Microbiota Characterization of Agricultural Green Waste-Based Suppressive Composts Using Omics and Classic Approaches
, Rob D. Finn 3 and Massimo Zaccardelli 1,*Round 1
Reviewer 1 Report
General comments
In general, I found several worthy points to underline such as the high level of significance of results, quality of presentation and interest to the readers of your manuscript. However, I observed on this manuscript an important flaw which decrease its scientific soundness. Authors used an approach to carry out the genomic analysis of fungal community biodiversity which is not as sound and powerful as the alternative approach targeting ITS regions of the 18S rRNA gene. I can understand why you did not use this alternative which may produce a higher resolution analysis of fungal community biodiversity and composition. I think you should have used this approach and I think this is inside of the study objectives. For this fact, I propose to resubmit the study using ITS approach for the genomic analysis of fungal community of both compost. If authors can manage this issue on a revised version of this study, the paper could be considered for publication in the Journal. Other more specific comment should be also addressed in that revised version that you can find listed under this paragraph.
Specific comments
L54: Explain what you mean when you write “matrices used for the composting process”.
L75: It would be interesting to include pile dimensions or amount (kg) of feedstock per pile.
L99-100: I think the experimental controls would be those unamended with both composts in order to test their biocontrol effect for comparison with those amended with those composts.
L101: Autoclaved peat is confusing. I think you wanted to mean peat inoculated with fungal pathogens, did not you?
L102: You should include how did you prepare the pots for the experimental control.
L112: Is the compost/water mixture added directly to the seeds? You should detail the following steps prepare the extract used in the germination assay.
L113-114: These CWE concentrations are found no consistent with that you have mentioned on line 112 (50:50 v/v).
L129: This equation is hard to understand. You should explain Spi. Is the sum of activities on all substrates equivalent to AWCD?
L142: Should not ITS1 and ITS2 regions of the 18S rRNA gene be the target of those primers? Include some bibliographic citation of this method.
L206-207: This sentence seems to be more suitable for the discussion section.
L249: Should not they be better ITS1 and ITS2 (internal transcribed spacer) region? You should insert “of high quality reads” before “for L5/6A and L2A samples.
L267: As you can observe in figure 4, you should delete Planctomycetes of this group of phyla being more abundant in the L5/6A compost.
L268: You should add Verrumicrobia, Gemmatimonadetes and Acidobacteria phyla to Planctomycetes as the most abundant in L2A compost.
L306: In figure 7, the results are not expressed in percentage but in number of annotations.
L319-320: In figure 7, the results are not expressed in relative abundance but in number of annotations.
L346-350: Please see general comments
L371: Change then by than
L380-381: This sentence is hard to understand. What do you mean when you write the both composts have the same number of genes involved in catabolic activity, but L5/6 compost has a higher functional expression?
Author Response
RESPONSE REVIEWER #1 COMMENTS
Point 1: In general, I found several worthy points to underline such as the high level of significance of results, quality of presentation and interest to the readers of your manuscript. However, I observed on this manuscript an important flaw which decrease its scientific soundness. Authors used an approach to carry out the genomic analysis of fungal community biodiversity which is not as sound and powerful as the alternative approach targeting ITS regions of the 18S rRNA gene. I can understand why you did not use this alternative which may produce a higher resolution analysis of fungal community biodiversity and composition. I think you should have used this approach and I think this is inside of the study objectives. For this fact, I propose to resubmit the study using ITS approach for the genomic analysis of fungal community of both compost. If authors can manage this issue on a revised version of this study, the paper could be considered for publication in the Journal. Other more specific comment should be also addressed in that revised version that you can find listed under this paragraph.
Point 1: The authors are grateful to the reviewer for these constructive comments and agree that an alternative approach targeting ITS regions of the 18S rRNA gene may have potentially produced a higher resolution analysis of fungal community biodiversity and composition. The Internal Transcribed Spacer (ITS) region one of the proposed barcode for fungi as it has species resolution for a very broad range of fungi compared to other fungal marker genes. However, many fungal taxa recovered by environmental ITS-sequencing can often be identified solely to kingdom or phylum level due to the lack of reference sequences or reference sequences annotated only to high taxonomic levels.
Nevertheless, we strongly believe the results of our study are mainly due to the bioinformatic pipeline used. The MGnify pipeline uses a highly conservative approach for taxonomic assignment, annotating reads only with the 99% of similarity to the reference database. This kind of approach leads to really reliable results, but with an identification only to the higher taxonomic levels, as occur in our case.
From the other hand, even though all the samples studied in this study are still available and stored at -80°C, a reanalysis targeting the ITS regions is not possible, due to the long storage time that can have altered the microbial composition during the time, potentially changing the results and not making them comparable with those presented in this paper.
Point 2: L54: Explain what you mean when you write “matrices used for the composting process”.
Point 2: Agreed and addressed. The text has been amended for clarity.
Point 3: L75: It would be interesting to include pile dimensions or amount (kg) of feedstock per pile.
Point 3: The authors thank the reviewer for these comment. Pile dimension are now provided in the text.
Point 4: L99-100: I think the experimental controls would be those unamended with both composts in order to test their biocontrol effect for comparison with those amended with those composts.
Point 4: We agree with the reviewer that this explanation was indeed missing an apologise for this oversight. Experimental controls were inoculated but not amended with both composts. The text has been amended for clarity.
Point 5: L101: Autoclaved peat is confusing. I think you wanted to mean peat inoculated with fungal pathogens, did not you?
Point 5: We agree with the reviewer that this explanation was indeed missing an apologise for this oversight. Peat was sterilized using autoclave and then added compost, to overcame effects of other contaminations. The text has been amended for clarity.
Point 6: L102: You should include how did you prepare the pots for the experimental control.
Point 6: We agree with the reviewer that this explanation was indeed missing an apologise for this oversight. The text has been improved including more information on the control pots preparation.
Point 7: L112: Is the compost/water mixture added directly to the seeds? You should detail the following steps prepare the extract used in the germination assay.
Point 7: The authors thank the reviewer for these comments and agree that more details is needed in the description of the method. The paragraph describing the biostimulation/phytotoxicity test has been improved with more details on the method.
Point 8: L113-114: These CWE concentrations are found no consistent with that you have mentioned on line 112 (50:50 v/v).
Point 8: We agree with the reviewer that this explanation was indeed missing an apologise for this oversight. CWE were prepared using a compost/water mixture (50:50 v/v) and, then, diluted and used at three concentrations (50, 16.6 and 5 g l−1). The text has been improved including more details.
Point 9: L129: This equation is hard to understand. You should explain Spi. Is the sum of activities on all substrates equivalent to AWCD?
Point 9: We agree with the reviewer that this formula is difficult to understand. Unfortunately, for a formatting mistake, Spi was converted in Spi. The formula has been corrected and the text improved with a better description.
Point 10: L142: Should not ITS1 and ITS2 regions of the 18S rRNA gene be the target of those primers? Include some bibliographic citation of this method.
Point 10: Those primers target NS1 – NS2 region of 18S rRNA as reported in the cited references [20, 21].
- Klindworth, A.; Pruesse, E.; Schweer, T.; Peplies, J.; Quast, C.; Horn, M.; Glöckner, F.O. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 2013, 41, e1–e1.
- White, T.J.; Bruns, T.; Lee, S.; Taylor, J.W. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics; Academic Press Inc: New York, 1990; pp. 315–322.
Point 11: L206-207: This sentence seems to be more suitable for the discussion section.
Point 11: Agreed and addressed. The text has been amended according to the reviewer’s comment.
Point 12: L249: Should not they be better ITS1 and ITS2 (internal transcribed spacer) region? You should insert “of high quality reads” before “for L5/6A and L2A samples.
Point 12: Agreed and addressed. The text has been amended according to the reviewer’s comment.
Point 13: L267: As you can observe in figure 4, you should delete Planctomycetes of this group of phyla being more abundant in the L5/6A compost.
Point 13: Agreed and addressed. The text has been amended according to the reviewer’s comment.
Point 14: L268: You should add Verrumicrobia, Gemmatimonadetes and Acidobacteria phyla to Planctomycetes as the most abundant in L2A compost.
Point 14: Agreed and addressed. The text has been amended according to the reviewer’s comment.
Point 15: L306: In figure 7, the results are not expressed in percentage but in number of annotations.
Point 15: We agree with the reviewer that this figure was indeed wrong and apologise for this oversight. For a mistake, during the manuscript preparation, the text was amended but the figure was not updated. The figure has now been updated.
Point 16: L319-320: In figure 7, the results are not expressed in relative abundance but in number of annotations.
Point 16: Agreed and addressed. As reported in the previous comment, Figure 7 has now been updated.
Point 17: L346-350: Please see general comments
Point 17: The authors thank the reviewer for these comments. A full answer has been reported in the point 1.
Point 18: L371: Change then by than
Point 18: Agreed and addressed. The text has been amended according to the reviewer’s comment.
Point 19: L380-381: This sentence is hard to understand. What do you mean when you write the both composts have the same number of genes involved in catabolic activity, but L5/6 compost has a higher functional expressionn?
Point 19: We agree with the reviewer that this explanation was hard to understand an apologise for this oversight. The text has been improved including a better discussion of the results.
Reviewer 2 Report
This paper compares the suppressive effect of two compost types (L5/6A and L2A) on two soil-born pathogens S. minor and R.solani. They found that rocket and fennel derived L5/6A effectively suppressed both pathogens (Fig. 1A). They compared both bacterial and fungal communities of these composts and found that L5/6A was less diverse in both bacterial and fungal communities (Fig. 3), but it was more abundant with specific groups Bacteroidetes, Proteobacteria, and Actinobacteria (Fig. 4). They explain that L5/6A was more abundant with Nocardiopsis or Pseudomonas species (Fig. 6), which led to a significant increase in pathogen suppressive effect. They also explain the different germination index (higher in L2A, Fig. 1B) by a higher abundance of Streptomyces and Flavobacterium. Overall the experimental design is comprehensive, results are informative, and the conclusion is supported by data. Below I suggest some comments polish the manuscript.
Major comments
The authors explain the difference in damping-off incidence by different abundance in two species Nocardiopsis and Pseudomonas, but it seems to be a bit overstatement. The differences in these species seem not so large, the abundance of Nocardiopsis and Pseudomonas was 1-1.99% in L5/L6A while 0-0.99% in L2A (log fold change seems to be large for Nocardiopsis). There were some other species which was significantly higher abundance in L5/L6A, but the authors ignore them. The authors should treat all species equally and estimate the relative contribution of each species on the difference in pathogen suppression effect or germination index. Or, the authors should mention on some other species like Sphingobacterium or Paracossus or Galbibacter and estimate their function in the composts. Is Pedomicrobium not important for germination?
The authors should focus more on secondary metabolites biosynthesis pathways in the WGS data. Especially, some specific pathways which Nocardiopsis, Pseudomonas, Streptomyces, and Flavobacterium possess should be tested in more detail.
Specific comments
L64 Explanation of why you chose these two pathogens, such as their importance, is needed.
Fig. 7 significantly different ones should be indicated as they did in Fig. 4.
L320-321 what do you mean for “almost the same level” ? Were they all not statistically significant?
L333-334 data needed.
L356-373 It would be better to use the abundance data here as they did in L281-291 to help the readers.
Author Response
RESPONSE REVIEWER #2 COMMENTS
Point 1: This paper compares the suppressive effect of two compost types (L5/6A and L2A) on two soil-born pathogens S. minor and R.solani. They found that rocket and fennel derived L5/6A effectively suppressed both pathogens (Fig. 1A). They compared both bacterial and fungal communities of these composts and found that L5/6A was less diverse in both bacterial and fungal communities (Fig. 3), but it was more abundant with specific groups Bacteroidetes, Proteobacteria, and Actinobacteria (Fig. 4). They explain that L5/6A was more abundant with Nocardiopsis or Pseudomonas species (Fig. 6), which led to a significant increase in pathogen suppressive effect. They also explain the different germination index (higher in L2A, Fig. 1B) by a higher abundance of Streptomyces and Flavobacterium. Overall the experimental design is comprehensive, results are informative, and the conclusion is supported by data. Below I suggest some comments polish the manuscript.
Point 1: The authors thank the reviewer for the very positive feedback on our work.
Point 2: The authors explain the difference in damping-off incidence by different abundance in two species Nocardiopsis and Pseudomonas, but it seems to be a bit overstatement. The differences in these species seem not so large, the abundance of Nocardiopsis and Pseudomonas was 1-1.99% in L5/L6A while 0-0.99% in L2A (log fold change seems to be large for Nocardiopsis). There were some other species which was significantly higher abundance in L5/L6A, but the authors ignore them. The authors should treat all species equally and estimate the relative contribution of each species on the difference in pathogen suppression effect or germination index. Or, the authors should mention on some other species like Sphingobacterium or Paracossus or Galbibacter and estimate their function in the composts. Is Pedomicrobium not important for germination?
Point 2: The authors are grateful to the reviewer for these constructive comment and agree that all the species should be taken into account when describing compost activity. In our case, we implemented the discussion with some other genera, like Galbibacter, Trupera and Luteimonas, that although their suppression activity has not been reported in literature yet, they could potentially play role.
In our opinion, take into account other taxa, with really low relative abundance (<1% in both composts), could be misleading and too speculative, without further specific investigations.
Point 3: The authors should focus more on secondary metabolites biosynthesis pathways in the WGS data. Especially, some specific pathways which Nocardiopsis, Pseudomonas, Streptomyces, and Flavobacterium possess should be tested in more detail.
Point 3: The authors agree with the comment. Unfortunately, the deep of the WGS sequencing allowed only to generated few MAGs, all of them confirming the 16S sequencing results, but none of them including the genera mentioned by the reviewer above. Moreover, the relative low number of reads included in each MAGs was not enough to cover a full description of the secondary metabolites biosynthesis pathways represented in the MAGs.
Point 4: L64 Explanation of why you chose these two pathogens, such as their importance, is needed.
Point 4: We agree with the reviewer that this information was indeed missing. The text has been amended providing an explanation of why we have chosen these two pathogens.
Point 5: Fig. 7 significantly different ones should be indicated as they did in Fig. 4.
Point 5: We agree with the reviewer that this information should be included in the figure, unfortunately no statistically significant differences were reported for the functional annotations between the two composts.
Point 6: L320-321 what do you mean for “almost the same level” ? Were they all not statistically significant?
Point 6: See previous comment
Point 7: L333-334 data needed.
Point 7: Agreed and addressed. The text has been amended according to the reviewer’s comment.
Point 8: L356-373 It would be better to use the abundance data here as they did in L281-291 to help the readers.
Point 8: Agreed and addressed. The text has been amended according to the reviewer’s comment.
Round 2
Reviewer 1 Report
Authors have suitably addressed all comments that I previously reported and they have elaborated an improved version of their original manuscript. Thus the manuscript in the current form could be accepted for publication in the Journal.
