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. 2024 May 1;16(3):e13271. doi: 10.1111/1758-2229.13271

In vitro interactions between Bradyrhizobium spp. and Tuber magnatum mycelium

Simone Graziosi 1,✉, Federico Puliga 1, Mirco Iotti 2, Antonella Amicucci 3, Alessandra Zambonelli 1
PMCID: PMC11062863  PMID: 38692852

Abstract

Tuber magnatum is the most expensive truffle, but its large‐scale cultivation is still a challenge compared to other valuable Tuber species. T. magnatum mycelium has never been grown profitably until now, which has led to difficulties to studying it in vitro. This study describes beneficial interactions between T. magnatum mycelium and never before described bradyrhizobia, which allows the in vitro growth of T. magnatum mycelium. Three T. magnatum strains were co‐isolated on modified Woody Plant Medium (mWPM) with aerobic bacteria and characterised through microscopic observations. The difficulties of growing alone both partners, bacteria and T. magnatum mycelium, on mWPM demonstrated the reciprocal dependency. Three bacterial isolates for each T. magnatum strain were obtained and molecularly characterised by sequencing the 16S rRNA, glnII, recA and nifH genes. Phylogenetic analyses showed that all nine bacterial strains were distributed among five subclades included in a new monophyletic lineage belonging to the Bradyrhizobium genus within the Bradyrhizobium jicamae supergroup. The nifH genes were detected in all bacterial isolates, suggesting nitrogen‐fixing capacities. This is the first report of consistent T. magnatum mycelium growth in vitro conditions. It has important implications for the development of new technologies in white truffle cultivation and for further studies on T. magnatum biology and genetics.

Tuber magnatum mycelium, together with its associated bacteria (Bradirhizobium spp.), were isolated for the first time. Cultural and phylogenetic analyses revealed the specificity of this interaction.

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INTRODUCTION

True truffles are hypogeous ascomycetes within the genus Tuber. This genus includes more than 180 ectomycorrhizal species (Bonito et al., 2013), and some of them have the highest economic value among edible mushrooms (Luxury Columnist, 2022) due to their excellent organoleptic properties (Mello et al., 2006). Tuber magnatum Picco, Tuber melanosporum Vittad., Tuber aestivum Vittad., and Tuber borchii Vittad. are the most economically important species, but only the last three have been extensively cultivated until now. Their cultivation is achieved by planting truffle seedlings in appropriate soils and climates. The most common inoculum type used by nurseries to produce truffle seedlings is made by crushing fresh, frozen or dried fruiting bodies to obtain a spore slurry that is used to inoculate the root system (Iotti, Piattoni, & Zambonelli, 2012). Several authors demonstrated that it is also possible to produce Tuber mycorrhizas with mycelial cultures (Chevalier & Frochot, 1997; Sisti et al., 1997) and that the truffle plants obtained by mycelial inoculum can fructify like those obtained by spore inoculum (Iotti et al., 2016). However, not all truffle mycelia can be cultivated in vitro conditions and, when possible, they grow slower than the mycelia of other ectomycorrhizal species. T. borchii (Barbieri et al., 2005), Tuber rufum Picco, and Tuber macrosporum Vittad. (Iotti et al., 2002) are some of the species that can be successfully isolated and grown on agar media, whereas T. magnatum mycelium is hard to isolate and its development has been limited to a few hundred micrometres (Iotti, Piattoni, & Zambonelli, 2012). Fontana (1968) first reported the isolation of T. magnatum mycelium from a fragment of gleba transferred on agar plates. Later, Mischiati and Fontana (1993) affirmed that they had isolated T. magnatum mycelium from mycorrhizas, but several years later, Mello et al. (2001) genetically verified that this mycelium belonged to the whitish truffle Tuber maculatum Vittad.

During their development in soil, the mycelium of ectomycorrhizal fungi interacts with many microorganisms, some of which, as the mycorrhiza helper bacteria, may affect the fungal metabolism and growth (Frey‐Klett et al., 2007). Bacteria also play an essential role in the life cycle of truffles, and in particular, the microbiome associated with T. magnatum ascomata seems to have a crucial role in aroma biosynthesis (Vahdatzadeh et al., 2015), fruiting body formation and nutrition (Monaco et al., 2022).

Many taxa of bacteria live in the ascoma of T. magnatum (Barbieri et al., 2007, 2010; Citterio et al., 1995; Monaco et al., 2021; Niimi et al., 2021a) and other Tuber spp. The majority of them belong to Proteobacteria, in particular Gammaproteobacteria and Alphaproteobacteria. The last includes Bradyrhizobium, which is the most abundant bacterial genus found in truffle ascomata (Antony‐Babu et al., 2014; Benucci & Bonito, 2016; Niimi et al., 2021b; Sillo et al., 2022). Barbieri et al. (2010) hypothesised the involvement of Bradyrhizobium in the nitrogen nutrition of T. magnatum. They detected the nitrogenase gene nifH of Bradyrhizobium spp. inside the T. magnatum ascoma and found that the level of nitrogen fixation was comparable to that of early nodules of legumes associated with specific nitrogen‐fixing bacteria (Barbieri et al., 2010, 2012).

Until now, the role of the Bradyrhizobium spp. or other Proteobacteria living inside T. magnatum ascomata on its mycelium development in vitro conditions has never been investigated. However, a few years ago, Le Roux et al. (2016) identified Alphaproteobacteria belonging to Rhodopseudomonas growing associated with the mycelia of T. melanosporum and T. brumale, which seemed to maintain the vitality of these truffle mycelia after repeated subculturing. Since Tuber mycelia grow slowly in vitro conditions and the risk of losing them after the first subculture is very high (Giomaro et al., 2005), the improvement of their growth performances would be fundamental for both scientific studies and truffle cultivation applications.

In this work, we isolated and maintained in vitro the mycelium of T. magnatum for the first time thanks to the presence of Bradyrhizobium spp. living inside the ascoma. These bacteria were characterised by phylogenetic analyses of four genes, and their specificity for T. magnatum was assessed by co‐culture tests with other Tuber species.

EXPERIMENTAL PROCEDURES

Mycelium isolation

During 2021 and 2022, many attempts to isolate T. magnatum strains from fresh ascomata collected in Italy were carried out. Fragments of gleba, 1–2 mm in size, were aseptically excised from the inner part of the ascoma and cultured in Petri dishes on modified Woody Plant Medium (mWPM) (Iotti et al., 2005) at 22.5°C in the dark. Each ascoma was then dried and deposited in the herbarium of the ‘Centro di Micologia’ of Bologna (CMI‐UNIBO) (Table 1). All isolates were subcultured every 50–60 days on mWPM to stabilise the cultures.

TABLE 1.

List of Tuber magnatum ascomata used for mycelium isolation.

Strain Species Putative host Provenience a Date
TMG5072 b T. magnatum na Molinella (BO), Emilia Romagna, Italy 28 September 2021
TMG5299 T. magnatum na na 14 September 2022
TMG5300 b T. magnatum na na 14 September 2022
TMG5301 T. magnatum na na 14 September 2022
TMG5302 T. magnatum na na 14 September 2022
TMG5312 T. magnatum Populus alba L. Montefalcone nel Sannio (CB), Molise, Italy 15 November 2022
TMG5316 T. magnatum na na 09 November 2022
TMG5317 T. magnatum na na 09 November 2022
TMG5318 T. magnatum na na 09 November 2022
TMG5319 b T. magnatum Quercus cerris L. Città della Pieve (PG), Umbria, Italy 15 November 2022
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Abbreviations: BO, Bologna; CB, Campobasso; na, not available; PG, Perugia.

a

na = provided by Truffleland s.r.l, Sant'Anatolia di Narco, Perugia, Italy.

b

Strain with an active growing mycelium.

The identity of each T. magnatum isolate was molecularly confirmed by polymerase chain reaction (PCR) with the species‐specific primers TmgI and TmgII (Amicucci et al., 1998). All the PCR reactions were carried out by mixing 25 μL of 2× Phanta® Max Master Mix (Vazyme Biotech Co) with 2 μL of each primer (10 μM), 1 μL of 2.5% dimethylsulphoxide and nuclease‐free sterile water to a reaction volume of 50 μL. Some hyphae were put directly into the reaction volume in aseptic conditions. The reaction mixtures underwent an initial denaturation step of 94°C for 5 min, followed by 25 cycles of 20 s at 94°C, 15 s at 62°C, 1 min at 72°C and a final extension at 72°C for 7 min. PCR products were run on 1% agarose gel and visualised by staining with ethidium bromide.

Microscopic observations

The morphological characteristics of the hyphae of T. magnatum isolates were observed and measured under a Nikon Eclipse TE2000 U Inverted Microscope (Nikon Corporation, Tokyo, Japan) and images captured with a Nikon DS‐Fi3 (Nikon Corporation, Tokyo, Japan). The measures were collected with the NIS Elements BR software (ver. 4.6, Nikon Corporation, Tokyo, Japan).

T. magnatum hyphae from the inner part of the colony of each strain were first observed without any treatments and then observed again after washing in sterile water added with 0.5% Tween 20, followed by vortexing for 1 min to remove loosely attached bacteria. Blue lactophenol was used to stain the wall polysaccharides of hyphae and Gram‐negative bacteria cells (Ericksen, 2015).

The hyphal features selected to describe T. magnatum strains were hyphal diameter, septal distance, Hyphal Growth Unit (HGU) (Trinci, 1974) and Vesicle Production Ratio (VPR). Vesicles are common morphological features of Tuber spp. mycelium and are represented by hyphal swellings (Iotti et al., 2002). VPR is represented by the formula:

VPR=vesiscle numberseptal distance.

Antibiotic treatment

At first, preliminary analysis was carried out on T. borchii—CMI‐UNIBO, strain n. TBO5005 (Puliga et al., 2021)—to evaluate the effect of antibiotic addition on Tuber spp. mycelial growth. To this purpose, 200 μg/mL of streptomycin, ampicillin and chloramphenicol (Kuykendall et al., 1988) were added to mWPM plates. Five mWPM plates (9 cm in diameter) added with antibiotics were inoculated with 0.5 cm mycelium plugs from 50‐day‐old T. borchii cultures, and the other five mWPM plates without antibiotic addition were used as controls. The colony diameter of each plate was measured every 7 days until the stationary phase (10 weeks), along two preset diametrical lines. After this preliminary analysis, the same procedure was applied to evaluate the mycelial growth of T. magnatum in the absence of bacteria. T. magnatum mycelial plugs were taken from 60‐day‐old cultures of each fungal isolate containing wild bacteria strains.

The area (cm2) covered weekly by the mycelium was calculated according to Puliga et al. (2022), assuming an elliptical shape covered by the mycelium as reported by Tryfinopoulou et al. (2020) with the following formula:

FCA=R1×R2×π,

where FCA is the fungal colony area (cm2) and R 1 and R 2 are the two perpendicular radii, respectively.

The area growth rate of the mycelium (AGR) was calculated with the formula of Sinclair and Cantero (1989):

AGR=FCAf−FCAiTf−Ti

where FCAf and FCAi are the FCAs at the end and beginning of the exponential growth phase, respectively; T f and T i are the times (weeks) at the end and beginning of the exponential growth phase, respectively.

Tuber mycelia/bacteria co‐culture test

The growth of the three T. magnatum strains (TMG5072, TMG5300 and TMG5319) was determined by measuring the FCA with the same method described above (Puliga et al., 2022). The bacterial population of the strain with the faster growth was selected for the next test.

The ability of the bacterial community isolated from T. magnatum ascomata to promote the growth of Tuber borchii (TBO5005), and T. melanosporum—CMI‐UNIBO, strain n. TME2 (Iotti, Rubini, et al., 2012)—was evaluated by co‐culture tests in mWPM plates (Iotti et al., 2005). For each truffle species, five plates (6 cm in diameter) were inoculated with 0.5 cm mycelium plugs and 10 μL of bacterial suspension (~1 × 108 CFU/mL of the TMG5072 bacterial community) in yeast mannitol medium (YM) (Keele Jr et al., 1969). An additional five Petri dishes for each truffle species were inoculated only with the Tuber spp. mycelium as a control, added with 10 μL of liquid YM. Finally, other five Petri dishes were inoculated with 0.5 cm mycelium plugs of TMG5072 together with its native bacteria. The mycelium diameter of each strain was measured every 7 days until the stationary phase (10 weeks from inoculation) along two preset diametrical lines. FCA and AGR were calculated as previously reported.

Statistical analyses

The morphological data, FCA and AGR were analysed using R Studio 2023.09.1+494. The significant difference between treatments was tested by one‐way analysis of variance and the means were compared by Tukey's t‐test (p ≤ 0.05).

Bradyrhizobium isolation

The bacterial populations growing together with each isolated T. magnatum strain were transplanted into mWPM (Iotti et al., 2005) and yeast mannitol agar (YMA) and kept at both 22.5°C and 28°C in the dark.

Bradyrhizobium strain isolation and purification were made by streak‐planting (Sanders, 2012) on YMA at 28°C in the dark, which are the best conditions for Bradyrhizobium growth (Hafiz et al., 2021; Vincent, 1970). The isolation was carried out starting from the bacterial population of each fungal strain.

After bacterial growth, the identity of at least 10 colonies from each T. magnatum strain was verified by a direct PCR approach using the Bradyrhizobium spp.‐specific primers BRdnaKf–BRdnaKr (Menna et al., 2009). PCR reactions were performed with the thermal parameters specified in Table S1. Three bradyrhizobial colonies from each T. magnatum strain were randomly selected for phylogenetic analyses.

The isolated Bradyrhizobium strains were then transferred into YM liquid medium and grown on an orbital shaker at 180 rpm, at 28°C in the dark for 10 days (Iturralde et al., 2020). After that, 500 μL of these cultures were added with 500 μL of glycerol and preserved at –80°C.

Phylogenetic analyses

Phylogeny of bradyrhizobia strains was inferred by maximum likelihood (ML) and neighbour joining (NJ) in raxmlGUI 1.5b2 (Silvestro & Michalak, 2012) and MEGA11 software (Tamura et al., 2021) using the genes 16S rRNA, glnII, recA and nifH. The selected genes were amplified through direct PCR using the primer pairs and the conditions reported in Table S1. The nifH gene was amplified with the newly designed primers NifseqF (ATTCTGATCGTCGGTTGCG) and NifseqR (GGATCTTCTCGGCAAGGC) to avoid non‐specific amplicons. Amplified fragments were sequenced at Eurofins Genomics (Germany) in both directions. Sequences were edited and assembled by the Bioedit Sequence Alignment Editor (Hall et al., 2011) and then aligned with the MUSCLE algorithm implemented in MEGA11 software (Tamura et al., 2021). The sequences were deposited in GenBank, and their accession numbers were listed here: OR544965–OR544973 (16S rRNA), OR569722–OR569730 (glnII), OR569731–OR569739 (recA) and OR569740–OR569748 (nifH). For each accession number, the closest BLASTn result was reported in Table S2.

Single gene phylogenies were inferred for the 16S rRNA, glnII, recA and nifH gene sequences, while a concatenated dataset was generated with the sequences of glnII and recA genes (Chahboune et al., 2011; Delamuta et al., 2017). The sequences used to construct phylogenetic trees and the outgroup are listed in Table S3. ML analyses were performed with 1000 throughout bootstrap replicates (100 runs), applying the models of nucleotide substitution GTR + G + I either for the 16S rRNA and nifH genes or for glnII + recA concatenated dataset. Single gene phylogenies of glnII and recA were inferred with NJ analysis with 1000 throughout bootstrap replicates (100 runs) and applying the p‐distance model. ML and NJ trees were edited using MEGA11 (Tamura et al., 2021). Only bootstrap values greater than 75% were shown on branches. Genetic diversity (p‐distance) within and among the Bradyrhizobium supergroups of both glnII and recA genes was evaluated using MEGA11 (Tamura et al., 2021).

RESULTS

Mycelium isolation

The PCR with specific primers confirmed the identity of all T. magnatum isolates, which were characterised by active and consistent growth in subsequent subcultures (TMG5072, TMG5302 and TMG5319). The mycelia grew both on the surface and in the agar medium. After the isolation procedure, all strains showed a very long lag phase. The excised fragments of the gleba also took more than 1 month to generate the first hyphae. The same behaviour was also observed after the first subculturing. The growth rate seemed to increase in the following subcultures, although the inocula took 2–3 weeks to form an evident hyphal extension from the plug. On mWPM, T. magnatum strains generally take 10 weeks from inoculation to reach the stationary phase.

The mycelial colony appeared whitish at the beginning (Figure 1) and gradually changed to ivory and pale yellow 8–10 weeks after inoculation.

FIGURE 1.

FIGURE 1

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Example of Tuber magnatum mycelium culture on modified Woody Plant Medium (mWPM) at the first stage of growth (A). Bacterial biofilm around hyphae (B). Morphology of T. magnatum mycelium (C–E); bar = 15 μm. Bacterial distribution along the hypha (C), vesicles (D) hyphal coils and aggregates (E). Hyphae and bacteria were coloured with blue lactophenol staining.

Each mycelial isolate showed the co‐occurrence of a native bacterial population. During subcultures, the first days after inoculation, the bacteria grew around the inoculation point, forming a cream colony that remained circumscribed only in the inner area of mycelium growth. After the mycelium colonised almost all of the surface of the plate, the bacteria spread deep into the medium, which became a little opaque.

Microscopic observations

For each strain, several vesicles, rare anastomoses and hyphal coils were observed (Graziosi et al., 2022) (Figure 1). The hyphal diameter averaged from 4.45 ± 0.16 μm for the strain TMG5072 to 5.38 ± 0.19 μm for the strain TMG5300 (Table 2). The statistical analysis of hyphal diameter showed no significant differences between the strains TMG5072 and TMG5319, whereas it was found between the strain TMG5300 and the other two strains.

TABLE 2.

Hyphal morphological characteristics of the isolates.

Strain 1 Hyphal diameter (μm) Septal distance (μm) Branching angle (°) HGU (μm/no. of branches) VPR (μm/no. of vesicles)
TMG5072 4.45 ± 0.16a (2.53–7.98) 56.86 ± 4.34a (5.56–149.85) 61.80 ± 7.31 (34–90) 144.81 ± 24.53 (50.17–220.01) 63.96 ± 9.76 (17.33–105.61)
TMG5300 5.38 ± 0.19b (3.31–8.70) 45.56 ± 6.44b (12.59–93.79) 67.70 ± 8.56 (34–112) 178.50 ± 37.76 (54.23–441.71) 66.77 ± 12.30 (32.24–113.11)
TMG5319 4.59 ± 0.13a (2.78–6.26) 55.49 ± 3.10c (16.42–126.04) 68.70 ± 7.69 (24–92) 145.62 ± 8.09 (104.5–175.94) 76.35 ± 7.78 (42.15–140.32)
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Note: Data are the mean of 50 measures from three different Petri dishes. Within columns, different letters indicate difference between treatments according to p < 0.05 by Tukey's test.

Abbreviation: HGU, Hyphal Growth Unit.

1

Strain of Tuber magnatum used in this study.

The average septal distance ranged from 45.56 ± 6.44 for the strain TMG5300 to 56.86 ± 4.34 μm for the strain TMG5072, and statistical differences were observed among all the strains (Table 2).

The branching angle was very similar among the tested strains and no statistical differences were found. Strain TMG5072 showed the lowest average angle (61.80 ± 7.31°), whereas strain TMG5319 exhibited the greatest (68.70 ± 7.69°).

Regarding HGU, strain TMG5300 is characterised by the highest average value (178.50 ± 37.76 μm) in contrast with the TMG5072 and TMG5319 strains, which showed 144.81 ± 24.53 and 145.62 ± 8.09 μm, respectively. Thus, strain TMG5300 developed more linear hyphae and the lowest number of branches. Nevertheless, there were no statistical differences among strains.

The occurrence of vesicles was very similar among strains and no statistical differences were detected. Strain TMG5072 had the highest frequency of vesicles, with a VPR average value of 63.96 ± 9.76 μm followed by TMG5300 (66.77 ± 12.30 μm) and TMG5319 (76.35 ± 7.78 μm).

After staining, bacterial cells adhering to hyphae were evident, although it did not occur extensively along their entire length. Other bacterial cells remained spread in the cultural medium. After the washing treatment, all T. magnatum strains exhibited only some bacterial clusters that remained attached to the hyphae.

Antibiotic treatment and co‐culture test

The growth of TBO5005 mycelium was not significantly affected by antibiotic addition (Figure S1), whereas TMG5072 mycelium and the associated bacteria were completely inhibited.

Among T. magnatum isolates, the strain TMG5072 showed the fastest growth and maintained its vitality with subculturing (Figure S2). On the contrary, mycelia of strains TMG5300 and TMG5319 grew slower and weaker just after the first subculture.

The FCA covered weekly by the tested truffle species on mWPM in the co‐culture test is reported in Figure 2. The lag‐phases differed between Tuber species (from a few days to 3 weeks). T. borchii was the first species to grow new hyphae, just 1 week after inoculation. T. magnatum and T. melanosporum were characterised by the longest lag‐phases and the exponential growth phase started 4 weeks after inoculation. Nevertheless, TMG5072 showed significantly faster growth and within 5 weeks after inoculation, its mycelial area exceeded that of TME2 (1.27 ± 0.26 cm2). At the stationary phase (8 weeks after inoculation), the area covered by TMG5072 mycelium (7.32 ± 0.23 cm2) reached approximately the TBO5005 one (7.36 ± 0.16 cm2). The bacterial addition did not significantly affect the mycelial growth, both in the case of T. borchii and T. melanosporum, during all over the measurement period.

FIGURE 2.

FIGURE 2

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Growth trend of area covered weekly by mycelia of Tuber magnatum strain 5072 (TMG5072, black solid line), Tuber borchii control (TBO5005, black dashed line), T. borchii with bacterial addition (TBO5005_bac, black dotted line), Tuber melanosporum control (TME2, grey dashed line), T. melanosporum with bacterial addition (TME2_bac, grey dotted line). FCA, fungal colony area.

These results were also confirmed by the data of AGRs (Figure S3) during the exponential phase. The AGRs values of both these two species added with bacteria exhibited a non‐significant difference despite the control, according to p < 0.05 by Tukey's test. On the other hand, the AGR of the TMG5072 mycelium, about 1.66 ± 0.043 cm2/week, was significantly higher than other truffle species with or without bacteria addition, respectively: 0.69 ± 0.022 cm2/week for TBO5005_bac and 0.59 ± 0.024 cm2/week for TBO5005; 0.12 ± 0.011 cm2/week for TME2_bac and 0.22 ± 0.031 cm2/week for TME2.

Bradyrhizobium isolation

The bacteria were unable to grow on mWPM without the mycelium, and no colony was formed after 1 month of incubation at both 22.5 and 28°C in the dark. Nevertheless, at 22.5°C, they can develop abundantly in a few days in the presence of the mycelium. Bacteria formed colonies within 10 days after inoculation in the absence of T. magnatum mycelium only on a selective medium (YMA) and with strictly specific conditions (28°C in the dark).

Molecular characterisation and nucleotide sequence analyses

Preliminary analysis using Bradyrhizobium‐specific primers showed that all the bacterial colonies (10 from each T. magnatum strain) belonged to Bradyrhizobium. BLASTn analysis of the 16S rRNA gene sequences (Table S2) revealed that the bacterial isolates (three isolates from each T. magnatum strain) have the highest similarity (>99.8%) with Bradyrhizobium sp. strain SRL50 (MN134555), Bradyrhizobium sp. 170 (CP064703) and Bradyrhizobium sp. S12‐14‐2 (CP129212). ML analysis based on the 16S rRNA gene (Figure S4) placed the strains into a clade containing Bradyrhizobium spp. sequences from T. borchii (clone Cl‐19‐TB8‐II—AY599677) and T. magnatum (clone TM5_22—DQ303373; clone TM1_39—DQ303378) ascomata (Barbieri et al., 2005, 2007) but did not resolve the phylogenetic position of the bacteria isolated in this study within the Bradyrhizobium supergroups defined by Avontuur et al. (2019). In fact, the 16S rRNA gene sequences clustered together with species of both Bradyrhizobium elkanii supergroup (Bradyrhizobium viridifuturi and Bradyrhizobium embrapense) and Bradyrhizobium jicamae supergroup (B. jicamae and Bradyrhizobium erythrophlei). On the contrary, ML analysis of the concatenated glnII and recA gene dataset (Figure 3) grouped all bradyrhizobia strains isolated in this study in a monophyletic and well‐supported clade closely related to Bradyrhizobium valentinum (JX518575, JX518589) and species in the B. jicamae supergroup (Avontuur et al., 2019). The same topology of the ML tree was also obtained with NJ analysis (Figures S5 and S6).

FIGURE 3.

FIGURE 3

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Maximum likelihood phylogeny based on concatenated glnII‐recA gene sequences showing the relationships between the nine Tuber magnatum bradyrhizobial strains isolated in this work and other members of the Bradyrhizobium genus. Accession numbers are indicated within brackets. Bootstrap values >75% are indicated at the nodes. Bar = 5 substitutions every 100 positions.

The nifH phylogenetic tree (Figure 4) was congruent with the concatenated phylogeny inferred in this study by glnII and recA genes, placing all the nine T. magnatum bradyrhizobial strains in a separated clade, with a branch support of 100. Furthermore, these strains were strictly related to Bradyrhizobium sediminis S2‐20‐1 (CP076134).

FIGURE 4.

FIGURE 4

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Maximum likelihood phylogeny of nifH gene sequences. Accession numbers are indicated within brackets. Bootstrap values >75% are indicated at the nodes. Bar = 5 substitutions every 100 positions.

The genetic diversity for glnII (Table S4) and recA (Table S5) genes within and between groups confirmed the belonging of Bradyrhizobium strains isolated in this group in the B. jicamae supergroup. In fact, uncorrected p‐distances between T. magnatum bradyrhizobia and the species of B. jicamae supergroup are on average always lower than the values calculated among the different supergroups for both glnII (0.08 versus >0.11) and recA (0.06 versus >0.08).

DISCUSSION

Mycelia of different Tuber species have been successfully isolated by many authors and used for a variety of scientific purposes (Ceccaroli et al., 2001; Iotti et al., 2002, 2016; Leonardi et al., 2017; Li et al., 2012; Liu et al., 2009; Nadim et al., 2015; Poma et al., 1999; Saltarelli et al., 2003; Sbrana et al., 2002; Vahdatzadeh & Splivallo, 2018). However, viable and stable mycelial cultures of T. magnatum have been obtained for the first time only by this work, despite the numerous attempts made over the years. In this study, it was necessary to wait more than a month before the gleba fragments used as inoculum produced the first hyphae. This long lag phase may partly explain why T. magnatum mycelium has never been successfully isolated before. Furthermore, the recurrent development of bacteria on the inoculated gleba fragment might have led the researchers to discard isolation plates before the hyphal growth became evident (A. Zambonelli, personal communication, September 1, 2023). In our study, bacteria co‐isolated from the gleba proved to be essential for the growth of T. magnatum mycelium on mWPM, which is one of the most suitable media for Tuber mycelium (Iotti et al., 2002). As these bacteria were not able to improve the growth of T. borchii and T. melanosporum mycelia in the same conditions, it is possible to hypothesise a taxon‐specific dependence. Similarly, Le Roux et al. (2016) identified a specific interaction between bacterial strains belonging to the Rhodopseudomonas genus and the mycelia of T. melanosporum and T. brumale.

Even in the presence of the bacteria, all T. magnatum strains isolated in this study had slower growth than most of the saprotrophic cultivated basidiomycetes and ascomycetes (Badalyan et al., 2023; Puliga et al., 2022) but similar to other ectomycorrhizal fungi (Iotti et al., 2005). Considering the Tuber genus, the growth of the strain TMG5072 calculated as FCA was similar to that of T. borchii and higher than that of T. melanosporum. The mycelium of this strain appeared more branched (lowest HGU value) than the other strains although the HGU means were not statistically different. An increase in branching rate can be related to a more consistent growth, as demonstrated previously for T. borchii (Amicucci et al., 2010). Significant differences between strains were found for hyphal diameter and septal distance. In particular, the strain TMG5300 showed shorter and larger hyphal cells with respect to the other two strains but a lower branching rate. These morphological differences could be due to T. magnatum strain genetic differences or to the specific interaction with the bacteria lineages.

Molecular analyses showed that all the isolated bacteria belonged to Bradyrhizobium, which have previously been found to be common members of the bacterial community inhabiting the ascomata of Tuber spp. (Antony‐Babu et al., 2014; Barbieri et al., 2005, 2007, 2010, 2012; Benucci & Bonito, 2016; Citterio et al., 1995; Frey‐Klett et al., 2007; Graziosi et al., 2022; Marozzi et al., 2023; Monaco et al., 2021, 2022; Niimi et al., 2021a, 2021b; Pavić et al., 2013; Sillo et al., 2022). In particular, our 16S rRNA gene sequences clustered together with those of bradyrhizobia found in T. borchii and T. magnatum by Barbieri et al. (2005, 2007), with identities ranging from 98.2% to 99.7%. Similarly, the bacteria identified by Benucci and Bonito (2016) in the ascomata of several hypogeous ascomycetes (Kalapuya brunnea M.J. Trappe, Trappe & Bonito, Leucangium carthusianum (Tul. & C. Tul.) Paol., Terfezia claveryi Chatin, Tuber indicum Cooke & Massee, T. melanosporum, Tuber lyonii Butters, Tuber gibbosum Harkn. and Tuber oregonense Trappe, Bonito & P. Rawl.) and by Antony‐Babu et al. (2014) in T. melanosporum ascomata, have 16S rRNA gene sequence identities always >98.7%. Our findings confirm the hypothesis of the existence of a ubiquitous Bradyrhizobium taxon that is part of the core microbial community of Tuber ascomata (Benucci & Bonito, 2016). Considering the effects of these bacteria on Tuber mycelia, it can be assumed that the species of this taxon may promote gleba formation during ascoma maturation.

However, the 16S rRNA gene is too conserved in bradyrhizobia to discriminate between species and is not able to discriminate also between Bradyrhizobium and closely related genera (Willems et al., 2001). For this reason, we conducted a multilocus phylogeny using the genes glnII and recA, which allowed the best resolution of the evolutionary relationships of our nine bradyrhizobial strains within the Bradyrhizobium genus. The concatenated tree placed all nine strains in a strongly supported clade closely related to the species in the B. jicamae supergroup (Avontuur et al., 2019). Unfortunately, no sequences of glnII and recA from bradyrhizobia inhabiting truffle ascomata are available in GenBank. Intriguingly, the three strains from the ascoma TMG5300 were grouped together in the same subclade (bootstrap value = 99), whereas the six strains isolated from TMG5319 and TMG5072 were paraphyletic and divided into four independent lineages.

The analyses on genetic divergence within and among Bradyrhizobium supergroups seem to confirm the inclusion of T. magnatum bradyrhizobia group into the B. jicamae supergroup. The B. jicamae supergroup contains nitrogen‐fixing bacteria commonly associated with leguminous plants but also included soil free‐living bacteria (Avontuur et al., 2019; Ormeño‐Orrillo & Martínez‐Romero, 2019). We successfully amplified the nifH gene in all isolated strains and the tree generated with their sequences also grouped the nine T. magnatum bradyrhizobial strains in a separate clade with high bootstrap support. The nifH genes of T. magnatum bradyrhizobia appear to be evolutionarily closer to that of the free‐living, nitrogen‐fixing and non‐nodulating B. sediminis isolated from freshwater sediment (Jin et al., 2022) rather than the nifH sequences of the root symbiotic and nodulating species.

The detection of nifH genes in the ascoma‐inhabiting bacteria leads to speculation on their role in nitrogen nutrition of T. magnatum (Barbieri et al., 2010). Moreover, the inability of the co‐isolated bradyrhizobia to grow in pure culture on mWPM suggests a possible mutualistic interaction with T. magnatum. Le Roux et al. (2016) made the same assumption of symbiotic interaction between the mycelia of T. melanosporum and T. brumale and bacteria in the Rhodopseudomonas, although these authors did not detect nifH gene in the analysed bacterial strains.

The role of the mycelia of ectomycorrhizal fungi on bacterial growth was already demonstrated by Rigamonte et al. (2010) and it may be related to the production of trehalose and various polyols (e.g., mannitol and arabitol). Mannitol is the main carbon source of Bradyrhizobium within a selective medium (Keele Jr et al., 1969; Kuykendall, 2015). These sugars are commonly produced during the active growth of ectomycorrhizal basidiomycetes (Hampp & Schaeffer, 1999; Ineichen & Wiemken, 1992; Martin et al., 1984, 1998; Söderström et al., 1988) and ascomycetes (Martin et al., 1985, 1988) including Tuber spp. (Ceccaroli et al., 2003, 2011). For instance, the utilisation of mannitol and trehalose exudated by Cantharellus cibarius Fr. mycelium was common among Pseudomonas spp. from different environments (Rangel‐Castro et al., 2000; Rangel‐Castro, Danell, & Pfeffer, 2002; Rangel‐Castro, Danell, & Taylor, 2002). Also, the high level of trehalose accumulated in Laccaria bicolor (Maire) P.D. Orton hyphae chemoattracted and promoted the growth of the helper bacteria (Deveau et al., 2010).

The intriguing relationship between bacteria and mycorrhizal fungi might have arisen at the origin of their evolution and taxon‐specific interaction may have evolved over time (Frey‐Klett et al., 2007). Seneviratne and Jayasinghearachchi (2003) reported the mycelial colonisation of some soil fungi by Bradyrhizobium spp. The fungal partner provided a site for cell adhesion and its exudates served as a source of nutrition for bradyrhizobia. Microscopic observations carried out by Seneviratne and Jayasinghearachchi (2003) showed an extended hyphal adhesion between bacterial and fungal cells similar to that observed in T. magnatum mycelium, although in our case the interaction seems to be closer. In fact, T. magnatum and the co‐isolated bradyrhizobia benefit each other and cannot grow separately, at least in mWPM, suggesting a mutual dependency. This unique association seems to have become essential during the evolutionary process of this truffle species. The genetic closeness of the bacteria inhabiting truffle ascomata could be due to a co‐evolution process between some bradyrhizobia taxa and the Tuber spp. This co‐evolutional process between truffles and bradyrhizobia might have induced the differentiation of some bacterial strains extremely specific to certain Tuber spp.

The strict relations between Bradyrhizobium spp. and T. magnatum mycelial growth could have practical implications in truffle cultivation. In fact, the application of these bradyrhizobia could affect the success of the spore inoculation in the greenhouse mycorrhization process and, later, the maintenance of T. magnatum soil colonisation after plantation in the field. Moreover, cultivating in vitro T. magnatum mycelium opens up the possibility of obtaining mycorrhized plants with T. magnatum mycelial cultures.

For a better exploitation of the beneficial effects of these bacteria on T. magnatum cultivation, further studies will be necessary to characterise their physiology and the exact nature of their relationship with T. magnatum mycelium.

AUTHOR CONTRIBUTIONS

Simone Graziosi: Conceptualization (equal); data curation (equal); investigation (lead); methodology (equal); software (lead); writing – original draft (equal); writing – review and editing (equal). Federico Puliga: Conceptualization (supporting); investigation (supporting); methodology (supporting); writing – review and editing (equal). Mirco Iotti: Data curation (equal); formal analysis (equal); methodology (equal); supervision (supporting); visualization (equal); writing – original draft (equal); writing – review and editing (equal). Antonella Amicucci: Conceptualization (supporting); methodology (equal); visualization (supporting); writing – review and editing (equal). Alessandra Zambonelli: Conceptualization (equal); funding acquisition (lead); project administration (lead); resources (lead); supervision (lead); validation (lead); visualization (equal); writing – original draft (equal); writing – review and editing (equal).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflict of interest.

Supporting information

Data S1. Supporting Information.

EMI4-16-e13271-s001.docx (3.9MB, docx)

ACKNOWLEDGEMENTS

The authors are grateful to Truffleland s.r.l for providing ascomata (Tuber magnatum). This work was funded by the European Union—NextGenerationEU under the National Recovery and Resilience Plan (PNRR)—Mission 4 Education and research—Component 2 from research to business—Investment 1.1 Notice Prin 2022—DD N. 104 del 2/2/2022, from title ‘Interactions of the white truffle Tuber magnatum with soil microbiome and plants,’ proposal code K272X8—CUP J53D23010090006. This study is part of the activities of the 37th cycle of the Ph.D. course in ‘Health, Safety and Green System,’ PON call ‘Research and Innovation 2014–2020.’

Graziosi, S. , Puliga, F. , Iotti, M. , Amicucci, A. & Zambonelli, A. (2024) In vitro interactions between Bradyrhizobium spp. and Tuber magnatum mycelium. Environmental Microbiology Reports, 16(3), e13271. Available from: 10.1111/1758-2229.13271

DATA AVAILABILITY STATEMENT

All the sequences were deposited in GenBank database. All the other data are reported in the text and /or in supporting information.

REFERENCES

  1. Amicucci, A. , Zambonelli, A. , Giomaro, G. , Potenza, L. & Stocchi, V. (1998) Identification of ectomycorrhizal fungi of the genus Tuber by species‐specific ITS primers. Molecular Ecology, 7, 273–277. Available from: 10.1046/j.1365-294X.1998.00357.x [DOI] [Google Scholar]
  2. Amicucci, A. , Zambonelli, A. , Iotti, M. , Polidori, E. , Menotta, M. , Saltarelli, R. et al. (2010) Morphological and molecular modifications induced by different carbohydrate sources in Tuber borchii . Journal of Molecular Microbiology and Biotechnology, 18, 120–128. Available from: 10.1159/000297915 [DOI] [PubMed] [Google Scholar]
  3. Antony‐Babu, S. , Deveau, A. , Van Nostrand, J.D. , Zhou, J. , Le Tacon, F. , Robin, C. et al. (2014) Black truffle‐associated bacterial communities during the development and maturation of Tuber melanosporum ascocarps and putative functional roles. Environmental Microbiology, 16, 2831–2847. Available from: 10.1111/1462-2920.12294 [DOI] [PubMed] [Google Scholar]
  4. Avontuur, J.R. , Palmer, M. , Beukes, C.W. , Chan, W.Y. , Coetzee, M.P.A. , Blom, J. et al. (2019) Genome‐informed Bradyrhizobium taxonomy: where to from here? Systematic and Applied Microbiology, 42, 427–439. Available from: 10.1016/j.syapm.2019.03.006 [DOI] [PubMed] [Google Scholar]
  5. Badalyan, S. , Gharibyan, N. , Gianchino, C. , Iotti, M. & Zambonelli, A. (2023) Morphological observation and biomass formation in different edible medicinal Morchella collections (Pezizomycetes, Ascomycota). Italian Journal of Mycology, 52, 50–61. Available from: 10.6092/issn.2531-7342/16112 [DOI] [Google Scholar]
  6. Barbieri, E. , Bertini, L. , Rossi, I. , Ceccaroli, P. , Saltarelli, R. , Guidi, C. et al. (2005) New evidence for bacterial diversity in the ascoma of the ectomycorrhizal fungus Tuber borchii Vittad. FEMS Microbiology Letters, 247, 23–35. Available from: 10.1016/j.femsle.2005.04.027 [DOI] [PubMed] [Google Scholar]
  7. Barbieri, E. , Ceccaroli, P. , Palma, F. , Agostini, D. & Stocchi, V. (2012) Ectomycorrhizal helper bacteria: the third partner in the symbiosis. In: Zambonelli, A. & Bonito, G.M. (Eds.) Edible ectomycorrhizal mushrooms: current knowledge and future prospects, soil biology. Berlin; Heidelberg: Springer, pp. 125–141. Available from: 10.1007/978-3-642-33823-6_8 [DOI] [Google Scholar]
  8. Barbieri, E. , Ceccaroli, P. , Saltarelli, R. , Guidi, C. , Potenza, L. , Basaglia, M. et al. (2010) New evidence for nitrogen fixation within the Italian white truffle Tuber magnatum . Fungal Biology, 114, 936–942. Available from: 10.1016/j.funbio.2010.09.001 [DOI] [PubMed] [Google Scholar]
  9. Barbieri, E. , Guidi, C. , Bertaux, J. , Frey‐Klett, P. , Garbaye, J. , Ceccaroli, P. et al. (2007) Occurrence and diversity of bacterial communities in Tuber magnatum during truffle maturation. Environmental Microbiology, 9, 2234–2246. Available from: 10.1111/j.1462-2920.2007.01338.x [DOI] [PubMed] [Google Scholar]
  10. Benucci, G.M.N. & Bonito, G.M. (2016) The truffle microbiome: species and geography effects on bacteria associated with fruiting bodies of hypogeous Pezizales. Microbial Ecology, 72, 4–8. Available from: 10.1007/s00248-016-0755-3 [DOI] [PubMed] [Google Scholar]
  11. Bonito, G. , Smith, M.E. , Nowak, M. , Healy, R.A. , Guevara, G. , Cázares, E. et al. (2013) Historical biogeography and diversification of truffles in the Tuberaceae and their newly identified southern hemisphere sister lineage. PLoS One, 8, e52765. Available from: 10.1371/journal.pone.0052765 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ceccaroli, P. , Buffalini, M. , Saltarelli, R. , Barbieri, E. , Polidori, E. , Ottonello, S. et al. (2011) Genomic profiling of carbohydrate metabolism in the ectomycorrhizal fungus Tuber melanosporum . The New Phytologist, 189, 751–764. Available from: 10.1111/j.1469-8137.2010.03520.x [DOI] [PubMed] [Google Scholar]
  13. Ceccaroli, P. , Saltarelli, R. , Cesari, P. , Pierleoni, R. , Sacconi, C. , Vallorani, L. et al. (2003) Carbohydrate and amino acid metabolism in Tuber borchii mycelium during glucose utilization: a 13C NMR study. Fungal Genetics and Biology, 39, 168–175. Available from: 10.1016/S1087-1845(03)00006-9 [DOI] [PubMed] [Google Scholar]
  14. Ceccaroli, P. , Saltarelli, R. , Cesari, P. , Zambonelli, A. & Stocchi, V. (2001) Effects of different carbohydrate sources on the growth of Tuber borchii Vittad. Mycelium strains in pure culture. Molecular and Cellular Biochemistry, 218, 65–70. Available from: 10.1023/A:1007265423786 [DOI] [PubMed] [Google Scholar]
  15. Chahboune, R. , Barrijal, S. , Moreno, S. & Bedmar, E.J. (2011) Characterization of Bradyrhizobium species isolated from root nodules of Cytisus villosus grown in Morocco. Systematic and Applied Microbiology, 34, 440–445. Available from: 10.1016/j.syapm.2011.04.008 [DOI] [PubMed] [Google Scholar]
  16. Chevalier, G. & Frochot, H. (1997) La Maîtrise de la culture de la truffe. Revue Forestière Française, 49, 201–213. [Google Scholar]
  17. Citterio, B. , Cardoni, P. , Potenza, L. , Amicucci, A. , Stocchi, V. , Gola, G. et al. (1995) Isolation of bacteria from sporocarps of Tuber magnatum Pico, Tuber borchii Vitt. and Tuber maculatum Vitt. In: Stocchi, V. , Bonfante, P. & Nuti, M. (Eds.) Biotechnology of Ectomycorrhizae. Boston: Springer, pp. 241–248. Available from: 10.1007/978-1-4615-1889-1_21 [DOI] [Google Scholar]
  18. Delamuta, J.R.M. , Menna, P. , Ribeiro, R.A. & Hungria, M. (2017) Phylogenies of symbiotic genes of Bradyrhizobium symbionts of legumes of economic and environmental importance in Brazil support the definition of the new symbiovars pachyrhizi and sojae. Systematic and Applied Microbiology, 40, 254–265. Available from: 10.1016/j.syapm.2017.04.005 [DOI] [PubMed] [Google Scholar]
  19. Deveau, A. , Brulé, C. , Palin, B. , Champmartin, D. , Rubini, P. , Garbaye, J. et al. (2010) Role of fungal trehalose and bacterial thiamine in the improved survival and growth of the ectomycorrhizal fungus Laccaria bicolor S238N and the helper bacterium Pseudomonas fluorescens BBc6R8. Environmental Microbiology Reports, 2, 560–568. Available from: 10.1111/j.1758-2229.2010.00145.x [DOI] [PubMed] [Google Scholar]
  20. Ericksen, B. (2015) Quantification of polysaccharides fixed to gram stained slides using lactophenol cotton blue and digital image processing. F1000Research, 4, 1. Available from: 10.12688/f1000research.5779.5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fontana, A. (1968) Miceli di funghi ipogei in coltura pura. In: Proceedings of the 1st International Truffle Conference, Spoleto, Italy, 24–25 May 1968. pp. 127–134.
  22. Frey‐Klett, P. , Garbaye, J. & Tarkka, M. (2007) The mycorrhiza helper bacteria revisited. The New Phytologist, 176, 22–36. [DOI] [PubMed] [Google Scholar]
  23. Giomaro, G.M. , Sisti, D. & Zambonelli, A. (2005) Cultivation of edible ectomycorrhizal fungi by in vitro mycorrhizal synthesis. In: Declerck, S. , Fortin, J.A. & Strullu, D.‐G. (Eds.) Vitro culture of mycorrhizas, soil biology. Berlin; Heidelberg: Springer, pp. 253–267. Available from: 10.1007/3-540-27331-X_14 [DOI] [Google Scholar]
  24. Graziosi, S. , Hall, I.R. & Zambonelli, A. (2022) The mysteries of the white truffle: its biology, ecology and cultivation. Encyclopedia, 2, 1959–1971. Available from: 10.3390/encyclopedia2040135 [DOI] [Google Scholar]
  25. Hafiz, M.H.R. , Salehin, A. & Itoh, K. (2021) Growth and competitive infection behaviors of Bradyrhizobium japonicum and Bradyrhizobium elkanii at different temperatures. Horticulturae, 7, 41. Available from: 10.3390/horticulturae7030041 [DOI] [Google Scholar]
  26. Hall, T. , Biosciences, I. & Carlsbad, C. (2011) BioEdit: an important software for molecular biology. GERF Bulletin of Biosciences, 2, 60–61. [Google Scholar]
  27. Hampp, R. & Schaeffer, C. (1999) Mycorrhiza—carbohydrate and energy metabolism. In: Varma, A. & Hock, B. (Eds.) Mycorrhiza. Berlin; Heidelberg: Springer, pp. 273–303. Available from: 10.1007/978-3-662-03779-9_12 [DOI] [Google Scholar]
  28. Ineichen, K. & Wiemken, V. (1992) Changes in the fungus‐specific, soluble‐carbohydrate pool during rapid and synchronous ectomycorrhiza formation of Picea abies with Pisolithus tinctorius . Mycorrhiza, 2, 1–7. Available from: 10.1007/BF00206277 [DOI] [Google Scholar]
  29. Iotti, M. , Amicucci, A. , Stocchi, V. & Zambonelli, A. (2002) Morphological and molecular characterization of mycelia of some Tuber species in pure culture. The New Phytologist, 155, 499–505. [DOI] [PubMed] [Google Scholar]
  30. Iotti, M. , Barbieri, E. , Stocchi, V. & Zambonelli, A. (2005) Morphological and molecular characterization of mycelia of ectomycorrhizal fungi in pure culture. Fungal Diversity, 19, 51–68. [Google Scholar]
  31. Iotti, M. , Piattoni, F. , Leonardi, P. , Hall, I.R. & Zambonelli, A. (2016) First evidence for truffle production from plants inoculated with mycelial pure cultures. Mycorrhiza, 26, 793–798. Available from: 10.1007/s00572-016-0703-6 [DOI] [PubMed] [Google Scholar]
  32. Iotti, M. , Piattoni, F. & Zambonelli, A. (2012) Techniques for host plant inoculation with truffles and other edible ectomycorrhizal mushrooms. In: Zambonelli, A. & Bonito, G.M. (Eds.) Edible ectomycorrhizal mushrooms: current knowledge and future prospects, soil biology. Berlin; Heidelberg: Springer, pp. 145–161. Available from: 10.1007/978-3-642-33823-6_9 [DOI] [Google Scholar]
  33. Iotti, M. , Rubini, A. , Tisserant, E. , Kholer, A. , Paolocci, F. & Zambonelli, A. (2012) Self/nonself recognition in Tuber melanosporum is not mediated by a heterokaryon incompatibility system. Fungal Biology, 116, 261–275. Available from: 10.1016/j.funbio.2011.11.009 [DOI] [PubMed] [Google Scholar]
  34. Iturralde, E.T. , Stocco, M.C. , Faura, A. , Mónaco, C.I. , Cordo, C. , Pérez‐Giménez, J. et al. (2020) Coinoculation of soybean plants with Bradyrhizobium japonicum and Trichoderma harzianum: coexistence of both microbes and relief of nitrate inhibition of nodulation. Biotechnology Reports, 26, e00461. Available from: 10.1016/j.btre.2020.e00461 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Jin, C.‐Z. , Wu, X.‐W. , Zhuo, Y. , Yang, Y. , Li, T. , Jin, F.‐J. et al. (2022) Genomic insights into a free‐ living, nitrogen‐fixing but non nodulating novel species of Bradyrhizobium sediminis from freshwater sediment: three isolates with the smallest genome within the genus Bradyrhizobium . Systematic and Applied Microbiology, 45(126), 353. Available from: 10.1016/j.syapm.2022.126353 [DOI] [PubMed] [Google Scholar]
  36. Keele, B.B., Jr. , Hamilton, P.B. & Elkan, G.H. (1969) Glucose catabolism in Rhizobium japonicum . Journal of Bacteriology, 97, 1184–1191. Available from: 10.1128/jb.97.3.1184-1191.1969 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kuykendall, L.D. (2015) Bradyrhizobium . In: Whitman, W.B. , Rainey, F. , Kampfer, P. , Trujillo, M. , Chun, J. , DeVos, P. et al. (Eds.) Bergey's manual of systematics of archaea and bacteria. New York: Wiley, pp. 1–11. [Google Scholar]
  38. Kuykendall, L.D. , Roy, M.A. , O'Neill, J.J. & Devine, T.E. (1988) Fatty acids, antibiotic resistance, and deoxyribonucleic acid homology groups of Bradyrhizobium japonicum . International Journal of Systematic and Evolutionary Microbiology, 38, 358–361. Available from: 10.1099/00207713-38-4-358 [DOI] [Google Scholar]
  39. Le Roux, C. , Tournier, E. , Lies, A. , Sanguin, H. , Chevalier, G. , Duponnois, R. et al. (2016) Bacteria of the genus Rhodopseudomonas (Bradyrhizobiaceae): obligate symbionts in mycelial cultures of the black truffles Tuber melanosporum and Tuber brumale . SpringerPlus, 5, 1–10. Available from: 10.1186/s40064-016-2756-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Leonardi, P. , Iotti, M. , Donati Zeppa, S. , Lancellotti, E. , Amicucci, A. & Zambonelli, A. (2017) Morphological and functional changes in mycelium and mycorrhizas of Tuber borchii due to heat stress. Fungal Ecology, 29, 20–29. Available from: 10.1016/j.funeco.2017.05.003 [DOI] [Google Scholar]
  41. Li, Y.‐Y. , Wang, G. , Li, H.‐M. , Zhong, J.‐J. & Tang, Y.‐J. (2012) Volatile organic compounds from a Tuber melanosporum fermentation system. Food Chemistry, 135, 2628–2637. Available from: 10.1016/j.foodchem.2012.07.013 [DOI] [PubMed] [Google Scholar]
  42. Liu, Q.‐N. , Liu, R.‐S. , Wang, Y.‐H. , Mi, Z.‐Y. , Li, D.‐S. , Zhong, J.‐J. et al. (2009) Fed‐batch fermentation of Tuber melanosporum for the hyperproduction of mycelia and bioactive Tuber polysaccharides. Bioresour. Technol., 100, 3644–3649. Available from: 10.1016/j.biortech.2009.02.037 [DOI] [PubMed] [Google Scholar]
  43. Luxury Columnist . (2022) The most expensive food in the world: 20 costly food items. Available at: https://luxurycolumnist.com/the-most-expensive-food-in-the-world/ [Accessed 25th December 2022]
  44. Marozzi, G. , Benucci, G.M.N. , Turchetti, B. , Massaccesi, L. , Baciarelli Falini, L. , Bonito, G. et al. (2023) Fungal and bacterial diversity in the Tuber magnatum ecosystem and microbiome. Microbial Ecology, 85, 508–521. Available from: 10.1007/s00248-021-01950-1 [DOI] [PubMed] [Google Scholar]
  45. Martin, F. , Boiffin, V. & Pfeffer, P.E. (1998) Carbohydrate and amino acid metabolism in the Eucalyptus globulus‐Pisolithus tinctorius ectomycorrhiza during glucose utilization. Plant Physiology, 118, 627–635. Available from: 10.1104/pp.118.2.627 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Martin, F. , Canet, D. & Marchal, J.‐P. (1984) In vivo natural abundance 13C NMR studies of the carbohydrate storage in ectomycorrhizal fungi. Physiologie Vegetale, 22, 733–743. [Google Scholar]
  47. Martin, F. , Canet, D. & Marchal, J.P. (1985) 13C nuclear magnetic resonance study of mannitol cycle and trehalose synthesis during glucose utilization by the ectomycorrhizal ascomycete Cenococcum graniforme . Plant Physiology, 77, 499–502. Available from: 10.1104/pp.77.2.499 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Martin, F. , Ramstedt, M. , Söderhäll, K. & Canet, D. (1988) Carbohydrate and amino acid metabolism in the ectomycorrhizal ascomycete Sphaerosporella brunnea during glucose utilization: a 13C NMR study. Plant Physiology, 86, 935–940. Available from: 10.1104/pp.86.3.935 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Mello, A. , Fontana, A. , Meotto, F. , Comandini, O. & Bonfante, P. (2001) Molecular and morphological characterization of Tuber magnatum mycorrhizas in a long‐term survey. Microbiology Research, 155, 279–284. Available from: 10.1016/S0944-5013(01)80005-7 [DOI] [PubMed] [Google Scholar]
  50. Mello, A. , Murat, C. & Bonfante, P. (2006) Truffles: much more than a prized and local fungal delicacy. FEMS Microbiology Letters, 260(1), 1–8. Available from: 10.1111/j.1574-6968.2006.00252.x [DOI] [PubMed] [Google Scholar]
  51. Menna, P. , Barcellos, F.G. & Hungria, M. (2009) Phylogeny and taxonomy of a diverse collection of Bradyrhizobium strains based on multilocus sequence analysis of the 16SrRNA gene, ITS region and glnII, recA, atpD and dnaK genes. International Journal of Systematic and Evolutionary Microbiology, 59, 2934–2950. Available from: 10.1099/ijs.0.009779-0 [DOI] [PubMed] [Google Scholar]
  52. Mischiati, P. & Fontana, A. (1993) In vitro culture of Tuber magnatum mycelium isolated from mycorrhizas. Mycological Research, 97, 40–44. Available from: 10.1016/S0953-7562(09)81110-6 [DOI] [Google Scholar]
  53. Monaco, P. , Bucci, A. , Naclerio, G. & Mello, A. (2021) Heterogeneity of the white truffle Tuber magnatum in a limited geographic area of central‐southern Italy. Environmental Microbiology Reports, 13, 591–599. Available from: 10.1111/1758-2229.12956 [DOI] [PubMed] [Google Scholar]
  54. Monaco, P. , Naclerio, G. , Mello, A. & Bucci, A. (2022) Role and potentialities of bacteria associated with Tuber magnatum: a mini‐review. Frontiers in Microbiology, 13, 1–7. Available from: 10.3389/fmicb.2022.1017089 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Nadim, M. , Deshaware, S. , Saidi, N. , Abd‐Elhakeem, M.A. , Ojamo, H. & Shamekh, S. (2015) Extracellular enzymatic activity of Tuber maculatum and Tuber aestivum mycelia. Advances in Microbiology, 05, 523. Available from: 10.4236/aim.2015.57054 [DOI] [Google Scholar]
  56. Niimi, J. , Deveau, A. & Splivallo, R. (2021a) Geographical‐based variations in white truffle Tuber magnatum aroma is explained by quantitative differences in key volatile compounds. The New Phytologist, 230, 1623–1638. Available from: 10.1111/nph.17259 [DOI] [PubMed] [Google Scholar]
  57. Niimi, J. , Deveau, A. & Splivallo, R. (2021b) Aroma and bacterial communities dramatically change with storage of fresh white truffle Tuber magnatum . LWT, 151, 112125. Available from: 10.1016/j.lwt.2021.112125 [DOI] [Google Scholar]
  58. Ormeño‐Orrillo, E. & Martínez‐Romero, E. (2019) A genomotaxonomy view of the Bradyrhizobium genus. Frontiers in Microbiology, 10, 1334. Available from: 10.3389/fmicb.2019.01334 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Pavić, A. , Stanković, S. , Saljnikov, E. , Krüger, D. , Buscot, F. , Tarkka, M. et al. (2013) Actinobacteria may influence white truffle (Tuber magnatum Pico) nutrition, ascocarp degradation and interactions with other soil fungi. Fungal Ecology, 6, 527–538. Available from: 10.1016/j.funeco.2013.05.006 [DOI] [Google Scholar]
  60. Poma, A. , Pacioni, G. , Colafarina, S. & Miranda, M. (1999) Effect of tyrosinase inhibitors on Tuber borchii mycelium growth in vitro. FEMS Microbiology Letters, 180, 69–75. Available from: 10.1111/j.1574-6968.1999.tb08779.x [DOI] [PubMed] [Google Scholar]
  61. Puliga, F. , Illice, M. , Iotti, M. , Leonardi, P. , Baghdadi, A. , Mozafari, A.A. et al. (2021) True truffle diversity in Iran. Italian Journal of Mycology, 50, 52–62. Available from: 10.6092/issn.2531-7342/12822 [DOI] [Google Scholar]
  62. Puliga, F. , Leonardi, P. , Minutella, F. , Zambonelli, A. & Francioso, O. (2022) Valorization of hazelnut shells as growing substrate for edible and medicinal mushrooms. Horticulturae, 8, 214. Available from: 10.3390/horticulturae8030214 [DOI] [Google Scholar]
  63. Rangel‐Castro, J.I. , Danell, E. , Borowicz, J. & Martin, F. (2000) Cantharellus cibarius: carbon and amino acid metabolism in relation to its fruitbody‐inhabiting fluorescent Pseudomonas. In: van Griensven (Ed.) XV th International Congress on the Science and Cultivation of Edible Fungi, Balkema, Maastricht, The Netherlands. pp. 87–93.
  64. Rangel‐Castro, J.I. , Danell, E. & Pfeffer, P.E. (2002) A 13C‐NMR study of exudation and storage of carbohydrates and amino acids in the ectomycorrhizal edible mushroom Cantharellus cibarius . Mycologia, 94, 190–199. Available from: 10.1080/15572536.2003.11833224 [DOI] [PubMed] [Google Scholar]
  65. Rangel‐Castro, I.J. , Danell, E. & Taylor, A.F. (2002) Use of different nitrogen sources by the edible ectomycorrhizal mushroom Cantharellus cibarius . Mycorrhiza, 12, 131–137. Available from: 10.1007/s00572-002-0160-2 [DOI] [PubMed] [Google Scholar]
  66. Rigamonte, T.A. , Pylro, V.S. & Duarte, G.F. (2010) The role of mycorrhization helper bacteria in the establishment and action of ectomycorrhizae associations. Brazilian Journal of Microbiology, 41, 832–840. Available from: 10.1590/S1517-83822010000400002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Saltarelli, R. , Ceccaroli, P. , Polidori, E. , Citterio, B. , Vallorani, L. & Stocchi, V. (2003) A high concentration of glucose inhibits Tuber borchii mycelium growth: a biochemical investigation. Mycological Research, 107, 72–76. Available from: 10.1017/S0953756202007062 [DOI] [PubMed] [Google Scholar]
  68. Sanders, E.R. (2012) Aseptic laboratory techniques: plating methods. Journal of Visualized Experiments, 63, e3064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Sbrana, C. , Agnolucci, M. , Bedini, S. , Lepera, A. , Toffanin, A. , Giovannetti, M. et al. (2002) Diversity of culturable bacterial populations associated to Tuber borchii ectomycorrhizas and their activity on T. borchii mycelial growth. FEMS Microbiology Letters, 211, 195–201. Available from: 10.1111/j.1574-6968.2002.tb11224.x [DOI] [PubMed] [Google Scholar]
  70. Seneviratne, G. & Jayasinghearachchi, H.S. (2003) Mycelial colonization by bradyrhizobia and azorhizobia. Journal of Biosciences, 28, 243–247. Available from: 10.1007/BF02706224 [DOI] [PubMed] [Google Scholar]
  71. Sillo, F. , Vergine, M. , Luvisi, A. , Calvo, A. , Petruzzelli, G. , Balestrini, R. et al. (2022) Bacterial communities in the fruiting bodies and background soils of the white truffle Tuber magnatum . Frontiers in Microbiology, 13(864), 434. Available from: 10.3389/fmicb.2022.864434 [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Silvestro, D. & Michalak, I. (2012) raxmlGUI: a graphical front‐end for RAxML. Organisms Diversity & Evolution, 12, 335–337. Available from: 10.1007/s13127-011-0056-0 [DOI] [Google Scholar]
  73. Sinclair, C.G. & Cantero, D. (1989) Fermentation modelling. In: McNeil, B.L. & Harvey, M. (Eds.) Fermentation: a practical approach. New York: IRL Press, pp. 65–112. [Google Scholar]
  74. Sisti, D. , Giomaro, G. , Rossi, I. , Ceccaroli, P. , Citterio, B. , Stocchi, V. et al. (1997) In vitro mycorrhizal synthesis of micropropagated Tilia platyphyllos Scop. plantlets with Tuber borchii Vittad. mycelium in pure culture. Acta Horticulturae, 457, 379–388. Available from: 10.17660/ActaHortic.1998.457.47 [DOI] [Google Scholar]
  75. Söderström, B. , Finlay, R.D. & Read, D.J. (1988) The structure and function of the vegetative mycelium of ectomycorrhizal plants IV. Qualitative analysis of carbohydrate contents of mycelium interconnecting host plants. The New Phytologist, 109, 163–166. Available from: 10.1111/j.1469-8137.1988.tb03704.x [DOI] [PubMed] [Google Scholar]
  76. Tamura, K. , Stecher, G. & Kumar, S. (2021) MEGA11: molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution, 38, 3022–3027. Available from: 10.1093/molbev/msab120 [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Trinci, A.P.J. (1974) A study of the kinetics of hyphal extension and branch initiation of fungal mycelia. Microbiology, 81, 225–236. Available from: 10.1099/00221287-81-1-225 [DOI] [PubMed] [Google Scholar]
  78. Tryfinopoulou, P. , Chourdaki, A. , Nychas, G.‐J.E. & Panagou, E.Z. (2020) Competitive yeast action against Aspergillus carbonarius growth and ochratoxin a production. International Journal of Food Microbiology, 317, 108460. Available from: 10.1016/j.ijfoodmicro.2019.108460 [DOI] [PubMed] [Google Scholar]
  79. Vahdatzadeh, M. , Deveau, A. & Splivallo, R. (2015) The role of the microbiome of truffles in aroma formation: a meta‐analysis approach. Applied and Environmental Microbiology, 81, 6946–6952. Available from: 10.1128/AEM.01098-15 [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Vahdatzadeh, M. & Splivallo, R. (2018) Improving truffle mycelium flavour through strain selection targeting volatiles of the Ehrlich pathway. Scientific Reports, 8, 9304. Available from: 10.1038/s41598-018-27620-w [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Vincent, J.M. (1970) A manual for the practical study of the root‐nodule bacteria (IBP handbook no. 15). London, UK: International Biology Program. [Google Scholar]
  82. Willems, A. , Coopman, R. & Gillis, M. (2001) Phylogenetic and DNA–DNA hybridization analyses of Bradyrhizobium species. International Journal of Systematic and Evolutionary Microbiology, 51, 111–117. Available from: 10.1099/00207713-51-1-111 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1. Supporting Information.

EMI4-16-e13271-s001.docx (3.9MB, docx)

Data Availability Statement

All the sequences were deposited in GenBank database. All the other data are reported in the text and /or in supporting information.

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