Effect of Bacillus velezensis GHt-q6 on Cucumber Root Soil Microecology and Root-Knot Nematodes
Round 1
Reviewer 1 Report (Previous Reviewer 1)
Comments and Suggestions for AuthorsThe authors improved the manuscript. However, i left some corrections in the manuscript - pls see the attached PDF.
Comments for author File: 
Comments.pdf
The English is fine, but it can be given a readthrough to further improve it by an English native speaker.
Author Response
Thank you for your significant reminding. According to your suggestion, we corrected the above grammatical errors and made an effort to correct the spelling and grammar errors and polish the whole manuscript.
Reviewer 2 Report (Previous Reviewer 2)
Comments and Suggestions for AuthorsThe manuscript presents a well-designed study investigating the potential of Bacillus velezensis GHt-q6 as a biocontrol agent against cucumber root-knot nematodes (RKNs) and its impact on soil microecology. The study is relevant to the field of sustainable agriculture and provides valuable understandings for the use of biocontrol agents as alternatives to chemical pesticides. The findings are supported by robust experimental data, including colonization studies, soil microbial community analysis, enzyme activity measurements, and nematode control efficacy.
The authors are suggested to read and cite the following latest articles to strengthen different sections of the manuscript especially introduction with regard to information on root-knot nematodes:
Abbas M, Saadedin S, Suleiman A (2024) Cloning and overexpression of Zea mays cystatin 2 (ccii) gene in Bacillus subtilis to reduce root-knot nematode infection in cucumber plants in Iraq. Plant Prot 8(4):621–633.
Yaseen I, Mukhtar T (2024) Impact of sequential and concurrent inoculations of Meloidogyne incognita and Fusarium oxysporum f.sp. vasinfectum on the growth performance of diverse okra cultivars. Plant Prot 8(2):303–313.
The descriptions of statistical methods are adequate, but the presentation of results (especially in figures and tables) could be improved for clarity. Ensure all error bars are present on graphs, and that all statistical significances are clearly marked.
The sample size of n=4 is rather low, and should be increased if possible.
The authors are suggested to address the following issues.
The methods section needs to benefit from more detailed descriptions, particularly regarding soil sampling and nematode extraction.
The connection between the in vitro testing of the strain, and the in vivo field testing should be made clearer.
The justification for the chosen time points for measurements should be provided.
The presentation of high-throughput sequencing data is complex and should be simplified for better understanding.
The discussion should be more focused and concise. Avoid excessive repetition of results.
The authors should strengthen the discussion by comparing their findings with more recent and relevant studies.
The discussion around the mechanisms of action of the bacteria should be more thorough.
The manuscript contains numerous grammatical errors and difficult phrasing. Careful editing is essential.
By addressing these points, the authors can significantly improve the quality of their manuscript and increase its suitability for publication.
Comments on the Quality of English LanguageThe manuscript contains numerous grammatical errors and difficult phrasing. Careful editing is essential.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report (Previous Reviewer 3)
Comments and Suggestions for AuthorsThe authors have revised
Author Response
Many thanks to the reviewers for your valuable comments that have improved the article
Reviewer 4 Report (Previous Reviewer 4)
Comments and Suggestions for AuthorsThis is a new version of an old manuscript that was rejected. A significant improvement is clear in the new version. However, the experimental design is still vague. For instance, it is written: “they were irrigated with various treatments (100 mL per plant): GHt-q6 (Treat1), B. subtilis (Treat2), clean water (Treat3), GHt-q6-Rif.” So, in which treatment lies GHt-q6-Rif ?.
The research is generally important and has significant results, but I call for rewriting it again slowly and carefully to clarify the part related to Materials and Methods before re-presenting it again, please.
Author Response
Thank you very much to the reviewers for their valuable comments, we have revised the material methods of the manuscript and marked them in red. In page 2 and page3。 And added an explanation of the GHt-q6-Rif.
Reviewer 5 Report (Previous Reviewer 5)
Comments and Suggestions for AuthorsFrom a nematology view, the lack of information on the population density of Meloidogyne in soil before planting is a serious shortcoming of the work. Furthermore, the experiment run only once. Therefore, the manuscript is not suitable for publication
Author Response
Many thanks to the reviewers for their comments. There is no information on the population density of root-knot nematodes before planting, because the location of our experiment was a greenhouse in Jinzhong, Shanxi Province. In winter, root-knot nematodes mostly overwinter with eggs, and rotary tillage is required two to three times before planting, so the population density is low and relatively uniform before treatment. So there is no statistical information.
The experiment run only once，We did not pay attention to this aspect when we did the experimental references.But we chose the greenhouses in the perennial disease, and the experiment had clear water as the control group, so the experimental data were reliable. However, we will improve these problems in future experiments.
Round 2
Reviewer 2 Report (Previous Reviewer 2)
Comments and Suggestions for AuthorsOnly minor grammatical errors remain
Author Response
Thank you very much for your valuable comments, we have revised the long sentences and grammatical issues in the manuscript. Hope to improve the quality of the manuscript。
Reviewer 5 Report (Previous Reviewer 5)
Comments and Suggestions for AuthorsThese type of experiments on biological control should be repeated as there is a high variation in results. Since the experiment run only once I insist in my previous statement which is rejection.
Author Response
Thank you very much for your valuable comments, which will be of great help to our future experimental design.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe revised version of the manuscript “Bacillus velezensis GHt-q6 as a promising agent for biocontrol 2 of root-knot nematodes on cucumber” has addressed most of my comments and suggestions provided in the 1st review process. The manuscript now is improved. However, there are still some inconsistencies noticed and minor refinements are still needed before potential publication. I also have added some remarks in the pdf file to be considered, please.
1- The introduction is improved but it still lacks some basic foundations, especially in the objective/hypotheses. In the revised version, the authors only provided the insights that could be generated from their study. Stating the objectives would follow the following format “Our study aims to (i) 1st objective; (ii) 2nd objective…” and so on. The same trend applied to the hypotheses “ Our study hypotheses that (i) 1st hypothesis; (ii) 2nd hypothesis…” and so on. Please adopt this in your 2nd revision.
2- The material and Methods section is improved as well. However, in the section: “2.3 GHt-q6-Rif and GHt-q6 strain fermentation broth treatment of cucumber seedlings”, I asked the authors to implement a visual scheme (as a figure), highlighting the main treatments used. This could be easily done and would make things more clear and understandable to the readers.
3- The authors worked very well on their results section. However, when stating the p-values (p <0.05) the authors should also add the Fisher index (Findex) and degree of freedom obtained (df) as recommended before. This is important for the statistical validity of the study.
4- The discussion is in good shape now. The authors are requested to refine the citation format aspect though.
5- Overall, the authors should invest some time in the English writing aspect of this paper, as many inconsistencies and mistakes were noticed in this version.
Comments for author File: Comments.pdf
Overall, the authors should invest some time in the English writing aspect of this paper, as many inconsistencies and mistakes were noticed in this version.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript provides a comprehensive analysis of the biocontrol potential of Bacillus velezensis GHt-q6 against root-knot nematodes in cucumber. The results are generally well-presented and the discussion is informative. However, there are areas where the manuscript could be improved to enhance its clarity, rigor, and overall impact.
The introduction includes several details about the economic importance of cucumbers and the impact of RKN that are somewhat extraneous to the core focus of the study. These details could be more brief.
There is some repetition in the explanation of the impact of RKN on crops, such as multiple references to nutrient deficiencies and yield loss. Streamlining these points would improve clarity and reduce redundancy.
The flow between paragraphs could be smoother. For example, the transition from the global importance of cucumbers to the discussion of the impact of RKN on agriculture feels abrupt. A more cohesive transition would help tie the concepts together.
From the “materials and methods” remove or condense some overly detailed procedural descriptions that are not essential for understanding the experimental setup.
Introduce the significance of rifampicin resistance earlier in the section and provide a brief explanation for its use.
Ensure each part of the section adds new information without repeating previous details. For example, the inoculation procedure could be explained once and cross-referenced when necessary.
Although the authors have used ANOVA and Duncan’s multiple range test for statistical analysis, it would be beneficial to provide more details about the experimental design (e.g., completely randomized design, randomized complete block design) and the specific statistical assumptions that were met.
Consider using a more robust statistical method like mixed-model analysis to account for potential variability between experimental units.
The authors have employed various techniques (e.g., α-diversity, β-diversity, MetagenomeSeq, LEfSe) to characterize the microbial community. However, a more in-depth discussion of the ecological effects of these changes would be valuable. For example, how do these changes in microbial community structure contribute to the biocontrol activity of GHt-q6?
The use of functional prediction tools (e.g., PICRUSt2, FAPROTAX) could provide further understandings for the metabolic potential of the microbial community and its role in biocontrol.
Although the authors have provided some insights into the potential mechanisms of action (e.g., competition, antibiosis), a more detailed investigation is needed to elucidate the specific mechanisms by which GHt-q6 exerts its biocontrol effect.
Although the overall writing is clear, certain sections could be further streamlined to improve readability. Consider using more concise language and avoiding unnecessary repetition.
Overall, the manuscript has the potential to make a significant contribution to the field of biocontrol. By addressing the points raised in this review, the authors can further strengthen the quality and impact of their work.
Comments on the Quality of English LanguageAlthough the overall writing is clear, certain sections could be further streamlined to improve readability. Consider using more concise language and avoiding unnecessary repetition.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe methods can be supplemented with the following data:
1. what species of root-knot nematodes were studied ?
2. did You know the population densities of each nematode species used in the experiment?
3. what cucumber variety was tested , what is the degree of its susceptibility or resistance to root-knot nematode species.?
Positives among others: grades of the numbers of nodules involved by root-knots nematode which are useful to the future surveys.
Reviewer 4 Report
Comments and Suggestions for AuthorsThe Manuscript ID: agronomy-3360662 examines Bacillus velezensis GHt-q6 as a promising agent for biocontrol of root-knot nematodes on cucumber. This study highlighted the significant potential of B. velezensis strain GHt-q6 to manage cucumber root-knot nematodes (RKNs) and improve soil health. The successful colonization of GHt-q6 in rhizosphere soils and cucumber roots could create a complex ecosystem with native microorganisms, and competition for space and resources in this ecosystem is critical for biocontrol effectiveness. The application of GHt-q6 had a positive effect on soil microbiology, enriched plant health, and reduced the RKN population density. Strain GHt-q6 mediated the interaction between soil microorganisms which ultimately decreased cucumber RKN disease. Eventually, the authors concluded that strain GHt-q6 can be involved in effective biocontrol strategies that provide sustainable alternatives to chemical pesticides, contribute to soil health, and agricultural productivity.
Generally, the subject is worth publication and the authors did a good job though typewriting the manuscript in two different colors is abnormal. Further insights should have improved this research. I’d suggest the following points:
1) The topic is original and relevant to the field as it addresses a specific gap in the field. However, it was much better to address the main question by the research via identifying these RKNs to the species level. So, in the present state, the main point addressing the RKN identification level (species/race) is quite ambiguous.
2) The authors defined their Treatments as follows: Treatment 1: irrigation with diluted GHt-q6-Rif fermentation broth was used for the determination of strain colonization; Treatment 2: irrigation with diluted GHt-q6 fermentation broth; Treatment 3: irrigation with 100 billion units/g of B. subtilis wettable powder diluted 100 times was used for the determination of the effect of biocontrol bacteria on soil microbial ecosystems and root-knot nematodes; Treatment 4: irrigation with clean water was used as a control. So, they should refer to each treatment number or the exact (re-state) treatment. On the contrary, several statements were quite ambiguous due to neglecting this rule. For instance, “After strain GHt-q6 treatment, the bacterial counts were all significantly higher than the control (p<0.05), and the promotion effect was better than the control agent” (which control agent? Please, clarify). Unclear statements occurred several times.
3) Figure 2. Effects of strain GHt-q6 on soil microbial and enzyme activities. You included treat 1, 2, and 3 as well without the control !! (clarify or add the control data, that is, Treatment 4).
4) It is much better to use the standard expressions, not new ones. For example, “Gall nematodes induce” should be root-knot nematodes or RKNs. Also, [In Grade 2, 25% to 50% of the total number of nodules “should be galls” in the root system were medium-sized…
Therefore, I would suggest resubmitting after major revision.
Reviewer 5 Report
Comments and Suggestions for AuthorsThe tittle is "B. v GHT-q6 as a promising agent for biocontrol of root-knot nematodes on cucumber" There is a high input on the study related on different aspects e.g the effect on soil microorganisms, recovery in plants etc but the part related on nematode control is quite poor and thin. There is no information on the population density of Meloidogyne in soil before planting, on the experimental design, the number of replicate plots etc. Furthermore, the experiment run only once. The Figure 6 and Table 1 do not contain the data of Treatment 4. Therefore, the manuscript is not suitable for publication
