PacBio Sequencing Unravels Soil Bacterial Assembly Processes along a Gradient of Organic Fertilizer Application
Round 1
Reviewer 1 Report
The manuscript titled: PacBio sequencing unravels soil bacterial assembly processes along a gradient of organic fertilizer application, by Wang et al., describes the bacterial community structure, ecological networks, and bacterial community assembly processes using the investment soil-cultivation test and PacBio sequencing. The manuscript is acceptable and the following points should be addressed:
L2: remove the word (Title) from beginning of title
L35: replace (and) with (,).
L41: replace (suggests) with (suggest)
L46: change (in identification in studies of) to (in identification studies of).
L63: add the reference after Sloan et al.
L72: add the reference after Ning et al. (not at the end of the phrase).
L86: add (consider) after (by multigradient fertilization).
L156: (Anderson and Walsh, 2013) reference is in wrong format (please put in numerical format according to the journal guidelines).
L209: In fig 1 legend, where is the details of D, E, F, G and H panels?
L219-220: explaining 77.3-78.7% and 71.2-73.5% of the community variance at the high and low fertilization levels, respectively. Do these values are related to HL, HH and RL, RH soils? If so, please add more details in the text to clarify the results.
L237: remove (Fig. 3A) at the end of the phrase.
L355-358: it’s a method phrase, please delete.
L375: put the reference (Wu et al. (2010) in the journal format.
The conclusion is too long, please reduce.
English is fine with minor editing required
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript describes the investigation of bacterial assembly processes in two types of soil with the application of an organic fertilizer gradient, using PacBio sequencing and robust statistical methods. In totality, the conceptualization, designing of experiments and the overall write up is good and clear. However, it needs minor corrections and there are some queries, which the authors should kindly respond to make it good.
1) Abstract (lines 14-16): The text requires revision, since there was a succession of the bacterial community in two types of soil.
2) Abstract (line 18): Authors need to briefly mention in the abstract the type of fertilizer used in the study (with organic matter ≥ 50% and N -P2O5-K2O ≥5%).
3) Introduction (lines 82-84): Although the authors take into account the neutral community model, it is important to describe which types of organic fertilizers have already been studied and the role of their composition in changes in microbial diversity in agricultural soils.
4) Results (figure captions): Authors must provide the meaning of H and R soil in all figure captions.
5) Results (lines 224-225): The authors need to explain this data better, since Figure S1 shows different values of generalist species.
6) Discussion (lines 337-339): Considering the text, the communities of pre-existing microorganisms in the natural organic fertilizer can strongly contribute to this alteration. A brief discussion of this should be considered. Mainly because the authors did not even mention whether the organic fertilizer used was not sterilized before experiments (materials and methods).
7) Discussion (lines 422-424): The text is confusing, it requires a reformulation of the way it was written.
8) Conclusion: Authors can add and revise this section for the better understanding of the topic and its future research.
Author Response
Response to Reviewer 2 Comments
Point 1: The manuscript describes the investigation of bacterial assembly processes in two types of soil with the application of an organic fertilizer gradient, using PacBio sequencing and robust statistical methods. In totality, the conceptualization, designing of experiments and the overall write up is good and clear. However, it needs minor corrections and there are some queries, which the authors should kindly respond to make it good.
Response 1: Thank you very much for acknowledging our research work. Your suggestions have helped to improve the quality of this manuscript, which we have carefully revised.
Point 2: Abstract (lines 14-16): The text requires revision, since there was a succession of the bacterial community in two types of soil.
Response 2: We have revised this sentence.
Point 3: Abstract (line 18): Authors need to briefly mention in the abstract the type of fertilizer used in the study (with organic matter ≥ 50% and N -P2O5-K2O ≥5%).
Response 3: We added a description of organic fertilizers in the Abstract section.
Point 4: Introduction (lines 82-84): Although the authors take into account the neutral community model, it is important to describe which types of organic fertilizers have already been studied and the role of their composition in changes in microbial diversity in agricultural soils.
Response 4: We reviewed the literature and added the following.
Commonly used organic fertilizers include composted animal manure, compost, sewage sludge, food processing wastes, and municipal biosolids [1]. Organic fertilizers often directly activate soil microbial biomass, gene abundance, taxonomic diversity and respiratory activity [2]. The application of organic fertilizers has long-term effects on soil microbial communities. However, the duration of this effect largely depends on the dose of fertilizer applied, as well as the period for which the fertilizer is applied [2].
Point 5: Results (figure captions): Authors must provide the meaning of H and R soil in all figure captions.
Response 5: We provided the meaning of H and R soil in all figure captions.
Point 6: Results (lines 224-225): The authors need to explain this data better, since Figure S1 shows different values of generalist species.
Response 6: We rewrote the sentences as follows.
“In general, generalist and specialist species were widely present in H and R soils, and the proportion of nonsignificant species was greater than 94% (Fig. S1). For the H soil, the proportion of generalist species in the low-fertilization group (HL, 0.7%) was higher than that in the HH group (0.6%, Fig. S1A/B). However, R soils had the opposite results, and the low-fertilization group (RL, 0.3%) was much lower than that in RH (0.6%, Fig. S1C/D).”
Point 7 Discussion (lines 337-339): Considering the text, the communities of pre-existing microorganisms in the natural organic fertilizer can strongly contribute to this alteration. A brief discussion of this should be considered. Mainly because the authors did not even mention whether the organic fertilizer used was not sterilized before experiments (materials and methods).
Response 7: The organic fertilizer used in this study was not sterilized before the experiments, and we described this fact in the revised manuscript.
Although the organic fertilizer used in this study was not sterilized before the experiments, the majority of organic fertilizer microorganisms do not survive in soil [2]{{{Semenov, 2021 #327}}}. After 2 weeks of application of organic fertilizer, the above microbial characteristics showed a downwards trend [2]. The vast majority of microorganisms associated with organic fertilizers, especially gram-negative bacteria, die almost immediately in the soil environment, and only a few genera can survive in soil for several months. Fungal abundance and diversity begin to decline after the 9th week of application of organic fertilizers [2].
Point 8 Discussion (lines 422-424): The text is confusing, it requires a reformulation of the way it was written.
Response 8: We rewrote this sentence.
“This shows that soil type has an important influence on the dominant bacterial community assembly process, which involves a wide spectrum of bacterial phyla.”
Point 9 Conclusion: Authors can add and revise this section for the better understanding of the topic and its future research.
Response 9: We have rewritten and streamlined the conclusion section.
In conclusion, high-dose application of organic fertilizer caused significant succession of the bacterial community in 2 different soil types. This succession is also closely related to the physical and chemical changes in soil caused by fertilization. The NCM and iCAMP analysis demonstrated that an increase in the amount of fertilizer applied changed the mechanism of bacterial community assembly, in turn increasing the potential importance of stochastic processes. With an increase in the fertilization dose, the importance of drift (DR) processes increased significantly, and the importance of homogeneous selection (HoS) decreased significantly. The presented results provide new insights for understanding the process of bacterial community assembly driven by the application of high doses of organic fertilizers and for studying the mechanism of community assembly of the same microbial lineages in different soils. Further research is required to verify the contribution of the high-dose application of organic fertilizer on the community construction process of rhizosphere microbial communities, root endophytes, and functional genes, especially on a larger geographical scale.
- Khan, M.N.; Mobin, M.; Abbas, Z.K.; Alamri, S.A. Fertilizers and Their Contaminants in Soils, Surface and Groundwater. In Encyclopedia of the Anthropocene, Dellasala, D.A., Goldstein, M.I., Eds.; Elsevier: Oxford, 2018; pp. 225-240.
- Semenov, M.V.; Krasnov, G.S.; Semenov, V.M.; Ksenofontova, N.; Zinyakova, N.B.; van Bruggen, A.H.C. Does fresh farmyard manure introduce surviving microbes into soil or activate soil-borne microbiota? Journal of Environmental Management 2021, 294, 113018, doi:https://doi.org/10.1016/j.jenvman.2021.113018.
Reviewer 3 Report
Organic fertilizers have potential advantages over inorganic fertilizers in improving soil fertility and crop yield without harmful side-effects. However, the effects of organic fertilizers on the soil microbiome and metabolome remain largely unknown, especially in the context of used doses and interactions with soil type and fertilization. In this study, bacterial community structure, ecological networks and bacterial community assembly processes were assessed using the investment soil cultivation assay and PacBio sequencing. The presented results provide new insights for understanding the process of bacterial community accumulation driven by the application of high doses of organic fertilizers and for studying the mechanism of community accumulation of the same microbial lineages in different soils. I do not comment on the reviewed manuscript, but I have a question whether both files, supplementary file(s) and non-published material are needed.
Author Response
Response to Reviewer 3 Comments
Point 1: Organic fertilizers have potential advantages over inorganic fertilizers in improving soil fertility and crop yield without harmful side-effects. However, the effects of organic fertilizers on the soil microbiome and metabolome remain largely unknown, especially in the context of used doses and interactions with soil type and fertilization. In this study, bacterial community structure, ecological networks and bacterial community assembly processes were assessed using the investment soil cultivation assay and PacBio sequencing. The presented results provide new insights for understanding the process of bacterial community accumulation driven by the application of high doses of organic fertilizers and for studying the mechanism of community accumulation of the same microbial lineages in different soils. I do not comment on the reviewed manuscript, but I have a question whether both files, supplementary file(s) and non-published material are needed.
Response 1: Thank you very much for acknowledging our research work. We checked both files, supplementary files and nonpublished material to confirm their completeness. Because some of the tables were too large, we presented them as separate Excel files and explained them in the supplemental materials document.
