Applied and Environmental Microbiology, June 2004, p . 3600-3608, Vol . 70, No . 6

Vertical Transmission of Endobacteria in the Arbuscular Mycorrhizal Fungus Gigaspora margarita through Generation of Vegetative Spores

V . Bianciotto,^1, A . Genre,^1, P . Jargeat,² E . Lumini,¹ G . Bécard,² and P . Bonfante^1*

Dipartimento di Biologia Vegetale dell'Università and Istituto per la Protezione delle Piante, CNR, 10125 Turin, Italy,¹ UMR 5546 CNRS/University Paul Sabatier, 31326 Auzeville, Castanet-Tolosan, France²

Received 8 December 2003/ Accepted 3 March 2004

One of the crucial points of bacterial associations with animal hosts is the transmission mechanism (15): some associations are so intimate that the bacteria are transmitted from mother to offspring through the eggs, in a manner parallel to that of mitochondria . This vertical mode of transmission has important evolutionary consequences (19) and is seen as a key factor for reduction of symbiont virulence . The evolutionary theory predicts that mutualistic symbioses evolve from parasitism, thanks to the reduction of parasite virulence (19) . As a consequence, pathogenic symbionts would tend to be transmitted horizontally, while mutualistic symbionts would be selected for vertical transmission (37) . Mathematical models support the idea that an efficient mutualistic relationship depends on a high vertical transmission rate (the acquisition of the symbiont is assured) (37) . A second interesting point is that the direct transfer of bacterial symbionts from a parent host to its progeny through a unicellular stage (usually the egg cell) causes a reduction in the size of the bacterial population: this "bottleneck" event has important consequences in the ecology of symbiotic bacteria (27) .

Not all mutualisms have evolved toward vertical transmission (17, 18) . In some associations, e.g., legumes and nitrogen-fixing bacteria, the bacteria must repeatedly reenter their host cells . In this case, the microbes are horizontally transmitted thanks to the presence of molecular mechanisms, like the type III secretion system, which assist them in host cell entry (16) . Other mathematical models have well defined the conditions under which mutualistic symbiosis should evolve without vertical transmission (20) .

Since 1990, a bacterial population has been constantly observed in the cytoplasm of the Gigaspora margarita isolate BEG 34, which is routinely obtained under pot culture conditions in our laboratory (30 fungal generations) . The transmission mechanism of these endobacteria is unknown . While in the case of G . pyriforme, cyanobacteria have been demonstrated to cyclically enter bladders through an endocytosis event (31), the fact that G . margarita itself requires an obligate plant association to accomplish its life cycle has so far hampered any experimental investigation . In this study, we report the development of an experimental system that allowed us to demonstrate that Candidatus G . gigasporarum is vertically transmitted through the fungal spore generations (SG0 to SG4) . For this analysis, Ri T-DNA-transformed carrot roots were inoculated with single spores of G . margarita, and the presence of endobacteria was visualized and quantified through several fungal generations by using confocal microscopy and PCR techniques .

Other microorganisms (AM fungal and bacterial isolates) were used as controls in PCR specificity tests for 23S primers (Table 1) .

 
Conditions for G . margarita spore production . (i) Petri dish cultures.
A clone of root-inducing T-DNA-transformed carrot roots, established by Bécard and Fortin (6), was routinely propagated on minimal medium in petri dishes .

For use as the plant partner in interactions with the fungus, roots explants were standardized and prepared as described by Bécard and Piché (7) .

Spores (SG0) of G . margarita BEG 34 Dijon were collected from pot cultures, surface sterilized, stored, and used as a fungal inoculum as previously described (6) .

A single spore of G . margarita per petri dish was used to inoculate the transformed root to initiate mycorrhizal cultures . The dishes were sealed thoroughly with Parafilm to confine the internal atmosphere and incubated in the dark at 26°C . They were stood up vertically so that the germ tubes of G . margarita spores elongated upward and contacted the transformed root as a result of their geotropic mode of growth . In each petri dish, new spores (SG1) were produced within 2 months as a result of mycorrhizal colonization . These spores were used individually for morphological observations, molecular characterization, or to inoculate new root cultures in order to obtain SG2 . Using the same protocol, SG3 and SG4 spores were produced successively .

(ii) Pot cultures.
Parallel experiments were performed by using a multispore inoculum (50 SG0 spores per pot culture) in order to colonize clover seedlings grown in plastic pots in a climatic growth chamber at 21°C, with a photoperiod of 14 h and a daylight intensity of 170 µmol m^–2 s^–1 . After 3 months, new spores (SG1) were produced; they were checked for the presence of bacteria and used to inoculate new clover seedlings in order to obtain further spore generations .

In vitro and pot culture conditions are summarized in Fig . 1 .

 
Molecular analysis . (i) Amplification of ribosomal DNA (rDNA) genes and design of primers for 23S rDNA of Candidatus G . gigasporarum.
The 16S rDNA is highly conserved, and its sequences are used in bacterial taxonomy . Comparative analysis of 16S rDNA is considered the "gold standard" for the phylogenetic affiliations of potentially novel and poorly classified organisms (29) . The 23S rDNA molecule, which is twice the size of 16S rDNA and contains regions that are more variable, has an even greater overall phylogenetic information content . For this reason, many authors have chosen it as an alternative target to develop specific probes and to type closely related strains (34, 36) . In the present study, a partial 23S rRNA sequence of the Candidatus G . gigasporarum of G . margarita BEG34 was obtained and screened for a signature specific to this endobacterium .

Genomic DNA from 50 SG0 spores of G . margarita BEG34 was extracted following the protocol described by Lanfranco et al . (23) . An 1,030-bp portion of the 23S rRNA gene was amplified by using primers ML1 and ML4 designed by White et al . (35) . The PCR was performed with a GeneAmp PCR System 9700 (Perkin-Elmer Corp., Norwalk, Conn.) under the following conditions: denaturation for 5 min at 94°C and then 40 cycles of 45 s at 94°C, annealing at 55°C for 1 min, and elongation at 72°C for 2 min . The reaction mixture (50 µl) contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.1 mM MgCl₂, 0.01% gelatin, 200 µM deoxynucleoside triphosphates (dNTPs), 1 µM each primer, 1 to 2 µl of the DNA preparation, and 1.5 U of Taq polymerase (Sigma, Milan, Italy) . A direct sequencing approach was used with the same primer pair, ML1-ML4 . The sequence was compared with the complete 23S rDNA sequences available in the EMBL-GenBank database and aligned with members of the ß subdivision of the Proteobacteria . The sequence was then manually searched for regions specific to the endobacteria of G . margarita BEG34 .

Two primers, GlomGIGf (5'-GGGTCCATTGCGGATTACTTC-3') and GlomGIGr (5'-GGGACCAGGACTTCCATCCCCC-3'), were designed to amplify an expected fragment of 565 bp . In particular, the primer GlomGIGr targeted a unique sequence for Candidatus G . gigasporarum . The specificity of the GlomGIGf-GlomGIGr primer pair was checked on DNA extractions from different bacterial strains and AM fungi (Table 1) . No PCR products were obtained from either bacterial strains or fungal isolates of Gigaspora rosea, known to be endobacterium free (10); by contrast, the expected fragment was amplified from two different cultures of the same isolate of G . margarita BEG34 maintained in Turin, Italy, and Dijon, France (see Fig . 3 below) .

 
(ii) Identification of Candidatus G . gigasporarum through spore generations.
To monitor the fate of Candidatus G . gigasporarum through spore generations, the specific primer sets GlomGIGf-GlomGIGr for 23S rDNA (this study) and BLOf-BLOr for 16S rDNA (9) were used in PCR assays .

Spore samples of each generation (SG0, SG1, SG2, SG3, and SG4) produced in petri dishes and in pot cultures were used (Table 2) . Extreme care was taken to avoid bacterial contamination, and all steps were carried out in a laminar flow hood (10) . Single-spore DNA extraction was performed by crushing each spore in a 30-µl volume containing 100 mM Tris-HCl, pH 8.3, 500 mM KCl, 11 mM MgCl₂, and 0.1% gelatin . After incubation at 95°C for 15 min, the crude extract was centrifuged at 10,000 x g for 5 min, and the supernatant was stored at –20°C . rDNA was amplified in a 25-µl volume containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.1 mM MgCl₂, 0.01% gelatin, 0.1 mM dNTPs, 0.5 µM each oligonucleotide, 1 to 2 µl of the DNA preparation, and 1 U of Taq polymerase . PCR amplifications were carried out in a GeneAmp PCR System 9700 . The thermocycler program for amplification was as follows: initial denaturation for 3 min at 95°C; 35 cycles of 45 s at 94°C, 1 min at 55°C, and 45 s at 72°C; and a final extension of 7 min at 72°C .

 
The amplification products obtained with both primer pairs on single spores of SG0, SG1, and SG4 were sequenced and aligned with the 16S rDNA sequence X89727 and with the 23S rDNA sequence AJ561042 (see above) . The alignment can be downloaded from http://www.bioveg.unito.it/files/beg34.htm .

(iii) PCR-restriction fragment length polymorphism analysis.
Genomic DNAs were extracted from 5 surface-sterilized SG0 spores and 10 in vitro-produced SG1, SG2, SG3, and SG4 spores by using the WIZARD Genomic DNA purification kit (Promega, Lyon, France) . The SG0 spores were surface sterilized three times with 4% chloramine T for 10 min each time and then rinsed with 0.02% streptomycin and 0.01% gentamicin and stored for 2 days in the antibiotics at 4°C . DNAs extracted from SG0, SG1, SG2, SG3, and SG4 spores were eluted in 20 µl of sterile water, and 2 µl was used for PCR amplification .

Bacterial 16S rDNA was amplified in a 25-µl volume containing 0.5 µM primer pair 27f-1495r (9), 2.5 µl of 10x buffer (Promega), 2.5 mM MgCl₂, 250 µM each dNTP, and 1 U of Taq polymerase . The PCR cycling conditions were as follows: denaturation at 93°C for 3 min; 35 cycles of 93°C for 30 s, 55°C for 1 min, and 72°C for 2 min; and final extension at 72°C for 2 min .

Ten microliters of the PCR products was digested in a 20-µl volume containing 2 µl of 10x buffer (Promega) and 0.5 U of restriction enzyme (SacII or RsaI) at 37°C for 4 h .

In a second set of experiments, restriction analysis was also performed with 16S rDNA amplified from DNA extracted from single spores and digested with AluI, SmaI, and MboI (data not shown) .

Morphological analysis . (i) Confocal microscopy.
G . margarita spores were laid on microscope slides in a drop (20 µl) of Bacteria Counting Kit component A (B-7277; Molecular Probes) diluted 1:1,000 according to the manufacturer's directions . The spores were then crushed with a coverslip, incubated in the dark for at least 5 min, and observed with an Olympus FluoView confocal microscope . Direct observation was performed under the built-in mercury arc fluorescence lamp with an excitation band-pass (BP) filter at 490 nm (blue) and an emission BP filter at 520 nm (green) . For confocal imaging, the 488-nm band of a Kr-Ar laser and a BP 510- to 540-nm emission filter were used .

The same conditions were used for the visualization of endobacteria in the cytoplasm of all the mycelial structures (germinating hyphae, extraradical mycelium, and auxiliary cells) .

(ii) Bacterial-cell quantification and size.
All images were acquired under the following conditions: 40x objective, 100-µm confocal aperture (pinhole), and constant photomultiplier tube settings (voltage, 525; gain, 3; offset, 10) . Grayscale images (12-bit) were acquired at a size of 1,024 by 768 pixels, with a real resolution of 0.4 by 0.4 by 0.8 µm in the x, y, and z axes .

Sample fields were chosen at random from the cytoplasm of the crushed spores, and seven serial optical sections along the z axis were acquired with a 3-µm interval across the cytoplasm thickness . The z-axis step was chosen according to our observations of the apparent bacterial cell size as described below, while the number of optical sections was determined by the slide-to-coverslip space, which was consistently 25 µm . Thanks to the Olympus FluoView software, a number of 100- by 100-µm squares were overlaid, in constant positions, on each optical section, and the bacterial cells contained in each square were counted manually . Each square's number was then summed with the numbers in the corresponding squares of all the optical sections within each z series in order to obtain the total number of bacteria in a volume of 100 by 100 by 21 µm (Fig . 2) .

 
The size of the fluorescence halo corresponding to each bacterial cell was estimated through high-magnification imaging (40x objective; 6x digital zoom) . Endobacterial cells are rod shaped, and their fluorescence halos under the observation conditions described above were 1 by 1 by 2 µm (see Fig . 5E below), with a larger vertical scattering due to the lower instrument resolution along the z axis . Based on these data and on the consideration that bacteria are randomly oriented in the spore cytoplasm, we assumed that a 3-µm interval between serial optical sections would avoid the possibility of counting a single cell twice while assuring that virtually all cells were visualized . Actually, a direct comparison between consecutive optical sections revealed that a few bacteria appeared in both images, probably because of local cytoplasmic movements . Bacteria appearing in the same position in two consecutive images were therefore counted only once, in order to avoid an overestimate of the total number .

 
Prior to quantitative analysis, an internal control of the method was performed by repeatedly counting the number of bacteria in the same volume of cytoplasm . Differences in the total number of bacteria counted were not statistically significant (data not shown) .

Ten spores for each generation produced in petri dishes were stained and analyzed according to the quantification protocol described above . Spores were harvested from four different petri dishes for each generation . Five 100- by 100- by 21-µm volumes were sampled for each spore . The average number of bacteria per sampled volume and the standard deviation were calculated . Then, the density of bacterial cells per cubic millimeter was deduced . Since such numbers were necessarily small decimals, for the sake of easier reading, the data were converted to the number of bacteria per 1,000,000 µm³, i.e., the number of bacterial cells contained in a cube of spore cytoplasm with 100-µm sides .

Differences in the numbers of bacteria among the different spore generations were tested for significance by analysis of variance and the Tukey post hoc test (Systat 10) .

Cultivability of Candidatus G . gigasporarum.
Assays for culturing the bacterium in pure culture were carried out by incubating 10 surface-sterilized crushed fungal SG0 spores on various media and under different conditions . Rich media, such as Luria-Bertani (Bio-Trypticase, 10 g liter^–1; yeast extract, 5 g liter^–1; NaCl, 10 g liter^–1; pH 7), TY (tryptone, 5 g liter^–1; yeast extract, 3 g liter^–1; CaCl₂ · H₂O, 0.87 g liter^–1), and BG (peptone, 10 g liter^–1; Casamino Acids, 1 g liter^–1; yeast extract, 1 g liter^–1; pH 7), were tested . Other media, such as minimal medium (6) and media for Rhizobium (mannitol or trehalose, 10 g liter^–1; yeast extract, 1 g liter^–1; K₂HPO₄, 0.5 g liter^–1; NaCl, 0.2 g liter^–1; MgSO₄, 0.2 g liter^–1) and for Pseudomonas fluorescens (tryptone, 20 g liter^–1; glycerol, 10 ml liter^–1; K₂HPO₄, 1.5 g liter^–1; MgSO₄, 1.5 g liter^–1), were also tested . All of these media were liquid in tubes (with and without agitation) or semisolid in tubes (agar, 2.5 g liter^–1) to submit the bacteria to partially anaerobic conditions or solid on plates (agar, 15 g liter^–1) . Two temperatures were systematically tested: 22 and 28°C . Trehalose was used in the Rhizobium medium as an alternative to mannitol because it stimulated growth of rhizospheric Pseudomonas and ectomycorrhiza helper bacteria (S . Delorme and P . Frey-Klett, personal communications) .

Nucleotide sequence accession number.
The partial 23S rDNA sequence from Candidatus G . gigasporarum has been deposited in the EMBL data library under accession no . AJ561042 .

Transmission of endobacteria . (i) Molecular analysis.
In order to verify the identity of endobacteria contained in all spore generations, PCR experiments with specific primers for 16S and 23S rDNAs were performed on single SG0, SG1, SG2, SG3, and SG4 spores harvested from both pot cultures and petri dishes . Figure 3 shows that fragments of the expected size (565 bp) were obtained from SG0, SG1, SG2, SG3, and SG4 spores that had multiplied in petri dish cultures by using the specific primers GlomGIGf-GlomGIGr (Table 2) . The direct sequencing of the PCR fragments confirmed the presence of Candidatus G . gigasporarum . As in vitro SG4 included spores whose bacterial DNA could not be amplified (with the 16S and 23S rDNA-specific primers [Table 2]), the reliability of DNA extraction was checked . PCR amplifications of SG4 spores that appeared to lack endobacteria were performed with the eukaryotic primers NS1-NS2 (24) . They systematically gave positive responses (data not shown) . Fragments of the expected size were consistently amplified from all spore generations (SG0 to SG4) obtained from pot cultures (Table 2) .

DNA amplification with the nonspecific 16S rDNA bacterial primers 27f and 1495r from the five spore generations (SG0, SG1, SG2, SG3, and SG4) consistently produced a single 1,500-bp fragment (data not shown) . Restriction analyses performed with a total of five restriction enzymes led to identical profiles across the spore generations, confirming the presence of the same bacterial population throughout the fungal generations . The sizes of the digested DNA fragments after in silico analysis correctly matched the profiles expected for the Candidatus G . gigasporarum 16S rDNA (X89727): three fragments of 840, 530, and 150 bp with SacII and four fragments of 900, 350, 150, and 130 bp for RsaI (Fig . 4) . Similar results were obtained with the restriction enzymes AluI, SmaI, and MboI, showing no difference between SG0 spores and the four in vitro-produced spore generations (data not shown) .

 
The results show that, at least in the four spore generations investigated here, only one bacterial population is present, and it corresponds to Candidatus G . gigasporarum . On this basis, the morphological analysis was performed with a universal bacterial fluorescent dye, with the advantage of a rapid, routinely usable, and highly efficient staining method .

(ii) Morphological analysis.
Endobacterial cells were consistently observed in all spore generations produced in both petri dish and pot cultures (Table 2) using the Bacteria Counting kit . In monospore-inoculated petri dish cultures, endobacteria were observed throughout all the mycelial structures produced during root colonization and spore production, i.e., germination hyphae, extraradical mycelium, auxiliary cells, and new spores (Fig . 5A to D) . Apparently dividing bacterial cells were observed in all these structures, and more frequently in germinating spores (Fig . 5E) . The detection of bacteria inside intraradical hyphae was hampered by the plant tissues, but in that case, their presence was demonstrated in ultrathin sections by electron microscopy observations (9) .

The regular observation of Candidatus G . gigasporarum in the cytoplasm of spores generated by a single starting spore in axenic petri dishes demonstrates the vertical transmission of the endobacteria without the participation of any external element (other spores or extracellular bacterial populations living in the soil) .

Quite interestingly, in a certain number of SG4 spores, endobacterial cells could not be observed . These data confirmed the results obtained by PCR amplifications in which bacterial DNA was not amplified from some of the SG4 spores (Fig . 3 and Table 2) . The total number of spores used in morphological and molecular analyses and the resulting observations confirming the presence of the endobacterial population are reported in Table 2 .

Bacterial-cell quantification.
The repeated observation of spore cytoplasm in successive generations revealed that the spores produced by monospore-inoculated petri dish cultures apparently harbored a progressively smaller population of endobacteria (eventually leading to some bacterium-free SG4 spores), while no decrease was evident through the spore generations produced by the multispore-inoculated pot cultures .

In order to better investigate this aspect, a quantification method was set up exploiting the capabilities of confocal microscopy .

Automatic quantification of the bacterial cells (based on particle size and counting algorithms) was discarded, as manual control of overestimates (due to double counting) proved to be necessary .

In addition, counting of bacteria in each optical section rather than in a projected image of the whole volume was introduced in order to allow the discrimination of each bacterium, since in many cases their high density resulted in confluent fluorescent areas in the projected image .

The average numbers of bacteria present in cubes of spore cytoplasm with 100-µm sides, calculated for each spore, are listed in Fig . 6 . From these values, an average bacterial density was calculated for each spore generation (Fig . 6, bottom right) . Though a wide range of variability was observed in these second-level average values, a general decrease occurred from SG0 to SG4 in monospore-inoculated petri dish cultures . The presence of a large ratio of SG4 spores where no endobacterial cells were observed of course lowered the average bacterial density for the generation and increased the standard deviation .

 
Cultivability of Candidatus G . gigasporarum.
No visible bacterial growth was observed in any of the culture media and growth conditions tested, even after 3 weeks of incubation .

Another possibility for bacterial transmission could involve the anastomosis of hyphae in the soil or within a root, even if anastomoses have only been described in Glomus (21) and not in Gigaspora species (G . Bécard, unpublished data) .

Transmission pathway.
Intracellular symbioses raise fascinating questions concerning the acquisition, transmission, and evolution of the endosymbionts (16) . Margulis and Chapman (26) discussed the importance of endosymbiosis as an evolutionary mechanism and distinguish between permanent and cyclical endosymbioses, the former remaining stable over time while the latter involves regular reassociation events . Most of the symbioses found in the plant kingdom are cyclical: each AM colonization event requires a reassociation of the fungal propagule with its host plant . Similar events are well described for rhizobia and other nitrogen-fixing microbes (14), as well as for G . pyriforme, in which cyanobacteria penetrate the fungal structures through an endocytotic process (31) .

In this respect, the permanent nature of the symbiosis between G . margarita and its endobacteria is surprising . However, it was already suggested by previous observations showing that the bacteria found in fungal spores isolated in very distant geographical areas are closely related (10), thus supporting the ancient establishment of the interaction . Bacterial acquisition by AM fungi during evolution would occur as rare events and would be conserved thereafter by strict vertical transmission (permanent symbiosis) . The asexual reproduction typical of AM fungi (22) and the coenocytic nature of the mycelium in Glomeromycota could facilitate this type of transmission .

This mechanism seems, therefore, to be different from that reported by Levy et al . (25), who showed how spores of Gigaspora decipiens might be invaded by some strains of Burkholderia . Their experiments, in fact, do not demonstrate the establishment of a permanent fungal-bacterial interaction but describe the invasion capacity of pathogenic microbes . Intracellular bacteria identified as Paenibacillus have also been detected in the mycelium of an ectomycorrhizal fungus (8) . Their simultaneous presence in the liquid culture of the fungus and inside the mycelium suggests horizontal transfer from the intracellular environment to the external medium or vice versa, as suggested by the authors .

Vertical transmission is, by contrast, very common for endobacteria associated with animal organisms . This mechanism is often described as maternal transmission (27) . However, since no evidence of sexual reproduction in AM fungi has been reported (22) and since our observations refer only to asexual spore production, this terminology was avoided .

In conclusion, to the best of our knowledge, the transmission mechanism of Candidatus G . gigasporarum appears to be unique among prokaryote-fungal associations .

Endobacterial propagation.
Morphological analyses show that spores and auxiliary cells are the preferred sites where bacteria accumulate during the fungal life cycle . They multiply in these structures, as well as in germinating hyphae, and they are also present in the intraradical hyphae (coils and inter- and intracellular hyphae), while they have never been observed in the thin arbuscule branches, probably also due to the smaller hyphal diameter (P . Bonfante, personal communication) .

We can hypothesize that bacteria move along the hyphae pushed by the strong cytoplasmic fluxes, described in Gigaspora as the driving forces moving lipid globules and nuclei across the symbiotic mycelium (1, 33) .

In addition, their accumulation inside the spores may be related to the fungal metabolism; Bago et al . (3) demonstrated the presence of a sugar flow from the intraradical mycelium toward the extraradical phase . Intraradical hyphae take up hexoses, from which they synthesize lipids and glycogen . These two main C and energy sources are supposed to be transferred to the extraradical phase, where they are used as metabolic fuel and stored in the newly formed spores (2, 4) . Spores are indeed known to be rich in lipids and glycogen (13), and recent molecular data have demonstrated that the lipidic stores are the starting point of gluconeogenesis, which turns them into ready-to-use sugars (4) . This makes spores rich sites for nutrients . Whether endobacteria actively move toward the sites where nutrients are more abundant or whether they reach them passively, following the cytoplasmic streams, they are found in an optimal niche for their reproduction in the cytoplasm of auxiliary cells, and particularly of spores .

Multispore versus monospore inocula and pot versus in vitro cultures.
An unexpected result was the finding of a significant difference between the artificial situation (transformed carrot roots-monospore inoculum-axenical conditions) and the more natural situation where spores are produced in pots from a multispore inoculum . In the former system, fewer bacteria were found in spores of each new generation, associated with a wider range of variability, as indicated by the standard deviations . Several SG4 spores were even found with no bacteria left, indicating that vertical transmission, under the experimental conditions used, was not completely effective . In order to discriminate between the effect of multispore inoculation and the effect of pot culture conditions on this variable bacterial decrease, we initiated five in vitro cultures with multispore inocula (10 spores each) (data not shown) . The resulting SG1 spores again showed a decrease in bacterial density (25%), but this decrease was not as strong as for the monospore cultures (83%) and was not statistically significant (Student t test) . This suggests that the key to the decreasing bacterial population is the single-spore inoculum .

Assuming that bacterial numbers in SG0 spores are variable and that endobacteria influence the mycorrhizal phenotype, we can propose that in the case of multispore inoculation, not all spores will be successfully involved in mycorrhization and sporulation . Spores containing the highest numbers of bacteria would achieve more effective colonization and produce new spores also containing high numbers of bacteria . By contrast, when using single-spore inoculation, the initial variability will not be averaged but rather amplified through the generations . Taken together, this would open a new and as-yet-undescribed scenario of competitive interactions among spores before the root colonization process .

P.J . was supported by the European Union GENOMYCA Project (project no . QLK5-CT-2000-01319) (http://www.dijon.inra.fr/bbceipm/genomyca/) . E.L . was supported by the SINAPSI grant SPTO-011/15/07/03/16.55 .

V.B . and A.G . contributed equally to this work .

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