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I would like to thank you for turning around comments of the reviewers so well. I hope you can agree with me that the paper has gained substantially in quality.
# PeerJ Staff Note - this decision was reviewed and approved by Valeria Souza, a PeerJ Section Editor covering this Section #
The quality of the manuscript has strongly increased. Two of the reviewers have provided additional suggestions for further improvement of the paper.
At line 55: signficant should be significant
Line 87: bag should be bags
Line 117: were presented in details in should be have been described in detail in
Line 138: permuations should be permutations
Line 163: The high quality reads ranged from 25,996 to 69,618 OTUs between samples (Table S1). I believe the amount of reads is described here.
Line 249: Dominate should be dominating
Line 251: exits should be exist
Line 256: cause a decline the should be cause a decline in the
Line 278: The activities of bacterial community should be The activities of the bacterial community
Line 282: The reduction in substrate total nitrogen, phosphorus and potassium at the elongation stage highly supported our results. I believe this claim should be clarified.
Line 284: Increase should be increased
Line 297: secreted should be secrete
Line 309: when the moisture content was in high level should be when the moisture content was high
Line 310: that microbes produced should be that microbes produce
Line 314: be associated should be are associated
Line 315: are the key players for bacterial adaptation This piece of text makes the sentence faulty
Line 319: in bacterial community should be in the bacterial community
Line 326: pathway should be pathway
Line 334: of in the bacterial community should be of the bacterial community
Line 358: stage be crucial should be stage is crucial
I appreciate that you included pictures showing the experimental setup, this way other researchers are able to replicate your experiment. In the current version, it also became more clear what type of samples you have analysed. Good job.
Line 320: colonization mechanisms of the bacterial settlers that entered into the substrate from air: Here it is written for the first time that the bacteria are coming from the air. In my opinion, the effect of the bacteria entering the substrate from air (or from worker’s hands) could be one of the strongest in microbial composition development. Since this is so crucial, I do not understand why this aspect is not mentioned earlier in the article. Please consider this for the final version.
Line 331: fungus and the associated microbiome: To my opinion, you can not say that the bacterial community is associated with the fungus or its growth. In line 17 you also speak about G. lucidum-associated bacterial communities as well as in the Background. Again, you can not state this. For example, in many insect studies some bacterial genera like Providencia or Proteus are associated with the insect’s presence (i.e. Singh et al., 2014*). In the current study there is not enough evidence for the association of the bacterial community with the fungus’ presence, so please do not make these statements or suggestions. What I can agree with, is the term “substrate-associated bacterial composition” which is also used in the title of the article.
No other comments
This was within guidelines.
This was within guidelines
Good
Overall, I found the revised manuscript cleaner and more engaging and insightful. The authors did a good job revising the manuscript, and it is more streamlined and reads well.
No comment
No comment
No comment
I would like to congratulate the authors on their very thorough revisions, which have greatly improved the quality of this article. I am very satisfied with the response to my previous points. However, I would request that the following minor alterations be made prior to publication:
Line 84: Remove the capital L from lucidum
Line 88: E, not Ei
Lines 128-129: The comment regarding barcoding should be in the section about PCR, rather than data processing
Line 239: Replace 'proved' with 'demonstrated'
Line 334: The phrase "indicating that they were the first settlers in the substrate" can be misleading. Although bacterial persistance does indicate that these bacteria were present early on, the more pertinant point is that they continued to be present rather than being replaced by other taxa.
Line 340-341: Fluorescent pseudomonads is a description, not a species name, so should not be in italics or capitalised
Table 4: Please provide P-values, rather than just significance codes
Fig 3: Relative abundance data may still cluster by sequencing depth on a NMDS plot (see Weiss et al 2017 Microbiome 5:27). Looking at Table S1, I am satified that this effect is not responsible for the primary pattern observed here. However, it is always advisable to check for sequencing depth effects by plotting. This may be done simply in R with the following code:
#Calculate depth (where samples are rows and OTUs columns)
depth<-(rowSums(OTUtable))
#Convert it to a value between 0-255, for later use in plotting
val<-max(depth)/255
grad<-depth/val
plot(NMDS$points[,2]~NMDS$points[,1], col=rgb(red=255,blue=0,green=0,alpha=grad,maxColorValue=255), xlab="NMDS1", ylab="NMDS2")
Dear authors,
All three reviewers found merit in your interesting study, but also provided a number of detailed suggestions to further improve the manuscript, especially regarding the approaches utilized, the scientific depth, including the presentation, interpretation and discussion of the data.
With pleasure I have been reading the article ‘Dynamic succession of substrate-associated bacterial structure and function during Ganoderma lucidum growth’. The article is well written in a clear and scientific manner. This study is in my opinion quite novel since it looks into the bacterial community that is growing together with the mushroom Ganoderma lucidum. In general, there have not yet been many studies that look into the bacterial composition on mushrooms and on its substrate. In addition, the mushroom Ganoderma lucidum has for the past few decades attracted attention with scientists all over the world because of its medicinal properties, which makes a study on this mushroom species very interesting.
I do have a number of points of feedback. I believe these are necessary to address to make the article easier to understand for the reader. In addition, I have feedback on the scientific aspects of the study.
To start, the title should not include the term bacterial structure, but rather should include the term bacterial composition.
It would be nice to include a more extensive discussion/conclusion, in which for example available literature on bacterial cultures on mushrooms and substrates are studied. This can be nice to compare the current study with. Also, it would be nice to include speculations as to why particular genera are more or less abundant in later stages of the mushroom’s life. For example, it is possible that Ganoderma lucidum defends itself against bacteria, because it produces many compounds including antibacterial ones and as such might be able to shape its own bacterial community. This is just an example and does not have to be included in the article.
The bags were sterilized using autoclaving, how can it be that bacteria start to grow on the substrate/mushroom during the hyphal stage? Were the polypropylene bags open on one side or did the bags have filters that allow bacteria to enter? This is currently not clear and needs to be explained, since these aspects have a tremendous impact on bacterial composition. It would help if picture(s) can be included that show the reader what the growing conditions look like. Pictures of taking samples would also really help. For example, it is currently not completely clear from what parts of the substrate/mushrooms the samples are taken and what the material looked like. Also, the inclusion of information on the humidity in the room might be important, as moisture/condensation on the substrate/mushroom is a perfect breeding area for bacteria and as such can have a huge impact.
Keep in mind that with the information provided in this paper other researchers should be able to replicate this experiment.
In line 112 it is mentioned that 25-27 cycles are used during the PCR. Since the impact of the number of PCR cycles is high for the outcome, it might be nice to indicate which samples received what number of cycles.
Were the PCR products purified before being pooled? If yes, please include this in the methods.
It would be nice to include a method of quantifying the amount of bacteria, instead of only having compositional data.
It is very nice that a chemical analysis was performed on the substrate material, but what I am missing is the measurement of acidity over time. It is commonly known that mushroom substrates become more acidic over time because of digestion of the substrate and as such particular bacterial species will thrive on this more acidic substrate. This is why I expect acidity to be a large confounder and it would be nice to include in the current study. Same applies to the moisture content of the substrate.
It is very nice that a KEGG pathway analysis was performed, but what I would like to see is not only the dry observations of the pathways changing over the growing phases, but rather also include what the biological reasons might be that drive the change in pathways.
In figure 2, a cladogram is shown including 12 samples in 4 groups. This results in 3 samples per group, which I believe is a very low number to make conclusions.
From the article I understand that one single fruiting body was used for sampling, which means that the power number is 1 (N = 1). With such a low power number, it is hard to come up with meaningful conclusions.
In the heat map it seems that for each group of samples, the average is being presented. I think it would be better to include all samples, rather than the average. This will give the reader more information in terms of similarity between samples within a group and will give an idea of the variability of particular bacterial groups.
At the y-axis of the barcharts it says Richness, I believe this should be Relative abundance.
Interesting research and good luck with the revisions!
Overall, the purpose of the study was to investigate the interactions between fungal growth stages and soil bacterial community, how soil bacterial community shapes fungal growth and vice versa. The objective is to be commended and provides new insights into fungal-bacterial dynamics.
However, there are several issues with the approaches utilized and the presentation of the data.
Why was the substarte autoclaved? Higher diversity at later stages may be due to bacterial growth after colonization at cultivation sites..
Line 84: It is not clear what and where the cultivation sites are.
Lines 87-99: three replicates per growth stage makes 4 fungal samples. How many substrate samples were obtained? Throughout the results and discussion, references are made to differences between substrates and between substrates and growth stages, yet no results to indicate this.
Line123: why the 0.001% cut off?
Lines 139-144. State the statistical values for results that are stated to be significant and vice versa, in the sections.
Lines 147-148: What does 295 OTUs shared in substrate between four growth stages mean? Again, state the associated statistical values for reports that are significantly different or not.
The results are present for both phyla and genera. Pick one. And revise discussion and other results sections accordingly.
Lines 168-174: There were no significant differences in fungal bacterial communities in across stages, despite the clear separation in the nmds plot. How does this relate to Table 2? Again, state the associated statistics.
Lines 187-199: Not entirely sure the heatmap analysis is required. Also, how did the 50 top bacterial OTUs get determined? Are these the OTUs that differed among samples from the LEfSe analysis?
Difficult to track results, for a variety of reasons.
1. There are phyla and genera results, but discussion crried out at phyla level.
2. There are different things mentioned but not presented. Substrates, substrate and growth stages, and growth stages. However, only growth stages are shown. If these were needed, they should be in results and not in supplementary materials
In the discussions, limit the speculations regarding functions of bacteria ( which were done at the phyla level), and avoid using words like co-evolved ( line 283) in this instance. The results do not speak to this.
Overall, I found the paper of values. I however, don't like the combination or rather a partial combination of substrate and fungal associated data. Partial because we don't see any actual substrate data. I would recommend focussing on only the fungal stages, doing the analysis at the genera level and discussing the results at the genera level.
Sterilizing the substrate prior to inoculation ensures a common starting point, but this impacts how bacteria can be said to improve fungal growth. However, in the 35 days it takes for the spores to germinate, colonization of the substrate would have occurred. I believe, just tracking the differences ( alpha and beta diversity) among soil samples at each sampling point and doing the same for fungal samples is a better approach, than comparing whats in the soils with whats in the fungus. You do not have any direct evidence of how this might be the case. Explain the sampling process for fungi and substrate properly. Minimize speculation of function based solely on this descriptive study, unless u can add quantitative data (RT-qPCR or qPCR) on the abundances of some of these bacterial genes in fungal samples.
Overall, this paper is well-written, well-structured and interesting. Detailed comments are provided on the pdf of the manuscript, but some overarching comments are given below.
The choice of which figures and tables have been placed within the main body of the text versus the supplementary material would benefit from revision. Figs S1 and S3, and Table S1, would be more appropriate within the main body, along with a figure similar to Fig S2. Fig 2, on the other hand, could be placed in supplementary material.
Please make sure that in-text citations list papers in date order, from oldest to most recent.
I have some concern over aspects of the analysis, particularly regarding whether and how differences in sequencing depth were controlled for. I assume that the data were not rarefied or subjected to other normalisation procedures before NMDS and PERMANOVA. What was the read distribution between samples? Both techniques are sensitive to differences in sampling depth, which can produce false clustering (see Weiss et al. 2017 Microbiome 5:27). In the case of PERMANOVA, this can be compensated for by including sample depth as a predictor; for NMDS, colouring the points by sequencing depth allows the plot to be checked by artefacts. Whilst I am not familiar with LefSE analysis, if it looks at differential abundance the data need to be normalised before analysis.
Some further detail is required in the Methods section – see notes on the manuscript.
The discussion focuses quite heavily on contextual information and restatement of results. I think it would be improved by digging further into interpreting the results, and hypothesising what may underlie them.
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