E, altitude 135 m ASL). All these truffières are closed to the public so the scientific data on production collected are more meaningful. The experimental areas, covering between 2 and 5 ha, are characterized by different habitats: a mixed broad-leaved forest in Tuscany and a man-
made park surrounding some buildings in Emilia-Romagna. This latter truffière is representative of the natural T. magnatum production areas in the Po valley and the putative host plants are poplar (Populus nigra L.) and linden (Tilia vulgaris Hayne). The soil is calcareous (13–19 % of total CaCO3) with a pH ranging from 8.0 to 8.3 in the different plots. The putative T. magnatum host plants in the Tuscan truffière are hornbeam (Ostrya carpinifolia Scop.), poplar (Populus alba L.), and oaks (Quercus cerris L., Q. petraea (Mattuschka) Liebl., Q. ilex L.). The soil has a CaCO3 content ranging from 4.1 to 9.3 % and a pH of 7.7–8.3 (Table 1).
Experimental design A Before–After–Control–Impact (BACI) design (Downes et al. 2002) to investigate the effects of soil tillage on T. magnatum development was applied in the studied truffières. The strength of a BACI design lies in the assumption that changes over time in the impact area, unrelated to the impact, are controlled for by changes over time in the control area because the time (before or after)×area (impact or control) interaction is analyzed (Downes et al. 2002). In order to apply BACI analysis, nine plots of about 500 m2 each, homogeneous for vegetation and soil parameters, were delimited in each truffière. The most important soil characteristics affecting the growth and fruiting of truffles were described for each Table 1 Main soil characteristics of the two truffières in L1 (tilled 1 year), L2 (tilled 2 years), and control (C) plot (CEC cationic exchange capacity, OM organic matter) Sand Silt (%) (%)
Argenta
Clay (%)
pH
Total CaCO3 (%)
CEC meq/ 100 g
OM (%)
C
54.30 31.90 58.80 L1 59.50 33.10 42.40 L2 60.00 31.60 36.70 Barbialla C 61.20 62.30
31.70 49.20 31.60 28.60 48.80 42.30 29.70 49.80 42.30 28.70 26.00
14.00 18.90 9.60 11.90 18.10 15.30 10.30 18.60 21.00 10.10 11.60
8.20 8.30 8.00 8.10 8.20 8.10 8.20 8.20 8.10 8.20 8.30
16.05 19.15 13.00 15.85 17.45 15.80 17.05 17.05 15.85 6.10 7.95
17.00 10.30 23.20 21.30 17.90 18.40 10.90 16.00 23.90 9.80 10.10
4.27 2.42 9.72 4.82 4.32 4.66 2.85 3.89 7.58 2.96 2.15
64.70 L1 67.60 63.70 62.70 L2 63.70 67.50 70.10
28.60 24.30 28.60 28.30 26.50 23.80 23.30
6.70 8.10 7.60 9.00 9.90 8.70 6.60
8.10 8.10 8.00 8.20 8.10 7.70 8.10
9.30 4.55 7.90 8.80 4.90 4.10 5.45
22.40 19.20 27.20 21.20 16.30 24.00 37.40
6.13 4.80 7.71 5.25 3.50 6.89 11.68
Mycorrhiza
plot using standard procedures (Soil Survey Staff 1993). Six plots (three per truffière) were tilled only once in 2009 (L1); six (three per truffiere) were tilled twice, one time each in 2009 and 2010 (L2); and six plots (three per truffière) were left as a control (C). Soil tillage was carried out at the end of March using a cutter operating up to a depth of 15 cm below ground, with respect to soil morphology (slope, rock outcrops, etc.). Assessment of bulk density and truffle production Soil sampling to assess bulk density was performed before and after soil tillage (in June 2008 and June 2011) by collecting two soil cores (0–5 cm depth) making three repetitions on diagonal of each plot (144 samples, 72 for each truffiére), taking care to remove litter and other coarse organic material from soil surface. Truffle production was assessed weekly from September to December for 3 years (2008–2010) (96 surveys in total, 48 in each truffière) by using trained dogs. All collected truffles were numbered, weighed, and recorded for each plot. Analysis of ECM fungal community ECM fungal communities of both experimental truffiéres before the treatments were analyzed in 2008 and reported in a recent study (Leonardi et al. 2013). In order to evaluate the effects of soil tillage on ECM communities also after the treatments, sampling was repeated in autumn 2010 in the same points of both experimental sites (40 samples in total, 20 for each truffière). After litter removal, soil cores (30 cm long and 6 cm in diameter) were taken both from the collection points of T. magnatum ascomata and from nonproductive patches as described by Leonardi et al. (2013). Samples were soaked overnight in tap water and then the roots were removed from the soil by careful washing over a 2-mm mesh sieve. Roots were examined under a dissecting stereomicroscope (×20) and ECM tips from each soil sample were sorted in morphotypes, counted, and washed from soil particles by three consecutive steps of vortexing (30 s) and centrifugation (1 min at 13,000 rpm). ECM morphotypes were stored at −20 °C in sterile water or at 4 °C in FAA (5 % of formaldehyde, 90 % of 70 %–ethanol, 5 % of acetic acid) until PCR amplification or morphological analysis, respectively. The relative abundance of ECM root tips was expressed in root tips density (no. of root tips per decimeter of soil). Each morphotype found in 2010 was morphologically compared to those described from the samples collected in 2008 and stored in FAA. Morpho-anatomical characteristics of the mantle, external elements (hyphae, rhizomorphs, and cystidia), as well as longitudinal and cross-sections of each morphotype, were examined under a microscope (Zeiss Imager Z1 Apotome, ×630) with differential interference
contrast. One ECM tip of each morphotype previously described and three representative ECM tips of each new morphotype were molecularly identified using a direct PCR approach as described by Iotti and Zambonelli (2006) with the primer pair ITS1F/ITS4 (White et al. 1990; Gardes and Bruns 1996). Identity of the previously characterized morphotypes was confirmed by treating their ITS1–ITS4 amplicons with the restriction enzymes AluI and HinfIII (Fermentas). They were then assigned to the operational taxonomic units (OTUs) identified by Leonardi et al. (2013). On the contrary, amplified fragments from the new morphotypes were sequenced using both the primers mentioned above and the obtained sequences were regarded as belonging to OTUs following the criteria reported by Landeweert et al. (2003) with the exceptions proposed by Leonardi et al. (2013). Analysis of T. magnatum soil mycelium Soil sampling to estimate T. magnatum mycelium was carried out in January 2009, 2010, and 2011, at the end of the annual white truffle season as reported by Iotti et al. (2012a). A set of nine equidistant soil cores (30 cm long and 16 mm in diameter) were taken from each plot along two diagonal lines, excluding a border 5 m wide all around. One sample per plot and year (54 samples in total, 27 for each truffiére) was made by pooling the nine soil cores after removal of all visible root fragments, stones, or litter compounds under a stereomicroscope. The soil was stored at −80 °C and then lyophilized for 3 days using the VirTis Benchtop 2 K freeze dryer (SP Industries). DNA extraction and T. magnatum quantification were carried out following the procedures and conditions set out by Iotti et al. (2012a). Briefly, isolation of total DNAs were performed with a CTAB-based protocol, followed by purification using a NucleoSpin Plant II kit (Macherey-Nagel, Düren, Germany), whereas TmgITS1for–TmgITS1rev primer pair and the TaqMan probe TmgITS1prob were employed for real-time PCR quantification. The thermal cycling conditions were: 10 min at 95 °C followed by 45 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The threshold fluorescence level was determined with the default adaptive baseline algorithm of the MxPro software (version 4.10) (Agilent Technologies) and the resulting Ct values were automatically converted to quantities of T. magnatum DNA using the standard curve method. A standard curve was generated for each run with a series of 10-fold dilutions of genomic DNA from T. magnatum (from 107 to 102 fg per reaction) as standards. Calibration curves were also generated to convert DNA concentration values resulting from real-time PCRs to absolute quantities of T. magnatum mycelium in soil. A total of seven serial dilutions of immature gleba tissue (from 2 to 2 * 10−6 mg) per gram of soil free from T. magnatum mycelium were processed in triplicate for each truffle ground. Gleba tissue and soil were lyophilized and grinded as described
Mycorrhiza
above to prepare the serial dilutions. An immature ascoma (without spores) was selected over mycelium because suitable pure cultures of T. magnatum are not available (Iotti et al. 2012b). Moreover, the presence of spores might be to the detriment of calibration curve reliability because DNA extraction from spores is difficult (Selosse et al. 2013). In turn, the soils used to generate the calibration curves were taken from unproductive truffle patches of each experimental site. Absence of T. magnatum mycelium within these soil samples was confirmed by species-specific PCRs (Amicucci et al. 1998) on soil DNAs previously isolated with the NucleoSpin Soil kit (Macherey-Nagel). Noninoculated soil samples were used as negative controls. DNA extractions and real-time PCRs were performed as previously described. Statistical analysis ECM fungal communities were described for each plot by the following: richness (total number of distinct OTUs detected), Pielou's index (describes how evenly the individuals are distributed among the OTUs) (E =0→1, where E =1 if all species occur at the same proportion), and Shannon–Wiener index (take into account the taxa richness and their relative abundance) (H ′=0→∞, increase of such value is due to additional unique species and/or a greater species evenness) (Magurran 2004). These indices were calculated using the software package Vegan version 1.17-9 (Oksanen et al. 2011), within the R system for statistical computing (version 2.12.2) (R Development Core Team 2011). Analysis of variance (ANOVA) and Tukey's HSD analysis were adopted to verify if the mean control– impact differences in bulk density, soil mycelium, truffle production (number of carpophores and total weight), and ECM fungal community in the “before” period were significantly different from the mean differences in the “after” period. Normality was checked using Shapiro–Wilk test and the homogeneity of variance was tested using Levene's test (Neter et al. 1996).
Table 2 ANOVA table for the effects of soil tillage on bulk density, ECM fungal communities, number of truffle, total weight, and soil mycelium
L1 and L2 soil tillage Bulk density Species richness ECM tip density (n./dm3) Number of truffle Total truffle weight Soil mycelium Before and after soil tillage Bulk density Species richness ECM tip density (n./dm3) Number of truffle Total truffle weight Soil mycelium Time (before or after)×treatment (L1/L2) Bulk density Species richness ECM tip density (n./dm3) Number of truffle Total truffle weight Soil mycelium
Type III, F
p level
7, 15,700 0, 28,919 0, 20,117
0.0029 0.7508 0.8189
0, 18,408 3, 77,645 2, 17,471
0.9068 0.0564 0.0996
21, 936 2, 54,961 1, 82,534 3, 69,231 0, 57,575 5, 36,787
0.0001 0.1202 0.1860 0.0591 0.6330 0.0237
3.365 0.35687 0.08289 1.247683 1.240824 318.333
0.0480 0.7028 0.9207 0.2999 0.3023 0.0297
10 %, respectively). Only slight differences were recorded in the control plots (Fig. 1). Moreover, Tukey's post hoc test revealed that the bulk density decreased significantly (p level0.05) (Tab. 2). 1.60
Results
Statistical differences were found between the bulk density values obtained before and after soil tillage and their interaction (Table 2). Soil tillage affected the soil bulk density in both the Argenta and Barbialla experimental truffières (Fig. 1). In the Argenta truffière (Emilia-Romagna), the bulk density measured before and after soil tillage decreased by approximately 34 % in the plots L2 and 15 % in those L1 (Fig. 1). Reductions in bulk density also occurred at Barbialla (Tuscany), although with lower percentage values (21 and
av. bulk density (g/cmc)
Effects of soil tillage on bulk density and truffle production
1.40 1.20 1.00 0.80
before soil tillage after soil tillage
0.60 0.40 0.20 0.00
C
L1 Argenta
L2
C
L1 Barbialla
L2
Fig. 1 Average bulk density and standard error (error bar) measured before and after soil tillage in the two truffières (Argenta and Barbialla)
Mycorrhiza
communities which remain dominated by Thelephoraceae and Sebacinaceae (Fig. 4).
Effects of soil tillage on ECM fungal communities Out of the 40 soil cores collected in autumn 2010, seven in the Argenta truffière and one in the Barbialla truffière, had no ectomycorrhizae, but only the roots of non-ECM trees (Aesculus hippocastanum , Acer campestre, Celtis australis, and Cornus sanguinea), as found in autumn 2008 (Leonardi et al. 2013). More than 7,000 colonized root tips were included in the analysis: 3,258 tips from the sampling performed in the autumn of 2008 and 4,141 tips from the 2010 sampling. In general, ECM root tip density increased in almost all plots through the experiment and, particularly, in the plots L2, even if no significant differences were found. In the Barbialla truffière, the ECM fungal community consisted of 45 species before soil tillage, whereas afterwards, only 27 were found. In Argenta truffière, the number of species decreased from 22 to 18 after the soil tillage. Seven new OTUs were molecularly identified with respect to the ECM fungal community characterized in 2008. Even if no statistical differences were found, the highest reduction in species richness was recorded in plots L1 and L2 of Barbialla and Argenta truffières, respectively (Table 3). Following soil tillage, the Shannon and Pielou's indices decreased (Table 3). In the L2 community of Barbialla, in particular, the Shannon index dropped from 3.39 to 3.21, confirming a loss of diversity. The Kruskal–Wallis test (α = 0.05) was performed between the Shannon and Pielou's indices to compare the different soil tillage treatments. Significant differences were only found with respect to the L2 soil tillage (p =0.049). The rank–abundance curves (Figs. 2 and 3) obtained from the control and the two differently tilled plots showed that the ECM fungal community structures differ between the truffières. After tillage treatments, ECM fungal communities tend to be dominated by a lower number of species. However, this reduction did not affect the composition of fungal Table 3 ECM fungal diversity in the total two truffières (Argenta and Barbialla), in L1, L2, and control C plot before and after soil tillage
In bold significant differences by the Kruskal–Wallis test (α =0.05)
Assessment of T. magnatum soil mycelium Real-time PCR data showed that T. magnatum mycelium markedly increased in the soil of all tilled plots in both truffières by the end of the last year of treatment (Fig. 5). ANOVA analysis demonstrated that the amount of T. magnatum mycelium was significantly higher (p
