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The overall magnitude of changes in SOC, TN, and C:N in response to chemical nitrogen fertilizers reductionThe results showed that chemical nitrogen fertilizers reduction significantly decreased SOC and TN by 2.76% and 4.19% respectively, while increased C:N by 6.11% across all database (Fig. 1). SOC mainly derives from crop residues and secretions which closely related to crops growths, and crops growths were affected by fertilization, especially nitrogen fertilization20,21. The reduction of chemical nitrogen fertilizer led to poor crop growth, which reduced the amount of crop residues return, and then decreased SOC. Similarly, TN from crops was reduced due to poor crop growth. In addition, the reduction of chemical nitrogen fertilizers directly reduced the input of soil nitrogen. The increase of C:N was the result of the decrease of TN being greater than that of SOC. The responses of C:N to chemical nitrogen fertilizers reduction enhanced the comprehension of the couple relationship between SOC and TN, which was beneficial to the evolution of the C-N coupling models. Moreover, the accuracy of C-N coupling models depends on the precise quantification of the responses of SOC and TN to nitrogen fertilization. And our results accurately quantified the difference responses of SOC and TN to different nitrogen fertilization regimes, thus optimizing the C-N coupling models.Figure 1The weighted response ratio (RR++) for the responses to chemical nitrogen fertilizers of soil organic carbon (SOC, a), total nitrogen (TN, b), and their ratios (C:N, c). Bars denote the overall mean response ratio RR++ and 95% confidence intervals (CI). The star (*) indicates significance when the 95% CI that do not go across the zero line. The vertical lines are drawn at lnRR = 0. The value represents independent sample size.Full size imageResponses of SOC, TN and C:N to chemical nitrogen fertilizers reduction magnitudeWhen grouped by chemical nitrogen fertilizers reduction magnitude, SOC showed a significant increase by 6.9% in medium magnitude, while SOC was significantly decreased by 3.10% and 7.26% in high and total magnitude respectively (Fig. 1a). Liu and Greaver22 also stated the reduction of medium nitrogen fertilizer increased the average microbial biomass from 15 to 20%, thereby increasing the SOC content. Previous studies had reported that there were strong positive correlations between soil organic matter and soil microbial biomass in both the agricultural ecosystem and natural ecosystem23,24. Numerous researchers have demonstrated the significance of nitrogen availability in soil for the plant biomass across most ecosystems25,26. Moreover, nitrogen deficient would inhibit the activity of extracellular enzymes and root activities27. Generally, soil degradation caused by continuous rising chemical nitrogen fertilizers application may inhibit the growth of crops and ultimately reduce the SOC28.TN showed no significant difference in low and medium chemical nitrogen fertilizers reduction magnitude (p  > 0.05), while TN in high magnitude and total chemical nitrogen fertilizers reduction magnitude exhibited a decrease with 3.10% and 9.37% respectively (Fig. 1b). Numerous studies described that the amount of nitrogen fertilizers used in China was higher than the demand of N for crop, which caused serious N leaching and runoff29,30. Chemical nitrogen fertilizers in low and medium magnitude would not decrease the TN of soil by reducing N leaching and runoff. However, the residual nitrogen in soil cannot meet the requirement for the sustainable growth of plant with litter or without exogenous nitrogen supplement, which resulted in the decrease of TN in high and total chemical nitrogen fertilizers magnitude. Consequently, optimal nitrogen fertilizers application rates will take into account crops yield and environment friendliness.Additionally, C:N had a significant increase with ranging from 1.82% to 8.98% under the four chemical nitrogen fertilizers reduction magnitude (Fig. 1c), suggesting the relative increase of SOC compared to TN. Previous studies have revealed that C:N had significantly influence on the soil bacterial community structures31. And there were also considerable studies indicated that chemical nitrogen fertilizers have impact on the soil bacterial communities32,33. We may speculate that the change of C:N would bring about the variations of soil bacteria communities under the chemical nitrogen fertilizers regimes.Responses of SOC, TN, and C:N to chemical nitrogen fertilizers reduction durationNegative response of SOC to short-term chemical nitrogen fertilizers reduction was observed in our study, which was consistent with the study of Gong, et al.34 that chemical nitrogen fertilizers reduction decreased SOC by reducing crop-derived carbon by one year. However, SOC was significantly increased by 1.06% and 4.65% at mid-term and long-term chemical nitrogen fertilizers reduction respectively, which was similar with the findings of Ning, et al.11 that SOC was significantly increased under more than 5 years of chemical nitrogen fertilizers reduction treatment. TN was significantly decreased by 1.96% at short-term chemical nitrogen fertilizers reduction duration, while the results converted at mid-term chemical nitrogen fertilizers reduction duration. The effect of long-term chemical nitrogen fertilizers reduction on TN was not significant (p  > 0.05). The divergent response of TN to different chemical nitrogen fertilizers duration was mainly caused by the various treatments. In terms of C:N, a greater positive response was observed at short-term chemical nitrogen fertilizers duration (9.06%) than mid-term and long-term duration (1.99%). Moreover, with the prolongation of the chemical reduction time of nitrogen, the response ratio tends to zero, suggesting that the effect of chemical fertilizers gradually decrease. This may be ascribed to the buffer capacity of soil to resist the changes from external environment, including nutrients, pollutants, and redox substances35.Responses of SOC, TN, and C:N to different chemical nitrogen fertilizers reduction patternsUnder the pattern of chemical nitrogen fertilizers reduction without organic fertilizers supplement, SOC and TN significantly decreased by 3.83% and 11.46% respectively, however, chemical nitrogen fertilizers reduction with organic fertilizers supplement significantly increased SOC and TN by 4.92% and 8.33% respectively. Moreover, C:N significantly increased under the two chemical nitrogen fertilizers patterns (p  0.05), but there was a negative effect on SOC in high and total magnitude (p  0.05). The no significant decrease at mid-term duration might result from the limited information reported in original studies of this meta-analysis36. TN showed no significant response to chemical nitrogen fertilizers without organic fertilizers supplement in the low and medium magnitude (p  > 0.05). However, TN was significantly decreased by 8.62% and 16.7% respectively in the high and total magnitude. When regarding to chemical nitrogen fertilizers reduction duration, TN was significantly reduced at all of the categories, ranging from 3.13% to 13.4% (Fig. 2c). In the pattern of chemical nitrogen fertilizers reduction with organic fertilizers supplement, chemical nitrogen fertilizers reduction at medium, high, and total magnitudes significantly increased SOC by 13.85%, 13.03%, and 5.46%respectively, however, the response of SOC in the low chemical nitrogen fertilizers magnitude was not significant. Chemical nitrogen fertilizers reduction duration significantly increased SOC by 7.01%, 1.71%, and 22.02% in the short-term, mid-term, and long-term respectively. Comparatively, TN showed a significantly increase in most chemical nitrogen fertilizers categories expect for the long-term chemical nitrogen fertilizers duration, with an increasing from 4.90% to 14.69% (Fig. 2d).Figure 2The weighted response ratio (RR++) for the responses to chemical nitrogen fertilizers of soil organic carbon (SOC, a), total nitrogen (TN, b), and their ratios (C:N, c) under the two patterns (with organic fertilizers ; without organic fertilizers). Bars denote the overall mean response ratio RR++ and 95% confidence intervals (CI). The star (*) indicates significance when the 95% CI that do not go across the zero line. The vertical lines are drawn at lnRR = 0. The values represent independent sample size.Full size imageOrganic fertilizers were mainly derived from animal manure or crops straws, which contained large amount of organic matter and nitrogen elements37,38. The application of organic fertilizers increased the input of SOC and TN directly. Moreover, organic fertilizer could promote the growth of crops by releasing phenols, vitamins, enzymes, auxins and other substances during the decomposition process, thus the SOC derived from crops would be increased37,39. In addition, organic fertilizers provide various nutrients for microbial reproduction, which increase the microbial population and organic carbon and total nitrogen content37. More importantly, the application of organic fertilizers could improve organic carbon sequestration and maintain its stability in aggregates, thereby reducing losses of SOC and TN40.C:N showed an increase under all of the chemical nitrogen fertilizers reduction with organic fertilizer supplement. The positive response of C:N to organic fertilizer supplement may be related to the higher C:N of organic fertilizer than soil. The average values of C:N of the commonly used organic fertilizers including animal manure, crop straws and biochar were 14, 60 and 30 respectively, while the C:N of soil was lower than 10 in average according to extensive literature researches41. Therefore, organic fertilizers would be a favorable alternative of chemical fertilizers for the sustainable development of agriculture.The correlation between the response of SOC, TN, and C:N and environmental variablesThe analysis of linear regression was conducted to analyze the environmental variables including mean annual temperature (MAT), mean annual precipitation (MAP), accumulated temperature above 10 °C (MATA), which may exert influence on SOC, TN and C:N. No significant correlation among the lnRR of SOC, TN, C:N and environmental variables were observed among the whole database (p  > 0.05; Fig. S1). Rule out the interference of organic fertilizers supplement, we analyzed the relationship between lnRR of SOC, TN, C:N and environmental variables as the Figures showed in Figs. 3 and 4 respectively. Under chemical nitrogen fertilizers without organic fertilizers supplement, there was a significant negative correlation between lnRR of SOC and MAT (p  More

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ChemicalsErbium (III) oxide nanoparticles (Er2O3-NPs) were purchased from Sigma-Aldrich Chemical Company (Saint Louis, USA) with pink appearance and product number (203,238). Powders of Er2O3-NPs with 99.9 trace metals basis were suspended in deionized distilled water to prepare the required concentrations and ultra-sonicated prior use.Cell lineHuman hepatocellular carcinoma (Hep-G2) cells were obtained from Nawah Scientific Inc., (Mokatam, Cairo Egypt). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) media supplemented with streptomycin (100 mg/mL), penicillin (100 units/mL) and heat-inactivated fetal bovine serum (10) in humidified, 5% (v/v) CO2 atmosphere at 37 °C.Characterization of Er2O3-NPsThe purchased powders of Er2O3-NPs were characterized using a charge coupled device diffractometer (XPERT-PRO, PANalytical, Netherlands) to determine its X-ray diffraction (XRD) pattern. Zeta potential and particles’ size distribution of Er2O3-NPs were also detected using Malvern Instrument Zeta sizer Nano Series (Malvern Instruments, Westborough, MA) equipped with a He–Ne laser (λ = 633 nm, max 5mW). Moreover, transmission electron microscopy (TEM) imaging was done to detect the shape and average particles’ size of Er2O3-NPs suspension.Sulforhodamine B (SRB) cytotoxicity assaySulforhodamine B (SRB) assay was conducted to assess the influence of Er2O3-NPs on the proliferation of cancerous Hep-G2 cells12. Aliquots of 100 µl of Hep-G2 cells suspension containing 5 × 103 cells were separately cultured in 96-well plates and incubated for 24 h in complete media. Hep-G2 Cells were then treated with five different concentrations of Er2O3-NPs (0.01, 0.1, 1, 10 and 100 µg/ml) incubated for 24 h or (0.1, 1, 10, 100 and 1000 µg/ml) incubated for 72 h. After 24 or 72 h of Er2O3-NPs exposure, cultured cells were fixed by replacing media with 10% trichloroacetic acid (TCA) and incubated for one hour at 4 °C. Cells were then washed five times with distilled water, SRB solution (0.4% w/v) was added and incubated cells in a dark place at room temperature for 10 min. All plates were washed three times with 1% acetic acid and allowed to air-dry overnight. Then, protein-bound SRB stain was dissolved by adding TRIS (10 mM) and the absorbance was measured at 540 nm using a BMG LABTECH-FLUO star Omega microplate reader (Ortenberg, Germany).Cells treatmentCancerous Hep-G2 cells were cultured at the appropriate conditions and dived into control and treated cells. The control cells were treated with an equal volume of the vehicle (DMSO; final concentration, ≤ 0.1%), while the treated cells were treated with the IC50 of Er2O3-NPs. All cells were left for 72 h after nanoparticles treatment and were harvested by brief trypsinization and centrifugation. Each treatment was conducted in triplicate. Cells were washed twice with ice-cold PBS and used for different molecular assays.Estimation of genomic DNA integrityThe impact of Er2O3-NPs exposure on the integrity of genomic DNA in cancerous Hep-G2 cells was estimated using alkaline Comet assay13,14. Treated and control cells were mixed with low melting agarose and spread on clean slides pre-coated with normal melting agarose. After drying, slides were incubated in cold lysis buffer for 24 h in dark and then electrophoresed in alkaline electrophoresis buffer. Electrophoresed DNA was neutralized in Tris buffer and fixed in cold absolute ethanol. For analysis slides were stained with ethidium bromide, examined using epi-fluorescent microscope at magnification 200× and fifty comet nuclei were analyzed per sample using Comet Score software.Estimation of intracellular ROS generationThe effect of Er2O3-NPs exposure on intracellular ROS production in cancer Hep-G2 cells was studied using 2,7-dichlorofluorescein diacetate dye15. Cultured cells were washed with phosphate buffered saline (PBS) and then 2,7-dichlorofluorescein diacetate dye was added. Mixed cells and dye were left for 30 min in dark and spread on clean slides. The resultant fluorescent dichlorofluorescein complex from interaction of intracellular ROS with dichlorofluorescein diacetate dye was examined under epi-fluorescent at 20× magnification.Measuring the expression levels of apoptotic and anti-apoptotic genesQuantitative real time Polymerase chain reaction (RT-PCR) was conducted to measure the mRNA expression levels of apoptotic (p53 and Bax) and anti-apoptotic (Bcl2) genes in control and treated Hep-G2 cells. Whole cellular RNA was extracted according to the instructions listed by the GeneJET RNA Purification Kit (Thermo scientific, USA) (Thermo scientific, USA) and using Nanodrop device purity and concentration of the extracted RNAs were determined. These RNAs were then reverse transcribed into complementary DNA (cDNA) using the instructions of the Revert Aid First Strand cDNA Synthesis Kit (Thermo scientific, USA). For amplification, RT-PCR was performed using the previously designed primers shown in Table 116,17 by the 7500 Fast system (Applied Biosystem 7500, Clinilab, Egypt). A comparative Ct (DDCt) method was conducted to measure the expression levels of amplified genes and GAPDH gene was used as a housekeeping gene. Results were expressed as mean ± S.D.Table 1 Sequences of the used primers in qRT-PCR.Full size tableAnalysis of cell cycle distributionDistribution of cell cycle was analyzed using flow cytometry. Control and treated cancer Hep-G2 cells with IC50 of Er2O3-NPs for 72 h were harvested, washed with PBS and re-suspended in 1 mL of PBS containing RNAase A (50 µg/mL) and propidium iodide (10 µg/mL) (PI). Cells were incubated for 20 min in dark at 37 C and analyzed for DNA contents using FL2 (λex/em 535/617 nm) signal detector (ACEA Novocyte flow cytometer, ACEA Biosciences Inc., San Diego, CA, USA). For each sample, 12,000 events are acquired and cell cycle distribution is calculated using ACEA NovoExpress software (ACEA Biosciences Inc., San Diego, CA, USA).Estimation of apoptosis inductionApoptotic and necrotic cell populations were determined using Annexin V- Fluorescein isothiocyanate (FITC) apoptosis detection kit (Abcam Inc., Cambridge Science Park Cambridge, UK) coupled with two fluorescent channels flow cytometry. After treatment with Er2O3-NPs for 72 h and doxorubicin as a positive control, Hep-G2 cells were collected by trypsinization and washed twice with ice-cold PBS (pH 7.4). Harvested cells are incubated in dark with Annexin V-FITC/ propidium iodide (PI) solution for 30 min at room temperature, then injected via ACEA Novocyte flowcytometer (ACEA Biosciences Inc., San Diego, CA, USA) and analyzed for FITC and PI fluorescent signals using FL1 and FL2 signal detector, respectively (λex/em 488/530 nm for FITC and λex/em 535/617 nm for PI). For each sample, 12,000 events were acquired and positive FITC and/or PI cells are quantified by quadrant analysis and calculated using ACEA NovoExpress software (ACEA Biosciences Inc., San Diego, CA, USA).Statistical analysisResults of the current study are expressed as mean ± Standard Deviation (S.D) and were analyzed using the Statistical Package for the Social Sciences (SPSS) (version 20) at the significance level p  More

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Dataset 1: Analysis of truffle growers archivesWe selected eleven T. melanosporum orchards located across the South-West France, from Montpellier (43°44′01.4″N 3°42′13.2″E) to Jonzac (45°27′17.7″N, 0°25′26.9″W; Fig. 2). These sites were selected for (1) the quality of the records of fruitbody production and practices by truffle growers (Table S1), including the detail of inoculations since plantation (amount and frequency of added crushed sporocarps), (2) the use of truffle traps by the owners and the quality of the record from these devices, and (3) the presence of oaks (Quercus ilex, Q. pubescens and Q. suber) as the only hosts tree species. Based on the archives of truffle growers, including a systematic recording of truffle production within and outside traps, we reported at each study site the contribution of truffle traps to the annual fruitbody production of the entire truffle grounds, by using number and/or weight of collected fruitbodies within (Pin) and outside (Pout) truffle traps.Dataset 2: In situ experiment tracing the inoculation effectThree orchards located near Angoulème (45°74′35.5″N, − 0°63′78.4″W), Jonzac (45°44′09.8″N, 0°43′96.7″W), and Arles-sur-Tech (42°45′44.9″N, 2°62′89.4″W), hereafter referred to Site 1 to 3 (Fig. 2) were selected for testing both disturbance effect and inoculum effect on fruitbody production in truffle traps. These sites presented a high fruitbody production and a high Pin/Pout ratio, thus optimum conditions to test mechanisms underlying how truffle traps influence fruitbody production. Host trees were between 5 and 18 years old at the beginning of the experiment (Fig. 2). At each site, we selected three non-adjacent trees (four on Site 3) that displayed a continuous fruitbody production over the three previous years. Under each selected tree, we excavated, at two-thirds of the distance between the tree trunk and the limit of brûlé (a vegetation-poor zone that shows the extension mycelia in the soil40, eight equidistant truffle traps [20 × 20 cm large × 20 cm deep] as shown in Fig. 3a. Under each tree, two traps were filled with only a mixture of peat and vermiculite (hereafter referred as non-inoculated controls) to test for disturbance effect. The used mixture was identical to that which is currently applied in commercial orchards. In three other traps, 5 g of crushed material from a single black truffle fruitbody (including its gleba and spores) were added to the previous mixture (hereafter referred as one mating-type inoculum). In the three last traps, 5 g of crushed material from two ascocarps with gleba of opposite mating types (hereafter referred as two mating-type inoculum) were added to the previous mixture. We added the two mating-type condition to accurately test a potential contribution of the gleba (haploid and thus with a single mating type) on future production. As quoted in Introduction, maternal individuals with opposite mating types tend to exclude each other locally (spatial segregation of clusters of individuals of same mating types26. Thus, the two mating-type inoculum allows us to detect in each trap a maternal contribution by the introduced gleba, despite potential exclusion by pre-installed individuals of the locally dominant mating type in the surrounding. Moreover, it allows us to detect a paternal contribution by the introduced gleba of the mating type opposite to the locally dominant. The eight truffle traps were randomly arranged, so that two repetitions of same modality were always separated by a repetition of another modality (Fig. 3a).In March 2013, six freshly collected truffles (weighting  > 60 g) were molecularly analyzed for the mating type of their gleba as in18. On Site 1 and Site 2, the inoculum was made of fruitbodies collected at Site 1. On Site 3, fruitbodies used as inoculum originated from truffle grounds in Sarrion (Spain). In April 2013, truffles traps were installed as explained above (in all, 8 traps × 3 (or 4) trees × 3 sites) and monitored for two years by truffle growers. Harvesting was performed by trained dogs (one different dog per site) checking truffle traps and the surrounding brûlés at each visit of the orchard by truffle growers. When dogs detected truffles, a small hole was excavated to collect ascocarps without disturbing the trap further. At the end of January, 2015, all truffle traps were completely excavated, remnant truffles overlooked by dogs were systematically collected (Fig. 3b). Three soil aliquots were collected within all traps and pooled. All truffles and soil aliquots were frozen for subsequent DNA analysis.Molecular and genetic analysesDNA extractions, mating typing and genotyping were done as in18. Briefly, DNA was extracted from the gleba and from spores of each fruitbody to get access to the maternal and zygotic DNA, respectively. Simple sequence repeat (SSRs) genotyping was performed using 12 polymorphic markers and the mating-type locus as in18. Gleba extracts displaying apparent heterozygous genotypes, likely due to contamination by spore DNA were systematically discarded from further analyses. For each fruitbody, the haploid paternal genotype was then deduced by subtracting the haploid maternal genotype from the zygotic diploid genotype. This data set was used for relatedness estimations. We discarded from all further analysis the marker me11, which displayed more than 39% missing data, as well as all samples with missing data for at any locus.Multilocus genotypes comparisonsBased on the 11 remaining SSRs and the mating-type (Table S5 and Figure S2), MLGs were identified on all maternal and paternal haploid genomes using GenClone v.2.041, and the probability that MLGs represented more than once resulted from independent events of sexual reproduction was calculated (PSex41,42). On each site, clonal diversity was measured as R = (G − 1)/(N − 1) according to43, where N is the number of fruitbodies and G the number of MLGs. For testing whether the gleba of the inoculated fruitbody contributed, either paternally (H1) or maternally (H2) to the harvested fruitbodies (Fig. 1c), the inoculated maternal MLG was compared to the paternal and maternal MLG of the harvested fruitbodies.Relatedness estimationFor testing whether the spores of the inoculum, which carry many distinct haploid MLGs due to meiosis, had paternal or maternal contribution(s) to the harvested fruitbodies (H3; Fig. 1c), we used relatedness estimation.For testing whether spores of the inoculum had a paternal contribution, an individual relatedness estimate to the spore inoculum was computed for each paternal genome detected in truffle traps. Relatedness r here describes the expected frequency E[p_offpat] of each allele in a given genome, E[p_offpat] = p_pop + r * (p_inoc − p_pop), where p_pop is the allele frequency in the local population (here estimated from the glebas of other truffles collected under the focal tree), and p_inoc is the frequency of the allele in the inoculum. Thus, p_offpat takes values 0 or 1, and p_inoc takes values 0, 0.5 or 1, except when two fruitbodies were used as inoculum (two gleba mating types traps). Thus r = (p_offpat − p_pop)/(p_inoc − p_pop). An individual relatedness estimate for each genome is then obtained by summing over alleles and loci the observed values of the numerator and denominator in this expression. A population-level estimate is further obtained by summing numerators and denominators over the paternity events in each population.To test whether such estimates are compatible with the hypothesis that the paternal individuals are not from the inocula, we obtained the distribution of population-level relatedness estimates by simulating samples under this hypothesis: paternal genotypes were randomly simulated according to alleles frequencies in the local population. For each population, 10,000 samples were simulated, and p-values were estimated as the proportion of simulations with higher population-level relatedness with inocula than the observed one. Confidence intervals for these p-values were computed from the binomial distribution for 10,000 draws, and Bonferroni-corrected over the three populations.For testing whether spores of the inoculum had a maternal contribution (H4, Fig. 1c), we estimated the relatedness of the locally used spore inoculum to each maternal genome detected in truffle traps (deduced from the gleba), and we confronted it to simulated samples as previously but with one modification: if the focal fruitbody was harvested in a trap inoculated with the inoculum A1, all genomes of truffles from traps inoculated with the same inoculum (A1 or A1 + A2 + A3, see Fig. 3c.) were discarded from the estimation of p_pop.Assessment of T. melanosporum mycelium concentration in truffle trapsOn Sites 1, 2 and 3, soil samples were collected in all traps and in the surrounding brûlés at harvesting date (January, 2015). In collected soils, total DNA was extracted and quantified as in19. Briefly, after sieving and homogenizing soil collected in each trap and from out of the brûlés, aliquots (10 g) were analyzed as follows. After extraction with the kit Power Soil (MoBio Laboratories, Carlsbad, CA, USA), the extra-radical mycelium of T. melanosporum was quantified using quantitative Taqman™ PCR (qPCR) with the primers and probe described in44. Triplicate real-time PCR were performed on each sample using the same concentration of primer and the same thermocycling program as in19. Standards were prepared using fresh immature T. melanosporum ascocarp, and a standard curve was generated for each site by plotting serial tenfold dilutions against corresponding initial amount of ascocarp. Absolute quantification of mycelium biomass of T. melanosporum was expressed in mg of mycelium per g of soil.Statistical analysesStatistics were done using R version 4.0.445.Effect of truffle traps on fruitbody production—The contribution of truffle traps to the overall production of orchards was assessed by (1) data mining of truffle growers’ archives (Dataset 1) and (2) comparing the density of truffles harvested in traps (expressed in number of truffles per m2 per orchard; for each sampled tree, traps correspond to an investigated soil surface of s = 8 × 0.2 x 0.2 = 0.32 m2) with the density measured within surrounding brûlés (Dataset 1). On Dataset2, at each site, the area occupied by brûlés was evaluated by measuring in the field the surface of soil devoid of vegetation consecutively to spontaneous T. melanosporum brûlé.Fruitbody production under different conditions (i.e. non-inoculated controls versus one gleba mating type traps versus two gleba mating type traps) were compared using generalized linear mixed models with negative binominal family and log link (R, spam package46). The full model included the logarithm of the sampled area as offset to account for variations in this sampled area, interactions of trap-modality effects with site effect. Formal likelihood ratio tests are based on one-step deletions from this full model, applied to subsets of the data relevant for each hypothesis tested. Additional bootstrap tests (1000 iterations) were run to correct any bias in small sample likelihood ratio tests.Concentrations of T. melanosporum mycelium in soil—Similarly as above, the inoculum effect on mycelium concentrations was compared using generalized linear mixed models with Gamma log family.Plant materialThe use of plants in the present study complies with international, national and/or institutional guidelines. All permissions to collect T. melanosporum fruitbodies in truffle orchards were obtained. The formal identification of biological material used in the study (T. melanosporum fruitbodies) was undertaken by F. Richard and E. Taschen. Voucher specimens of all collected fruitbodies have been deposited in the Centre d’Ecologie Fonctionnelle et Evolutive herbarium in Montpellier (France).Ethical approvalAll co-authors approve the ethical statement regarding the submitted manuscript.Consent to participateAll co-authors consent to participate to the research and agree with the content of the submitted manuscript. All authors reviewed and submitted manuscript. More