Molecular Diversity of Arbuscular Mycorrhizal Fungi Associated with the Rhizosphere of Vachellia seyal Del. from Selected Saline Soils in Senegal

Comments 1: Line 70: Please, provide another example, as phosphorous was already mentioned

Response 1: [phosphorus has been replaced by nitrogen in the revised version. Thank you for pointing this out. We also cited a new author concerning nitrogen :

“[8.      Koegel, S.; Boller, T.; Lehmann, M.F.; Wiemken, A.; Courty, P.E. Rapid nitrogen transfer in the Sorghum bicolor-Glomus mosseae arbuscular mycorrhizal symbiosis. Plant Signal Behav. 2013 8(8):e25229. doi: 10.4161/psb.25229.]”

Comments 2: Line 123: It is not clear if soil DNA extractions were performed from the original composite soil from the three different sites, or from soil of the trap cultures.

Response 2: DNA extractions were performed from the original composite soil from the three different sites. We have added a new sentence in the revised manuscript for more clarification. You can check please the section : [DNA extraction from soils]

Comments 3: Line 137 : It is not clear if root DNA extractions were performed from field plants of the three different sites, or from roots of the trap cultures.

Response 3: Root DNA extractions were performed from the roots sampled in the rhizosphere of the three different sites. We have also added this new sentence in the revised manuscript for more clarification. You can check please the section : [DNA extraction from roots]

Comments 4: Line 157 : DNA amplifications were only performed on DNA extracted from the soils? Original or trap cultures? The sequence of the procedure in the methods used is not clear

Response 4: DNA amplification was performed on DNA extracted from rhizospheric soils and roots sampled from the three sites, as well as from spores isolated after trap cultures. We have also added this new sentence in the revised manuscript for more clarification. You can check please the section : [Nested PCR amplification]

Comments 5: [Line 192: Twenty samples from each soil type? How did you reach the 75 sequences mentioned in line 224?]

Response 5: Several batches consisting of twenty samples of plasmid DNA randomly taken from clones meeting these criteria were sequenced, using the Dye Chain Terminator method (MWG-Biotech, Ebersberg, Germany). We have also added this new sentence in the revised manuscript for more clarification. You can check please the section : [Cloning and sequencing]

Not all samples were sent for sequencing at the same time. They were sent in batches of 20 samples.

Comments 6: Figure 2 : Please clarify the color codes used in the Figure 2. What are they about, or what do the legends in blue and green mean? There are sequences in two different tones of brown. Are both sequences recovered from roots? Use more contrasting colors.

Response 6: We have changed in the revised manuscript. Figure 2 has been renamed Figure 3 in the revised version. The legend has been modified to provide more detail on the colors used. The authors believe that these colors make it easier to visualize the difference in the origin of the sequences.

Check please: “Figure 3. Neighbor-joining tree of arbuscular mycorrhizal (AM) fungi (Glomeromycota taxa) based on alignment of 25S rDNA sequence fragments from the rhizosphere of V. seyal for Bam-bey (green), Ngane (Blue) and Ndiafate (orange), roots (brown) and spores (red) aligned with known sequences. Mortierella sp was used as relevant outgroup species”

Comments 7: Line 304 : Please rephrase the sentence. You mean that "Three lineages were recovered ONLY from Ngane"

Response 7: We Agree. We have rephrased the sentence :

“as showed in figure 5. Three specific lineages were recovered only from the moderately saline soil of Ngane”

4. Response to Comments on the Quality of English Language

Point 1: In general, the meaning of the sentences and paragraphs is understood. But there are some mistakes. I mentioned the more evident

Response 1: The English has been thoroughly proofread and corrected

5. Additional clarifications

Several important modifications have been made to the document, particularly concerning the statistical analyses, diversity indices, Venn diagram... The revised manuscript also includes two new figures, a new table, and five new bibliographic references.

Comments 1: L26-29 grammar

Response 1: [The grammar has been corrected in the revised version] Thank you for pointing this out. For clarity, we have split it into two sentences. “This study revealed several unidentified arbuscular fungi and a particularly high host specificity in V. seyal roots. The vast majority of OTUs isolated in this study had no homologous sequences in the databases.”

Comments 2: L46 A is not needed.

Response 2: Agree. We have changed. “A” has been removed

Comments 3: L51 scares- scarce.

Response 3: Agree. We have changed. It’s now “scarce”

Comments 4: L57 restauration –restoration

Response 4: Agree. We have changed. Now it’s written “restoration”

Comments 5: L93 16°05’O is it correct?

Response 5: Agree. “O” is now changed by “W”

Comments 6: Table 1 Give units of all parameters

Response 6: Agree. Units (%) have been added for Clay, coarse silt and coarse sand. Check Table 1 please

Comments 7: L107 polythene a typo?

Response 7: We have changed by “polyethylene”

Comments 8: L111-112 ??

Response 8: Agree. We have changed. This sentence has been removed to avoid repetition with the sentence written above: “The collected samples were pooled, mixed and analyzed after mixing all subsamples from 109 one site (composite samples).”

Comments 9: L135-136 Was there enough DNA to see on a gel?

Response 9: Agree. We have added figure 2 (page 8) concerning DNA size

Comments 10: L223 ?

Response 10: To explain more : After culturing the bacteria in several Petri dishes, a large number of colonies are obtained. Some are white and others are blue. A selection of the white colonies must then be carried out. You can check please the methodology in the section “Cloning and sequencing”

Comments 11: L419 hypogeal – hypogaea

Response 11: Thank you for pointing this out. We have changed. Now it’s written “hypogaea”

Comments 12: L419-424 I am lost. The sentence on lines 419-421 I understand as ‘in Bambey, we found some fungi that we found neither at Ngane nor at Ndiafate’. OK, why then you next say that agriculture (at Bambey) reduces fungi diversity? Please clarify this passage.

Response 12: We have added a Venn Diagram (figure 5) for more clarity. When you compare Bambey (cultivated soils) and Ngane (non cultivated soils), you have 5 species in Bammbey and 7 species isolated in Bambey.

4. Response to Comments on the Quality of English Language

Point 1: No comment

Response 1:  The English has been thoroughly proofread and corrected

5. Additional clarifications

Several important modifications have been made to the document, particularly concerning the statistical analyses, diversity indices, Venn diagram...

Comments 1: It is little bit stringer that authors do not assess root colonization by AMF? It is recommended to provide the following information’s related to root colonization for each site (F%: frequency of mycorrhization of root fragments, M%: intensity of root cortex colonization, m%: intensity of colonization within individual mycorrhizal roots). These information’s are useful to understand the effect of salt on the Vachellia seyal mycorrhizal association?

Response 1: This study was conducted in three parts, based on a previously defined research question:

1. Studies on morphological diversity

2. Studies focused on nutritional aspects, specifically the contribution of arbuscular fungi to improved salt stress tolerance in *Vachellia seyal* (syn. *Acacia seyal*)

3. The final part, which involves the molecular identification of arbuscular fungi associated with *V. seyal*. In fact, several authors have shown that a large portion of these fungi cannot be cultivated in greenhouses or through various available culture techniques. However, the use of molecular tools allows for the detection of their presence in the soil. The inability to culture them means that many of these species remain unknown, particularly in African soils. There are few publications that describe and highlight the molecular diversity of arbuscular fungi in African soils, which justifies this study.

The effects of root colonization (Intensity and frequency of root colonization) have been published separately in another article, which addressed the nutritional effects of the association between V. seyal and arbuscular fungi found at various sites along the salinity gradient. Please refer to the following article (page 12): Manga, A.G.B.; Ndiaye, M.; Ndiaye, M.A.F.; Sané, S.; Diop, T.A.; Diatta, A.A.; Bassene, C.; Min, D.; Battaglia, M.; Harrison, M.T. Arbuscular Mycorrhizal Fungi Improve Growth and Phosphate Nutrition of Acacia seyal (Delile) under Saline Conditions. Soil Syst. 2022, 6, 79. https://doi.org/ 10.3390/soilsystems6040079

Comments 2: Photos of endogenous structures of AMF colonization (intraradical hyphae, and spores) within a root of Vachellia seya should also be provided.

Response 2: The photos of spores isolated from the three study sites relate to point 1 of our study (morphological diversity). These data have already been published. Please refer to the article: Manga A, Dalpé Y, Soumaré A, Diop TA (2017). Diversity of Arbuscular mycorrhizal fungi associated to Acacia seyal (Delile) in semi-arid zone of Senegal. World Journal of Microbiology, 3(2): 120-127.

However, the photos primarily concern the exogenous parts. The authors did not find it necessary to photograph the endogenous parts, as the objective was to characterize the spores based on their morphological features, such as size, color, wall ornamentation, and the number of wall layers...

Comments 3: Additionally, enumeration of AMF spores from soil samples was not reported? Please provide the spores number for each site and photos of morphological appearance of some spores in a soil suspension of rhizospheric of Vachellia seyal under salinity.

Response 3: The authors remind that the main objective of the present study was focused on molecular diversity (as mentioned in the title). The authors believe that the enumeration of spores, and photos of morphological appearance of some spores falls outside the scope of this study and could be addressed in another study if necessary. Regarding the spore photos, please refer to the previously cited article : Manga A, Dalpé Y, Soumaré A, Diop TA (2017). Diversity of Arbuscular mycorrhizal fungi associated to Acacia seyal (Delile) in semi-arid zone of Senegal. World Journal of Microbiology, 3(2): 120-127.

Comments 4: Images of PCR products amplified by first and nested PCR should be provided

Response 4: Thank you for pointing this out. We agree with this comment. The images related to PCR products amplified by first and nested PCR have been added to the revised manuscript. You can find them on page 8, Figure 2.

Comments 5: Please remove the table 2 and replace it by a new one where distribution of AM fungal OTUs detected in studied sites could be performed in relation with Shannon–Wiener diversity index?

Response 5: Thank you for pointing this out. The authors believe that Table 2 is important for clearer visibility of the accession numbers and a better understanding of the origin of the OTUs (sites, rhizosphere, root, or soil). Reading Table 2 and the phylogenetic tree helps to better understand the origin of the isolated OTUs. Several articles include similar tables. Please refer, for example, to the article by Spruyt et al. 2014 : 11.           Spruyt, A.; Buck, M.T.; Mia, A.; Straker, C.J. Arbuscular mycorrhiza (AM) status of rehabilitation plants of minewastes in South Africa and determination of AM fungal diversity by analysis of the small subunit rRNA gene sequences. S. Afr. J. Bot. 2014, 94: 231–237. https://doi.org/10.1016/j.sajb.2014.07.006

 Therefore, in response to your suggestion, the authors have added Table 3 (page 14), which includes the Shannon–Wiener diversity index, to the revised manuscript.

Shannon–Wiener diversity index has been added to the document as requested (methodology, index calculation and analysis for the three sites and discussion). However, the authors point out that the Shannon-Wiener diversity index is being sometime less used for scientific reasons, which explain why the calculation of the index is biased. Please consult the following article :

W.L Strong, (2016) Biased richness and evenness relationships within Shannon–Wiener index values, Ecological Indicators, Volume 67, Pages 703-713, https://doi.org/10.1016/j.ecolind.2016.03.043

The authors have therefore chosen to use rarefaction curves, which show that not all species were identified during the study.

Comments 6: Venn diagram should be presented where you show clearly the Shared and site-specific OTU associated to Vachellia seyal..

Response 6: The Venn diagram has been added to the revised manuscript, showing the common and site-specific OTUs. You will find it on page 13, Figure 5.

Comments 7: It is necessary to perform Principal Component analysis of OTUs distribution along a soil salt and among soil properties. This would be really useful and could be used in your analysis. I also note that you discuss the genotypic data of the OTU with regard to soils, but it’s difficult to see the relevance of this if the soil data are not well-presented.

Response 7: In the manuscript, the authors considered that the most important element in the table presenting the physicochemical composition of the soil (Table 1) was the electrical conductivity, which indicates the level of salinity. The presentation in table form facilitates reading the salinity levels according to the sites. The calculation of different indices (Shannon, Simpson, Pierlou) seemed more relevant to estimate the diversity of the OTUs (Table 3).

Comments 8: I noticed that some OTUs (6) were obtained from bambey soil with no OTUs from spores from the same site? How to explain this result? Similarly, how could you explain data reported for Ngane where no OTUs from roots and spores?.

Response 8: During this study, some OTUs present were not detected for various reasons mentioned in the discussion section. This does not mean that they do not exist.

As explained in discussion (line 342 of the original manuscript) “This was performed to focus the study on this lineage of Glomeromycetes but this approach may underestimate the diversity within the group.” And in line 346-347 “Only members of the genus Glomus were detected by targeting the three soil samples. Other AMF, such as Gigaspora, Scutellospora or Acaulospora, were not found.”

Line 359-360: “Furthermore, all DNA-based studies suggest that the true diversity of AMF in the field is much higher than that currently represented in culture collections as sporulating AMF.”

Line 384- 388: “The number of clones analyzed for each soil was probably too small to allow us to  detect the maximum diversity within the soils. None of the soil-derived sequences were 385 identical, and many more soil samples need to be analyzed to monitor the full genetic diversity. The sequence groups described in this study, identified by phylogenetic analysis, are partly dependent on the limited number of clone samples.”

Comments 9: More statistical analysis such as Pearson correlation coefficients between soil properties, OTUs richness, and Shannon–Wiener diversity index among studied sites is needed.

Response 9: New subsections (Statistical and diversity analysis, Statistical analysis results… ) have been added in the methodology to address your suggestions. Pearson correlation, OTUs richness, and Shannon–Wiener diversity index (table 3) have been performed. The results were then analyzed and discussed. You can refer to pages 6 and 7 (methodology), page 14 (results), and pages 17 and 18 (discussion) of the revised manuscript.

4. Response to Comments on the Quality of English Language

Point 1: No comments

Response 1: The English has been thoroughly proofread and corrected

5. Additional clarifications

Overall, many modifications have been made to improve the quality of the revised manuscript. Statistical analyses and index calculations have been added, analyzed, and discussed. Two figures and one table have also been added, along with five new bibliographic references

Comments 1: As you already performed the phylogenetic tree, no add value will be reported by the table 2. So, please remove the table 2 and replace it by a new one (as suggested during the last reviewing round) where distribution of AM fungal OTUs detected in studied sites could be performed in relation with Shannon–Wiener diversity index?

Response 1: The table 2 is removed and replaced by a new one where distribution of AM fungal OTUs detected in studied sites have been performed in relation with Shannon–Wiener diversity index. You can check it in the new revised document in page 12 : “Relationships between OTUs and diversity indices”.

The new elements have been highlighted in green.

Comments 2: The accession numbers should be reported in the ''Data Availability Statement'' based on the journal instructions.

Response 2: Thank you. The accession numbers have been reported in the ''Data Availability Statement'' as requested. Check please the “Data Availability Statement” section of the revised mabuscript

Comments 3: The phylogeny should be re-computed using the Maximum likelihood estimation method as it is the most robust approach.

Response 3: Maximum Likelihood Estimation (MLE) is indeed one of the most commonly used methods for building phylogenetic trees, but whether it is the "best" method depends on the specific context, data, and requirements. MLE-based methods are generally very accurate for large datasets and complex evolutionary models. MLE is one of the best methods available for phylogenetic tree building, particularly in complex and data-rich scenarios. However, "strong" is subjective, and choosing the right method. Other methods like Neighbor Joining (NJ) or Parsimony are faster and may be preferable for simpler datasets, though they may not capture the complexity as effectively as MLE. Then MLE tends to be the preferred method when for example data is extensive and complex (e.g., whole-genome sequences). If computational resources or simplicity are priorities, NJ or Parsimony might be better choices for smaller datasets.

The authors therefore believe that the NJ method is the best for this study due to the limited number of OTUs.