Summary:

In this article (egusphere-2025-2078), the authors set out to investigate the role of black truffle (Tuber melanosporum Vittad.) in shaping the fungal community in the soil ecosystem it grows in. They focused solely on the soil fungal community (not considering prokaryotes) and compared the more "natural" forest soil system against the cultivated plantation system. They also compared samples from spring and autumn to gain insights into seasonal effects on the role of T. melanosporum. Based on previous studies and general knowledge of fungal ecology, the following hypothesis were put forth: 

1. a) soil fungal networks are richer and more complex in forests compared to plantations.

    b) a differential seasonal effect on soil fungal communities can be observed

2. T. melanosporum is strongly connected in the soil fungal network, possibly acting as a hub species

3. a) the prevalent functional fungal guilds differ when comparing forest and plantation systems     

    b) greater prevalence of ectomycorrhizal fungi vs saprotrophs can be observed in forests compared to plantations

To investigate these hypotheses, topsoil (0-20 cm) from inside the brûlé, i.e. presumably area affected by T. melanosporum, was obtained from both systems (four replicates) and in both seasons .The brûlé boundaries were determined visually and multiple samples from within the area were combined to a composite sample, but no negative controls from outside that area were taken. Some sampling sites were paired (forest and plantation in close proximity), while others were not. Fungal occurrence in the samples was determined by metabarcoding. Co-occurrence networks were created based on this and the role of T. melanosporum within these networks was studied. Soil functioning was proxied through the potential activities of eight exoenzymes related to carbon (β-glucosidase, β-cellobiohydrolase, β-xylosidase, β-glucuronidase, and laccase), nitrogen (chitinase and leucine-aminopeptidase), and phosphorus (alkaline phosphatase) cycling. These were measured for the soil samples and the potential role of different fungal guilds in explaining these activities was predicted by modeling.

Forest fungal communities showed significantly greater β-diversity, while α-diversity did not differ significantly between plantation and forest. Based on a single mixed co-occurrence network, OTU links and network complexity appeared significantly higher in plantations compared to the forest system (contrary to expectation from hyp. 1), while no significant difference between seasons was observed. In separately modeled co-occurrence networks for both ecosystems, T. melanosporum was not strongly connected to other OTUs and did not appear to act as a hub species (contrary to hyp. 2). Differing abundance of fungal guilds was observed between both systems and ectomycorrhizal were more prevalent in the forest (fitting hyp. 3). 

Key limitations of the study:

1. No control samples outside the brûlés were taken, meaning there was no true negative control. The authors themselves identify this limitation (l. 382 – 387), but do not sufficiently address it in their analyses. Co-occurrence works by checking shared patterns of presence or absence. Since only samples from truffle-dominated areas were used which would be expected to almost always contain T. melanosporum reads, positive connections would only be expected with other highly abundant taxa, since only these could match the truffles occurrence pattern. Negative connections would also not hold as much informative value in this specific sampling approach, since they would likely mainly depict less abundant / more rare taxa that occur in few samples. One reason for T. melanosporum not showing up as a hub species in the analysis could also be that it already modified the microbiome and reduced the abundance of some other fungi. Without the outside control, we are unable to compare to the “undisturbed” ecosystem without truffle dominance, which really limits what can be deduced about its actual role in the system.

In a similar vein, the negative control would have also allowed researchers to rule out environmental filtering as the main driver for co-occurrence, by depicting the community without truffle but in the exact same soil conditions. Some of the sampling sites appear at least somewhat paired, while others are completely singular, which makes it hard to disentangle the actual effect of T. melanosporum on the local soil microbiome, compared to differences purely based on abiotic factors. This issue could maybe be circumvented by comparing matched sampling sites. While there can be merit in combining all the data to finder larger underlying trends, some nuance will inevitably get lost by lumping these potentially diverse and unbalanced datasets together. Since detailed per-sample soil parameters are not supplied to reviewers, it is difficult to decide whether this would have been a sensible measure.

2.  T. melanosporum is described as being "dominant" in the brûlé (l. 31 – 33), leading to the distinct and visible vegetation pattern which also formed the basis for picking sampling spots. Based on this one would expect it to be found in almost every sample, especially since 4 subsamples from each tree were combined. However, fig. 4 shows that for plantations some and for forests a lot of samples appear to have ~0 Tmel reads.     This data is only presented as a plot, no table with the exact numbers (sample number + number of Tmel reads) is provided, but based on the figure it seems like a decent chunk of the soil samples per brûlé did not contain any T. melanosporum DNA, or not enough to be detected by the metabarcoding approach. This raises the question whether a simple phenotypic determination of truffle-dominated soil is sufficient for actually picking positive samples, or whether amplification efficiency of the ITS region is sufficient. In the current version of the manuscript, the authors do not address the zero Tmel abundance samples at all, which would be a critical point to discuss. 

3. Only the fungal perspective is considered, despite bacteria likely making up a large part of the soil microbial community, especially at the alkaline pH found at the sampling sites. This means that only the interactions with the small fungal subset of the soil microbiome are considered. While additional amplicon sequencing for bacteria would have likely exceeded the scope of this study, some less complex methods like a comparison between general 16S vs ITS qPCR could at least have helped quantifying how much of the overall community is not included in this analysis. 

Conclusion:

We understand that some of the mentioned limitations are hard to address without extensive resampling or sequencing, but we strongly urge the authors to reconsider which conclusions can be drawn from their data and which questions go beyond their scope. Especially the title (l. 1 – 3) as well as the statements about a stronger negative influence of black truffle on the fungal network in plantations (l. 382 – 387) should be carefully reevaluated and potentially rephrased. Without the negative controls that would depict the undisturbed network, these conclusions do not just require confirmation but lack strong proof altogether, especially since members of the community that might have been fully suppressed by T. melanosporum are not accounted for here. An approach of only using paired sites to counteract some of the study design limitations could be promising to investigate the influence of abiotic conditions, as well as forest vs plantation on the fungal community. The issue of brûlé samples without any detectable T. melanosporum reads should also be further investigated and put into the focus of the revision. 

The article "Managed black truffle-producing systems have greater soil fungal network complexity and distinct functional roles compared to wild systems" by Varou et al. characterizes the fungal community in truffle plantation soils of Spain (with an outstanding sample size) and compares it with the fungal community in the soil of wild truffle-producing forests, while providing in-depth insight into the role of Tuber melanosporum in the soil fungal community networks, as well as into the influence of the most important fungal guilds on soil enzymatic activity. Truffle cultivation has traditionally advanced by observing the ecology of truffle in the wild. However, comparisons of wild and cultivated truffle sites are rare. This makes the study relatively novel. Besides, the information on soil enzymatic activity of the truffle soils is also relatively novel, even more its study in relation to the composition of the soil fungal community. All this information is an incremental knowledge that helps to better understand the role of truffle within the soil fungal community of truffle plantations and to unravel the role of this fungal community into the productivity and sustainability of truffle cultivation. The introduction of the study provides a comprehensive review of the state of the art, as well as clear and specific objectives and hypothesis for the study. The materials and methods are sufficiently described. The results are clearly exposed and provide an exhaustive analysis of the data, delving into ecological relationships with potential agronomic interest. The discussion connects the study objectives and hypotheses with the results, thoroughly exploring the ecological and practical implications of the study. However, a paragraph with the study limitations, the potential lines for future research and/or the practical implications of the study for truffle cultivation could help emphasizing the relevance and novelty of the study ("Understanding the ecology, including the biodiversity and functioning, of black truffle-dominated soils will provide a stronger foundation for making informed decisions regarding the management of black truffle plantations" may not be the strongest sentence to end the manuscript).

However, a few minor issues should be clarified:

1) L125 Which were the dates of sampling? Besides, the final sampling size is 231 (explained in L137), but it is not clear which is the final size for wild/cultivated and for spring/autumn.

2) L129 "Soil functioning was proxy through the potential activities" Is this sentence ok?

3) L189 The tests for hypothesis 2 do not seem to correspond with the hypothesis 2 specified in L101, since no hypothesis talks about soil parameters. Besides, the study aims to find the differences in the fungal community between cultivated and wild truffières (L91). For this, the networks of both types should be compared, or alternatively, it should be tested whether the network for wild sites is different from a random network and whether the network for plantations is different from a random network.

4) L205. How does ENET methodology deal with proportion data (percentage of reads for a guild)? Proportion data are frequently not normal data (GLMM?). Besides, since 3-4 guilds (including "non classified OTUs") practically dominate the community, the percentage of the main guilds are most likely highly correlated. How do you assess that colinearity does not affect the results of ENET methodology? 

5) L239 Figure S4 seems closely related to the main specific objectives. Why is it not included in the main manuscript? Besides, why did you decide to characterize the alpha-diversity only with richness and not with indices of diversity such as Shannon or Simpson?

6) L240-241 Significant differences in the PERMANOVA can be related to both differences in the centroid location and in the dispersion of each group samples, which seems to be the case according to Fig. S4b. Contrary to what is said in the manuscript, and according to Fig. S4b, the communities do not seem clearly dissimilar, but only different in dispersion.

7) L243-244. Wouldn't it be more correct to say that plantations tended to show higher values of pH, K and active carbonate?

8) L244-245. Taking into account that both seasons are almost centered in the biplot center, wouldn't it be more correct to say that spring showed more extreme values of pH, OM, Fe, although not always in the same direction?

9) L305 Why did you use T. melanosporum No of reads for regressions and not relative abundance of T. melanosporum, which you previously chose as normalized variable (L149)?

10) Careful with British/American English (e.g. normalised/normalized).

11) L321 "To test if soil ecological fungal guilds could explain soil carbon and nutrient cycling". L407 "When the different fungal guilds were further tested for their contribution in soil functioning, saprotrophs did significantly predict most of the soil enzymatic activities tested in both truffle-producing systems". Does the ENET provide correlation values or a partition of the variance? The manuscript suggests the latter. If so, which proportion of the variance in enzymatic activities do the fungal guilds explain?

12) L346-347 "Our results agree with the differences in β-diversity of soil fungal community previously observed in

Mediterranean and temperate climate sites, sampled across four seasons (Piñuela et al., 2024)". How do they agree?

13) L356 Brûlé in italics?

14) L370 "Differences in soil fungal network structure among forest and plantations have been, however, regularly explained by the variation in soil properties (Wang et al., 2024b), or different vegetation cover..." Considering this, it would be interesting to discuss a little the differences in vegetation structure between wild/cultivated sites (age of trees, percent canopy cover, percent soil cover by litter, periodic tillage, percent soil cover by herbs/shrubs, etc.).