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MORPHOLOGICAL, PHYSICO-CHEMICAL AND MOLECULAR INVESTIGATIONS ON TUBER BELLONAE FROM BASILICATA-ITALY.

Byline: S. M. Mang, T. Zotta, I. Camele, R. Racioppi, M. D'Auria and G. L. Rana

ABSTRACT

Morphological, physico-chemical and molecular investigations were accomplished on ascomata of Tuber bellonae from Basilicata (southern Italy) identified on the basis of their macro-and microscopic features. Tuber aestivum and T. mesentericum were used as comparative truffle species. An accurate comparison of ascospore dimensions, in 4-spored asci, a morphological character previously used to distinguish the above described truffles, was also carried out. The volatile organic compounds (VOCs) from T. bellonae were investigated by solid phase microextraction technique coupled to gas chromatography and mass spectrometry (SPME-GC-MS). The following loci were used for molecular analysis: a) the beta-tubulin gene, b) the (GTG)5 microsatellite, c) the M13 minisatellite. The ascospore volume of T. bellonae in 4-spored asci resulted significantly bigger than those of T. aestivum and T. mesentericum. Twenty-three VOCs were detected.

Out of these, only 1,4-dimethoxy-2-methylbenzene, never found in T. aestivum and T. mesentericum, could be considered the main VOC marker discriminating T. bellonae. High polymorphism level was revealed in T. bellonae by both microsatellite and minisatellite markers. Finally, preliminary results on partial b-tubulin gene sequence of T. bellonae specimens suggest that T. bellonae is closely related to T. aestivum and T. mesentericum, probably holding an intermediate position between them.

Keywords: b-tubulin gene, molecular phylogeny, morphology, VOCs, Tuber bellonae.

INTRODUCTION

Basilicata, a region of southern Italy, is very rich in fungal biodiversity. In fact, about 83-85 taxa of hypogeous and semi-hypogeous fungi, including 29Tuber entities were discovered in its territory in the last 14 years (Rana et al., 2008, 2012, 2015 and unpublished data). Amongthe Tuber species, T. bellonae Quel. (1887) can be considered, at present, a very less distributed and rare taxon in either Basilicata (Rana et al., 2008) or in few other truffle producing countries. It was initially found and described by Quelet in Provence (France) and successively evidenced in Italy, South-East of France and Spain (Pacioni and Fantini,1997; Montecchi and Sarasini, 2000; Riousset et al., 2001) and, some years ago, in three humid and cold areas of central-southern Poland (Lawrynowicz et al., 2008).

Pacioni and Fantini (1997), who first studied and described the taxon in Italy, seemed to have used only very few ascomata originated in Sardinia and Spain and selected, among macro-and microscopic features useful for differentiating the species from T. mesentericum Vittad. and T. aestivum Vittad., the following ones:

a) the average ascospore volume in tetrasporic asci: 15,000 um3 instead of 10,000 um3 and 5,000 um3, the last two measured in the single last two Tuber species, respectively; b) the ascospores shape and ornamentation: spherical (22.5-55 um), rarely sub-spherical, enclosed by a reticulate-alveolate exosporium with net meshes delimited by 4-6 walls 5-7.5 um high; c) a peridium constituted by very small and flat warts, almost never transversely striated; d) the odour, at first phenolic (3-methylanisole), then grateful due to the presence of ethyl-methyl-chetone, without any phenolic component; e) thecolour of the gleba, light-brown with numerous, irregularly flexuous and subtle sterile veins; f) theovoid (77-95 x 7-115 um) asci, supported by a hooked pedicle and containing 1-6 spores.

The following additional and some times contrasting writings are, nevertheless, available on T. bellonae: 1) the odour of T. bellonae, according to Riousset et al. (2001) and Lawrynowicz et al. (2008), should be clearly phenolic and never grateful; 2) despite of the initial phenolic odour, Pacioni and Fantini (1997) though T. bellonae was more close to T. aestivum than to T. mesentericum, and also wrote that "some clones of T. aestivum, produce ascomata with a basal cavity and, in others, the relative amount of phenoliccomponents is rather high"; 3) about ascospore shape, Riousset et al. (2001) reported that,in T. bellonae it is "spherique". On the other hand, samples of T. bellonae from Poland (Lawrynowicz et al., 2008) do not seem characterized by ascospores that match with features underlined by the above mentioned authors because at least 40-50 % of them have a sub-spherical or ovoid form.

What above specified, along with the presence/absence of a cavity at ascoma's base and the existence, according to Pacioni and Fantini (1997), of hypothetical clones or variants of T. mesentericum with a striated peridium wart surface renders impossible to differentiate the above described taxaonly using morphological characters. Discriminating factors seem to be, at contrary, the ascospore volume in tetrasporic asci and, overall, the enzyme electrophoretic pattern (Pacioni and Fantini, 1997; Pacioni and Pomponi, 1991).

In Basilicata, several hundreds ascomata of T. aestivum [including T. aestivum (Chatin) Montecchi et Borelli 1990 fo. uncinatum] and T. mesentericum were macroscopically and microscopically examined since 1997-98 (Rana, unpublished data).Wart surface was always clearly and horizontally striated in the first species and, although less clearly also in the second one. Odour was always grateful in T. aestivum and in its genetically identical form uncinatum (Weden et al., 2005) whereas markedly phenolic and persistent in T. mesentericum. Ascospores were mostly ellipsoid or ovoid in mature asci of both species.The exosporium reticulum was rather regular in T. aestivum with pentagonal (but also rhomboid and hexagonal) meshes larger than those of T. mesentericum which, according to Montecchi and Sarasini (2000) and Riousset et al. (2001), were vice versa irregular in shape and often interrupted.

In addition, other ascomata, protected by a peridium with small and flat warts with surface weekly striated in horizontal sense, having a pleasant fungal odour, an exosporim reticulum like that of T. mesentericum and a percentage of spherical ascospores varying from 20 to 50-55 % were sometimes encountered. It has been hypothesized that these ascomata, could belong to T. bellonae which so far seems a not well defined black truffle taxon. For what above underlined and for verifying this hypothesis, it seemed opportune to accomplish some comparative micro-and macroscopic observations, VOCs studies and molecular analyses on typical ascomata of the three above mentionedTuber species.

Molecular studies seem particularly helpful to assess the genetic diversity within and among taxa and furnish valuable information to better classify truffles (Halasz et al., 2005; Frank et al., 2006; Bonitoet al., 2010). Markers employed to assess the genetic variation in truffles and to serve as tools for a correct species identification are: RAPDs (see, among others,Rossi et al., 2000; Bertault et al. 2001; Weden et al., 2004); RFLPs (Rossi et al., 2000; Bertault et al., 2001; Paolocci et al., 2004), AFLPs (Murat et al., 2008; Riccioni et al., 2008), microsatellites and minisatellites (see, among others,Rubini et al., 2004; Pomarico et al., 2007) and rDNA of the Internal Transcribed Spacer (ITS) [see, among others, Mello et al., 2002, 2011; Bonito et al.,2010; Chen et al., 2011; Schochet al., 2012; Zambonelli et al., 2012].

The genetic diversity of Tuber species was extensively investigated during the last three decades and generally resulted closely linked to species. The high intra-polymorphism, detected in eitherT. aestivum or T. mesentericum compared toother truffles, let Pacioni and Pomponi (1991) to hypothesize that they are two species complexes. Despite all published works, knowledge available for T. aestivum and T. mesentericum (see, among others,Pomarico et al., 2007; Sica et al., 2007; Lawrynowicz, 2009; Hobart, 2011; Stobbe et al., 2012)is still incomplete because it is unknown how many effective types/subspecies could be present within each of these species complexes. In fact, another species, i.e. T. sinoaestivum sp. nov.(Zhang and Liu 2012, in Zhang et al., 2013), was recently found within T. aestivum in southwestern China (Zhang et al., 2013).

Furthermore, presence of species complexes was also reported for T. excavatum Vittad. 1831, T. gennadii (Chatin) Patouillard 1903 (= Terfezia gennadii Chatin) and T. candidumHarkn. 1899 (Bonito, 2009). Apart from the previously revealed molecular markers, b-tubulin gene has been also extensively employed in species recognition and phylogeny of filamentous ascomycetes (Glass and Donaldson, 1995) and truffles (see among others, Paolocci et al., 2004; Jeandroz et al., 2008; Gryndler et al., 2011).

Due to its rarity in nature and its insignificant commercial value when compared to other truffles,T. bellonae has been less studied and thus, its classification is consequently still unclear and controversial. Furthermore, T. bellonae was compared to other Tuber species in only a very limited number of scientific papers (Gryndler et al.,2011; Bozac et al., 2012; Zambonelli et al., 2012).

In this context, the present study was undertaken with the following goals: 1) to cast light on the microscopic features and VOCs of T. bellonae present in Basilicata; 2) to estimate the genetic variation of T. bellonae and its related species T. aestivum and T. mesentericum using as molecular markers the beta-tubulin gene, the (GTG)5 microsatellite and the M13 minisatellite; 3) to assess phylogenetic relationships among the above mentioned truffles species and try to determine the taxonomic position of T. bellonae.

MATERIALS AND METHODS

Biological material: For morphological and molecular assays, ten mature ascomata for each of the three Tuber species i.e. T. bellonae, T. aestivum and T. mesentericum, collected in Potenza and Matera provinces of Basilicata in various years, were used (Table 1 and Figure 1). The host plants associated with the above mentioned truffle species were mainly Quercus spp. and/or Fagus sylvaticaL. In few cases oak and Pinus spp. were found together in natural beds ofT. bellonae and T. aestivum. For the appartenance to T. bellonae, only specimens containing more than 40 % spherical spores (observedat 100x) were selected.

Morphological analysis: Truffle species identification was achieved on the basis of spore morphological traits according to Montecchi and Sarasini (2000), Riousset et al. (2001), Pacioni and Fantini (1997). To avoid eventual changes in ascospore size, distilled water was always used as mounting medium for microscopic slide preparation. For acquisition of bright field images, an Eclipse 80i microscope (Nikon, Japan), equipped with a 5 mega-pixels colour cooled digital camera (DS-5Mc, Nikon) and a DS-L1 camera controller (Nikon) interfaced with a PC, were used. External surface of truffle peridium was observed at 25x with a Zeiss stereomicroscope. Peridium structure was observed at 1000x using an Axioscop Carl Zeiss optical microscope equipped with a DS-U1 camera (Nikon). Ascospores were observed with a 10x/0.25 Plan Nikonobjective to catch a greater number of spores in each focal field. Ascus and ascospore images were recorded employing a NIS-Element BR 3.0 software package (Nikon).

From each ascoma sample, two microscope slides were prepared and micrographs of ascus and spores contained in 5 randomly selected fields were acquired. The following parameters were measured for each truffle species: the length (minimum, maximum and average values) of the major and minor ascospore axes (without ornamentations); the percentage and dimensions of asci with different spore number; the percentage of spherical, sub-spherical and ellipsoid ascospores in 1-, 2-, 3-and 4-spored asci; in the case of T. aestivum5-spored asci were also considered; the ascospore volume (average value measured among spherical, sub-spherical and ellipsoid ascospores) in each ascus type (Table 2). The formula 4/3 p r3 (where r is the sphere radius) was used to calculate the volume of spherical ascospores; for sub-spherical and ellipsoid ones the formula 4/3 p a b2 (where a and b are the major and minor ellipsoid semi-axes, respectively) was preferred.

A total number of 915 ascospores (412 of T. bellonae; 207 of T. aestivum, and 296 of T. mesentericum,) was analysed and, on the basis of Q, i.e. length/width quotient (Pacioni and Fantini, 1997), classified as spherical (Q from 1.00 to 1.02), sub-spherical (Q from 1.03 to 1.29) and ellipsoid (Q > 1.29).

A more stringent valuation, with Q values ranging from 1.00 to 1.002 for spherical spores and from 1.003 to 1.29 for sub-spherical ones was also accomplished. However, for comparative purposes, the Q values established by Pacioni and Fantini (1997) were used.

Analysis of Volatile Organic Compounds: Analysis of VOCs in T. bellonae (Table 1) was accomplished by SPME-GC-MS as described in details by Mauriello et al. (2001) and D'Auria et al. (2006). VOCs were commonly determined using thin slices (0.5 g) of each collected sample after a maximum 48 h storage at 4 degCin hermetically closed vials.VOCs detected were finally compared to those found in ascomata of T. aestivum and T. mesentericum in the past (Mauriello et al., 2001).

DNA extraction, PCR and sequencing: Ascomata, previously brushed under tap water to detach soil debris, were superficially sterilized with alcohol (90 %), deprived of peridium, cut in thin slices and frozen at - 20 oC until molecular analyses were performed. Genomic DNA (gDNA) was isolated from 100 mg of gleba using DNeasy Plant Mini DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's directions. The extracted gDNA concentration was determined using a ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, Delaware, U.S.A.). Subsequently, the extracted DNA was used as a template for b-tubulin gene amplification using the Bt2a+Bt2b primer pair and following the protocol of Glass and Donaldson (1995). Finally, the amplicons were purified, concentrated and directly sequenced as reported for the ITS region by Mang and Figliuolo (2010) with aminor change, e.g. b-tubulin gene primers Bt2a+Bt2b were used for either amplification or sequencing reactions.

Partial b-tubulin gene sequences of T. bellonae were queried against those available in National Center for Biotechnology Information (NCBI) GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with the BLASTN algorithm (Altschul et al., 1997).

M13 minisatellite amplification and analysis:Minisatellite variation analysis was carried out using the universal primer M13 (GAGGGTGGCGGTTCT) and amplifying the repetitive gDNA sequence (Latouche et al., 1997). PCR reactions were performed as suggested by Williams et al. (1990) with the following minor modifications: the final volume of PCR reaction was 25 ul, and contained 1X PCR buffer, 2 mM MgCl2, 0.25 mM dNTPs, 1 U Taq DNA polymerase (Invitrogen, New York, U.S.A.), 0.2 uM M13 universal primer and 20 ng of gDNA template. Control experiments were included without template DNA.

Amplifications were performed with an iCycler (BioRad, Hercules, California, U.S.A.) and consisted of the following steps: 2 min at 95 degC, 1 min at 94 degC for 35 cycles, 1 min at 45 degC, 1 min at 72 degC and 5 min at 72 degC. Finally, amplified DNA fragments were separated in a 2 % agarose gel electrophoretic run in 1XTBE buffer at 100 V, stained with ethidium bromide (EB), visualized under an EurdegClone UV transilluminator (EurdegClone Ltd., Milan, Italy) and finally photographed using an OCD Video Camera Module. Every assay was performed at least twice. All clear bands at given molecular weight levels were considered while faint ones were not scored.

Fragment sizes were estimated using a 1-kb DNA Ladder (Invitrogen). After a preliminary visual analysis of the electrophoretic profile of each taxon, the relative mobility of the amplified fragments was recorded using a binary code. Finally, Nei's genetic distance indices (Nei and Li, 1979) were calculated to evaluate the intra-and inter-taxa polymorphism (Peakall and Smouse, 2006, 2012).

(GTG)5 microsatellite amplification and analysis: The (GTG)5 primer reported by Buscot et al.(1996) was used to amplify the gDNA of all Tuber taxa analyzed. PCR reactions were performed as described by Guenaoui et al. (2013). Control experiments were carried out without template DNA. An iCycler (BioRad) was employed for the microsatellites amplification using a "touchdown" program as described by Don et al. (1991). Amplification products were analyzed by electrophoresis in 2 % agarose gel run in 1X TBE buffer at 100 V, subsequently stained with EB and photographed. Each assay was performed at least twice. Finally, PCR products analysis was carried out in the same mode reported for the M13 minisatellite.

Statistical analysis of molecular markers data: The analysis of binary haploid genetic data obtained with M13 and (GTG)5 microsatellite markers was carried out with help of GenALEx 6.501 program (Peakall and Smouse, 2006, 2012). The Nei's genetic distance was calculated using a dataset containing combined data of both the above mentioned markers and considering three populations corresponding to the three analyzed tuber taxa. Subsequently, the distance matrix with all pair-wise comparisons was used as an input for a further principal components analysis (PCA) via covariance matrix with standardization to achieve a graphical output of the first two coordinates exhibiting the genetic relationships among the investigated taxa (Peakall and Smouse, 2006).

Phylogenetic analyses: Forty-one b-tubulin gene sequences were used inthe phylogenetical analysis. Eight sequences were generated from this research and other 33 sequences were retrieved from EMBL-EBI European Nucleotide Archive database as shown inTable 1. In order to determine the phylogenetic position of T. bellonae, the eight partial b-tubulin gene sequences obtained in this study were compared with the33 partial b-tubulin gene sequences downloaded from EMBL-EBI database. In addition, the partial b-tubulin gene sequence of T.maculatum Vittad. (FN555425), from NCBI database, was selected and used as the outgroup (Table 1). Phylogenetic analyses were carried out using MEGA version 6.0 software (Tamura et al., 2013), treating gaps as missing data, excluding ambiguous positions (complete deletion option) and using the CLUSTAL alignment method.

Following sequence alignment, the evolutionary distances interpreted as units of the number of base substitutions per site were computed using the Maximum Composite Likelihood method (Tamura et al.,2004). Codon positions included were 1st+2nd+3rd+Noncoding. The evolutionary history was inferred using the following methods: UPGMA (Sneath and Sokal, 1973), Neighbor-Joining (NJ) (Saitou and Nei, 1987), Minimum Evolution (Rzhetsky and Nei, 1992) and Maximum Parsimony (MP) (Eck and Dayhoff, 1966). A bootstrap consensus un-rooted tree (cut-off value 70 %) was inferred from 1000 replicates to represent the evolutionary history of the investigatedTuber taxa (Felsenstein, 1985).

RESULTS

Morphology:Macroscopic and microscopic features of ascomata of the three Tuber species investigated,except for spherical spores percentage found in T. bellonae (Table 2), were like those described by Montecchi and Sarasini (2000) and Riousset et al. (2001). Transversal striae of peridium pyramidal wart faces were very evident, less clear and almost absent in T. aestivum, T. mesentericum and T. bellonae, respectively (Figure 2A- C). Peridium structure was pseudo-parenchymatic in the Tuberspecies studied, with polygonal, spherical and oval elements dimensions of which decreased from T. aestivum through T. mesentericum to T. bellonae. Peridium of the last species exhibited some kidney-shaped cells and all the species showed dark brown outer peridium layer cells (data not shown).

Data regarding the other microscopic features of the truffles analysed in this study are reported in Table 2. Average ascus dimensions (width and length) (peduncle excluded) differed among the considered species: in particular, asci of T. aestivum had an ovate shape (axes: 66 and 98 u m) and the smallest size, while those of T. bellonae, oblong or ellipsoid (73 and 104 u m), and of T. mesentericum, elliptical (67 and 107 u m), were significantly larger. The smallest asci width generally characterized 1-spored asci of T. aestivum, while the major length was found in 4-spored asci of T. mesentericum (Table 2). Ascospore surface was, in T. aestivum, reticulate-alveolate with almost regular polygonal meshes, often containing one central crest, whereas appeared irregularly reticulate-alveolate in the other two species/taxa (Figure 3 A-C).

Compared to T. mesentericum (4 % spherical, 10 % sub-spherical and 87 % ellipsoid spores) and T. aestivum (9 % spherical, 32 % sub-spherical and 59 % ellipsoid), in which ellipsoid ascospores were predominant, T. bellonae was characterized by spherical (53 %) and sub-spherical (43 %) ascospores, even if a small percentage (4 %) of elliptical spores was found. Using a Q ranging from 1.00 to 1.002 for selecting spherical ascospores, their percentage in T. bellonae decreased to 41 % and that of sub-spherical ones consequently increased up to 55 %. The percentage of spherical, sub-spherical and ellipsoid spores varied among the ascus type (1-, 2-, 3-, 4-and 5-spored asci).

T.aestivum had, on average, ascospore dimensions slightly smaller than those of T. mesentericum, while T. bellonae had the ascospore length (average value of major axis) ranging from that of T. mesentericum to that of T. aestivum (Table 2). The average value of minor axis of T. bellonae spores, on the contrary, was significantly larger because of higher number of spherical and sub-spherical spores. In all three species, 2-and 3-spored asci were predominant. Among the analysed samples, a significant percentage (15.7 %) of 5-spored asci were only found in T. aestivum which, actually, exhibited also 1 % of 6-spored asci (data not shown).The average ascospore volumes in 4-spored asci of T. bellonae, T. mesentericum and T.aestivum were 49,379 u m3; 19,584 u m3 and 18,309 u m3, respectively.

The average ascospore diameter (42.1 u m), calculated in this study for T. bellonae, based on total spore number, is close to that obtained by Lawrynowicz et al. (2008) i. e. 38.7 u m (min 22.5 max 55 u m), even if these authors did not specify if they considered only the spherical spores.

VOCs:Fresh ascomata of T. bellonae used in this study had a fungal pleasant odour. When they were analysed by SPME-GC-MS showed 23 twenty three volatile compounds reported in Table 3. The main VOCs always found in T. bellonae were 1-methoxy-3-methylbenzene (peak area from 6.74 to 66.32 %) and 1,4-dimethoxy-2-methylbenzene (peak area from 3.70 to 19.43 %). Even if with a smaller peak area (ranging from 0.17 to 2.11 %), 3,5-dimethoxytoluene was found in 91 % of tested ascomata. Dimethylsulphide, a VOC found in the past in 100 % and 80 % of T. mesentericum and T. aestivum ascomata (Mauriello et al. 2001), respectively, was only detected in sample number 10. In one instance (sample number 2), SPME-GC-MS analysis, accomplished after 1 month storage at 4 degC, revealed the presence of hexanal as a probable oxidation product; 3-methylbutanal and 3-methylbutanol, which were not present in the fresh samples were also detected, whereas all other VOCs present in fresh samples exhibited a lower peak area.

Partial b-tubulin gene amplification and sequencing: Partial b-tubulin gene was efficiently amplified from gDNA of all three considered Tuber species always giving a PCR product of about 500 bp (Figure 4). Only amplification products of T. bellonae were directly sequenced and finally, eight b-tubulin gene sequences, were deposited into the EMBL-EBI database under the following accession numbers: HF947920, HF947921, HF947922, HF947923, HF947924, HF947925, HF947926, and HF947927 (Table 1).

(GTG)5 microsatellite length polymorphism: PCR amplification with the (GTG)5 primer produced fragments ranging from 250 to 1400 bp. An average number of 6 amplified fragments was detected using this marker. But, no species-specific profile was revealed in the three Tuber taxa analyzed. Microsatellite (GTG)5 analysis, due to the high number and different size of bands observed, indicated that there is a quite high genetic diversity among the specimens of either T. bellonae or the other two Tuber species. Finally, based on the (GTG)5 profile, at moment, it can be assumed that an elevated polymorphism degree could be present within the rare species T. bellonae although more ascomata (if possible considering the already mentioned rarity of this taxon in nature) should be analyzed to support this assumption.

M13 minisatellite variation: The pattern of M13 minisatellite marker consisted of fragments ranging in size from about 400 to 1200 bp.An average number of 4 electrophoretic bands was obtained with this marker. For all the Tuber taxa analysed, M13 was also polymorphic at either inter-or intraspecific level. Results of this preliminary molecular investigation are consistent with either those reported by Pomarico et al. (2007) who found a high expected heterozygosity for M13 locus in T. aestivum and T. mesentericum in respect of other threeTuber species studied (T. borchii Vittad.,T. magnatum and T. brumale Vittad.) or those of Pacioni and Pomponi (1991) who detected a high genetic heterogeneity in both T. aestivum and T. mesentericum based on multilocus isozyme patterns.

Even if M13 marker seemed useful to distinguish different Tuber species and different profiles were detected for the three taxa here investigated, it should not be considered species-specific as it also showed a high intra-specific polymorphism.

PCA:Genetic analysis using M13 and (GTG)5 markers data via PCA did not show any clear separation among the three Tuber taxa studied. Furthermore, as it is shown in Figure 5, only a scanty separation of T. mesentericum specimens from those of T. bellonae and T. aestivum was evidenced.

Phylogeny:A possible similar evolutionary history of T. bellonae was traced with all followed methods (UPGMA, NJ, ME and MP).Therefore, only thatof NJ is here presented. The consensus tree showed an unique group (Clade I) of T. aestivum-T. mesentericum complex which also includes T. bellonae with moderate statistical support (bootstrap values about 50 %). This clade resulted consistently separated from T. maculatum, which, as expected, performed as a distant species (Fig. 6). Furthermore, within the big clade I, the eight specimens belonging to T. bellonae occupied two positions, e.g. four were placed near T. aestivum (subgroup A) and the other four were positioned close to T. mesentericum samples (subgroup B). The only strong bootstrap support (97 %) detected was for 4 T. bellonae sequences placed within T. aestivum specimens (subgroup A) while a lower bootstrap support (< 50 %) was observed for the other 4 ascomata of T. bellonae (subgroup B) closer to T.mesentericum.

In this context, the precise phylogenetic relationship among T. bellonae, T. aestivum and T. mesentericum still remain unclear. This is perhaps, due to the fact that the nucleotide variation present on only b-tubulin gene is surely insufficient to allow separation of such closely related taxa.

Table 1.Tuber species employed in this study and their partial b-tubulin gene sequences GenBank accession number.

###Geographical###Collecting date###GenBank

###Species###origin (Place / Province)###(mdy)###accession no.

###T. bellonae###Muro Lucano / Potenza###12/14/2005###-

###T. bellonae###Campomaggiore / Potenza###01/12/2006###HF947920

###T. bellonae###Brienza / Potenza###01/13/2006###HF947921

###T. bellonae###Pignola / Potenza###11/22/2005###HF947922

###T. bellonae###Pignola / Potenza###03/06/2006###HF947923

###T. bellonae###Campomaggiore / Potenza###02/09/2006###HF947924

###T. bellonae###Tricarico / Matera###02/28/2006###HF947925

###T. bellonae###Muro Lucano / Potenza###01/18/2006###-

###T. bellonae###Abriola / Potenza###11/11/2011###HF947926

###T. bellonae###Pierfaone / Potenza###11/17/2011###HF947927

###T. aestivum###Pignola / Potenza###06/06/2006###-

###T. aestivum###Gorgoglione / Potenza###05/24/2006###-

###T. aestivum###Campomaggiore / Potenza###07/01/2006###-

###T. aestivum###Muro Lucano/ Potenza###05/31/2006###-

###T. aestivum###Pignola / Potenza###03/06/2006###-

###T. aestivum###Forenza / Potenza###07/03/2005###-

###T. aestivum###Tricarico / Matera###06/07/2005###-

###T. aestivum###Tricarico / Matera###04/24/2005###-

###T. aestivum###Ruoti / Potenza###05/31/2006###-

###T. aestivum###Abriola / Potenza###11/30/2009###-

###T. aestivum###-###-###AY226030*

###T. aestivum###-###-###DQ336290*

###T. aestivum###-###-###AF516813*

###T. aestivum###-###-###AF516810*

###T. aestivum###-###-###AY226029*

###T. aestivum###-###-###AF516811*

###T. aestivum###-###-###AF516812*

###T. aestivum###-###-###AY170361*

###T. aestivum###-###-###AF516801*

###T. aestivum###-###-###AY226035*

###T. aestivum###-###-###AY226033*

###T. aestivum###-###-###AF516802*

###T. aestivum###-###-###AF516807*

###T. aestivum###-###-###AF516809*

###T. aestivum###-###-###AY226031*

###T. aestivum###-###-###AF516806*

###T. aestivum###-###-###AY226034*

###T. aestivum###-###-###AF516805*

###T. aestivum###-###-###AY226032*

###T. aestivum###-###-###AF516803*

###T. aestivum###-###-###AF516804*

###T. aestivum###-###-###DQ336291*

###T. aestivum###-###-###GU979150*

###T. aestivum###-###-###GU979152*

###T. mesentericum###Gorgoglione / Potenza###12/26/2009###-

###T. mesentericum###Brienza / Potenza###02/16/2006###-

###T. mesentericum###Corleto Perticara / Potenza###12/13/2005###-

###T. mesentericum###Abriola / Potenza###11/11/2011###-

###T. mesentericum###Abriola / Potenza###12/06/2012###-

###T. mesentericum###Pignola / Potenza###12/18/2012###-

###T. mesentericum###Abriola / Potenza###11/30/2009###-

###T. mesentericum###Villa d'Agri / Potenza###09/06/2006###-

###T. mesentericum###Muro Lucano / Potenza###01/12/2006###-

###T. mesentericum###Pignola / Potenza###03/06/2006###-

###T. mesentericum###-###-###AY170362*

###T. mesentericum###-###-###AF516816*

###T. mesentericum###-###-###AF516817*

###T. mesentericum###-###-###AF516818*

###T. mesentericum###-###-###AY170364*

###T. mesentericum###-###-###AY170363*

###T. mesentericum###-###-###AF516814*

###T. mesentericum###-###-###AF516815*

###T. maculatum###-###-###FN555425*

Table 2. Ascospore axes and volume in 1-4 spored asci and ascus type dimension of Tuber bellonae, Tuber mesentericum and Tuber aestivum from Basilicata.

###Length of ascospore axes (um)

###Major O (min-max-average)###Minor O (min-max-average)

###22.2 61.2 41.6###21.7 58.6 40.1

###Ascus type dimension and %

###1-spored###2-spored###3-spored###4-spored

###(length=79###(length=104um;###(length=100 um;###(length=103 um;

###um;###width=75 um)###width=85um)###width=99 um)

T.###width=73 um)###26.0 %###39.0 %###17.1 %

bellonae###17.9 %

###Ascospore morphology and % in 1-4 spored asci

###27 % spherical###44 % spherical###60 % spherical###52 % spherical

###50 % sub-###51 % sub-###38 % sub-spherical###44 % sub-spherical

###spherical###spherical###2 % ellipsoid###4 % ellipsoid

###23 % ellipsoid###5 % ellipsoid

###Average ascospore volume in 1-4 spored asci

###50,276 um3###49,822 um3###49,490 um3###49,379 um3

###Length of ascospore axes (um)

###Major O (min-max-average)###Minor O (min-max-average)

###29.0 60.9 44.9###23.7 43.6 33.6

###Ascus type dimension and %

###1-spored###2-spored###3-spored###4-spored

###(length=75um;###(length=90 um;###(length=99 um;###(length=107 um;

###width=67 um)###width=76 um)###width=79 um)###width=87 um)

T.###17.0 %###26.6 %###36.2 %###20.2 %

mesentericum###Ascospore morphology and % in 1-4 spored asci

###0 % spherical###2.0 % spherical###1.0 % spherical###5.0 % spherical

###0 % sub-###3.4 % sub-spherical###17.4 % sub-###16.4 % sub-spherical

###spherical###94.6 % ellipsoid###spherical###78.0 % ellipsoid

###100 %###81.6 % ellipsoid

###ellipsoid

###Average ascospore volume in 1-4 spored asci

###20,676um3###20,297 um3###19,796 um3###19,584 um3

T.###Length of ascospore axes (um)

aestivum###Major O (min-max-average)###Minor O (min-max-average)

###21.1 59.8 40.4###17.0 47.2 32.2

###Ascospore morphology and % in 1-5 spored asci

###1-spored###2-spored###3-spored###4-spored###5-spored

###(length=70###(length=80 um;###(length=90um; width=68###(length=99um;###(length=98 um;

###um;###width=61 um)###um)###width=70um)###width=70 um)

###width=66 um)###27.1 %###25.7 %###16.1 %###15.7 %

###14.3 %

###Average ascospore volume in 1-5 spored asci

###0 % spherical###18 % spherical###13 % spherical###7% spherical###4 % spherical

###60 % sub-###21 % sub-###27 % sub-spherical###32 % sub-###42 % sub-

###spherical###spherical###60 % ellipsoid###spherical###spherical

###40 % ellipsoid###61 % ellipsoid###61 % ellipsoid###54 % ellipsoid

###Average ascospore volume in 1-4 (5) spored asci

###19,732 um3###18,636 um3###18,598 um3###18,309um3###17,056 um3

Table 3. Volatile organic aroma compounds determined in Tuber bellonae by SPME-GC-MS

Compound###Retention###Sample number

###time###Area (%) of the single compound

###(min)###1###2###3###4###5###6###7###8###9###10

2-methyl-1,3-###1.66###4.21###1.63###4.21###7.52###6.03

butadiene

Dimethylsulfide###1.68###14.95

3-methyl-butanal###2.51###0.46###0.46###4.48###2.45###1.96###2.46###0.58

3-methyl-1-butanol###3.55###0.12###0.12###0.51###0.18###0.18###0.38###0.12

Hexanal###4.70###0.20###0.20

2-heptanone###6.51###0.84###0.85

1-octen-3-ol###8.39###11.52###11.29

3-octanone###8.61###0.03###0.06###0.82

1-methoxy-3-###9.25###66.32###69.50###66.32###16.45###65.57###7.47###64.81###17.14###6.74###45.29

methylbenzene

1-undecene###10.62###0.19###1.29

3-methylphenol###10.75###0.14

3-ethyl-5-###11.12###0.88###0.90###0.32###0.08###0.22###2.17

methylphenol

2,3-dimethoxytoluene###12.37###0.51###0.21###0.51###0.22###0.05###0.49###0.15

3,4-dimethoxytoluene###13.47###1.35###1.94###1.35###1.84###0.24###1.34###0.28###0.21

1,4-dimethoxy-2-###13.65###13.57###19.43###13.57###3.55###18.36###9.43###13.47###3.70###9.62###4.75

methylbenzene

3,5-dimethoxytoluene###13.99###2.11###2.11###0.34###0.75###0.31###2.09###0.17###0.35###0.48

2,5-###14.98###14.98###0.07

dimetoxyethylbenzene

4-butylindan-5-ol###16.32###16.32###0.04

1,2,3-trimethoxy-5-###16.72###0.40###0.64###0.40###0.63###3.60###0.43###0.50

methylbenzene

1,3,5-trimethyl-2-###18.56###0.03###0.10###0.04

cyclopentylbenzene

2,6-###20.44###0.02###0.06###0.02###0.07###0.16###0.27###0.12###0.02

diisopropylnaphtalene

1,7-###20.52###0.04###0.06###0.04###0.07###0.13###0.02

diisopropylnaphtalene

1,3-###21.07###0.03###0.05###0.03###0.06###0.14###0.12###0.03###0.06###0.04

diisopropylnaphtalene

DISCUSSION

Compared to the previous works (Pacioni and Fantini, 1997; Lawrynowicz et al., 2008) and treatises (Montecchi and Sarasini, 2000; Riousset et al., 2001) on T. bellonae in which no precise numerical data (e.g. number of measured ascospores) are reported, a satisfactory large ascospore number (412) was considered in this study. Therefore, the results referring to T. bellonae ascospore morphology and dimensions should be considered statistically more effective. The average ascospore volume in 4-spored asci of T. bellonae ascomata from Basilicata was 49,379 u m3 instead of 15,072 +/-2.712 u m3 reported by Pacioni and Fantini (1997). In our study, only the 16.3 % of ascospores in 4-spored asci of T. bellonae, had an average volume of 21,781 u m3, value which is comparable with data of the above authors.

Only for these spores, the resulting average diameter, i.e. 35.0 u m, can be considered close to the value of 31.5 u m which can be extrapolated from the results of Pacioni and Fantini (1997) using the formula 4/3 p r3 reported in materials and methods.

VOCs analysis through SPME-GS-MS of T. bellonae ascomata was not accomplished in the past. Among the VOCs detected in this study, dimethylsulphide was found only in one of the examined ascomata whereas it had been detected by Mauriello et al. (2001) in 100 % and 80 % of samples of T. mesentericum and T. aestivum, respectively; 1-methoxy-3-methylbenzene, always present in aroma of T. bellonae, was found by the same authors in 78.5 % and 25 % of T. mesentericum and T. aestivum ascomata, respectively. Therefore, the above two VOCs could not be used to differentiate the three species of Tuber. Useful to recognize T. bellonae resulted, vice versa, the 1,4-dimethoxy-2-methylbenzene, which was always absent in aroma of T. mesentericum and T. aestivum.

Another compound, 3,4-dimethoxytoluene, detected in 82 % of T. bellonae samples and not present in any of the two othermentioned Tuber species, could be also used to discriminate T. bellonae.Oddly enough, 3-methylanisole, with a phenolic odor, and ethyl-methyl-chetone, reported as characteristic for less and more mature T. bellonae ascomata by Pacioni and Fantini (1997), were never detected in the samples analyzed in this study.

Existence of a marked intraspecific heterogeneity in T. bellonae (and the two other related species) was confirmed by these preliminary results of genotypic analyses. In fact, all three markers employed [M13 minisatellite, (GTG)5 microsatellite and b-tubulin] were suitable to prove the existence of high polymorphism in all the three analyzed taxa. First data obtained in this study using partial b-tubulin gene point out an intermediate position of T. bellonae between T. aestivum and T. mesentericum which does not exactly fit outcomes from earlier works based on morphology and isozyme patterns (Pacioni and Pomponi, 1991; Pacioni and Fantini, 1997). Furthermore, findings of this research demonstrate, for the first time, that a high molecular diversity is present in T. bellonae, a taxon not well investigated till now at molecular level.

Very differently from Gryndler et al. (2011) who studied, among other truffle species, also a single ascoma of T. bellonae but did not obtain for it any PCR amplification employing Glass and Donaldson's b-tubulin gene primers, the fungal universal primers Bt2a+Bt2b employed in this study, allowed an effective and straightforward approach to investigate for the first time the variation present on this gene into T. bellonae (Figure 4).

Microsatellite-primed PCR, using (GTG)5 as primer, showed that a clear polymorphism exists among thetested specimens of T. bellonae from Basilicata region. Nevertheless, further studies using various molecular markers with different evolution rates and an increased number of ascomata are needed to draw some steady conclusions. A high and a modest genetic diversities were previously reported by other authors in T. aestivum and in T. mesentericum, respectively (Pacioni and Pomponi, 1991; Mello et al., 2002; Weden et al., 2004; Weden et al., 2005; Pomarico et al., 2007; Sica et al., 2007). The elevate intra and inter-specific polymorphism found in this work well matches these results.The M13 minisatellite marker was also useful to illustrate the presence of an intra-and inter-polymorphism within and among the analyzed Tuber taxa.

Results of the present investigation confirmed those of Pomarico et al. (2007) who showed that analyzing different Tuber species, including T. aestivum and T. mesentericum, M13 marker can be a useful tool to explore intra-specific genetic variability.

The relationships found in this study among specimens belonging to the three Tuber taxa are not consistent with the results of Pacioni and Fantini (1997) who showed, by isozyme markers, the neat separation of T. bellonae from T. aestivum and T.mesentericum. In fact, based on minisatelite and microsatellite data, all ten T.bellonae specimens were not very clearly separated from the above two closely related taxa despite of a slight tendency of distinct clustering observed for eight of them. This may perhaps mean that M13 and (GTG)5 markers could have a weak statistical power to detect T. bellonae into T. aestivum-T. mesentericum species-complex.

Although, the outcomes of morphological observations and spores measurement appear useful to distinguish the three species, the preliminary molecular acquirements achieved with this study still suggest an incomplete separation of T. bellonae from T. aestivum and T. mesentericum. In fact, the analysis of the b- tubulin gene variation seems to confirm that the T. bellonae is part of the large T. aestivum-T. mesentericum complex and is closely related to both species, although, perhaps, due to the small sample size and single gene consideration, the definition of groups into clade I lacks of very high statistical support (bootstrap values less than 70 %). Even if, this work demonstrates the existance of a high polymorphism in T. bellonae and its proximity to T. aestivum and T. mesentericum, its exact phylogenetic position within the T. aestivum-T. mesentericum species complex can not be clearly defined.

In future, multiple loci analyses (MLA) already applied into truffle species phylogeny (Wang et al., 2006; Bonito et al., 2010; Bonuso et al., 2010) will be essential to confirm whether this taxon could be considered or not as a separate species.

Disclosure Statement: No potential conflict of interest was reported by the authors.

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