High-Throughput Sequencing Reveals the Effect of the South Root-Knot Nematode on Cucumber Rhizosphere Soil Microbial Community

Round 1

Reviewer 1 Report

The present manuscript is an interesting read and I have few recommendations that I believe, can help to improve the quality of the manuscript further. 

1. Lines 38-40 The statements put forth by the authors would be better if they had cited a reference to them. Please try adding supporting reference(s).

2. The nematode M. incognita (MI) lives well inside the roots of its hosts and final part of the introduction is a bit vague. It might be better to say that 'Infection with MI can influence rhizosphere microbial communities" rather than "MI affects microbial communities". I am not sure of the presented research can validate this. I request the authors to please consider revising their hypotheses and final part of the introduction.

3. I also have a major issue with MI affecting soil properties. The data presented are observations and cannot be termed as "cause and effect". My apprehensions are with how can MI directly influence soil properties when for a major part of their lifecycle, they are restricted inside the plant roots. If the authors want to continue with this argument, I would recommend to add other studies or references where direct effects of RKNs or other nematodes have been studies with respect to soil physicochemical & biochemical properties. Or you can include these data as observations. 

4. The legends of the panels in figure 3 are too small and difficult to read. I would recommend the authors to  make them larger in size or present them as separate figures in supplementary.

5. In section 3.4, the authors have compared fungal and bacterial taxa. I would like to know the reason for this comparison as I am unable to find its rationale in the discussion. 

6. Section 4.1. I am having the same apprehensions as discussed above in point 3. The observations are interesting given that all the greenhouses had the same soil type. However, arguments on RKNs directly affecting soil properties, that too to such an extend is far fetched. The important questions that I have is 'why & how?'. Also, the supporting citations are for Fusarium, a microbial plant parasite which is not pathologically or metabolically similar to MI. 

7. Some of the references are not appropriate. For example reference number 37 (line 328) does not even have the word pathogen in the publication. Similarly, reference number 42 (line 344) discusses about induced systemic resistance (ISR) and not the argument that Bacillus species can keep away pathogens. I understand there are a huge number of publications available on this regard and recommend the authors to use appropriate references.

8. Section 4.2. The first sentence (line 326-327) is too vague. I suggest the authors to be more specific or add appropriate reference to the argument.

 

 

 

1. Lines 51 & 52 I would recommend using the term 'nematicides'  as different terms such as insecticides and pesticides have been used for the same set of chemicals to control nematodes. Or please use a consistent term throughout the manuscript (in the Discussion section 'pesticide' has been used)

2. Line 302 it is of great value 'to research' can be  changed to something like 'to study' 

3. Section 4.2 has several instances where authors cite only one reference but mention the term 'studies' in their statements. Lines 327, 328, 342

Author Response

Comments and Suggestions for Authors

Point 1: Lines 38-40 The statements put forth by the authors would be better if they had cited a reference to them. Please try adding supporting reference(s).

Response 1: Thank you for your advice. We have added relevant references in the revised manuscript. (Line 41).

Point 2: The nematode M. incognita (MI) lives well inside the roots of its hosts and final part of the introduction is a bit vague. It might be better to say that 'Infection with MI can influence rhizosphere microbial communities" rather than "MI affects microbial communities". I am not sure of the presented research can validate this. I request the authors to please consider revising their hypotheses and final part of the introduction.

Response 2: Thank you for your comments. We agree with you and you have given good advice. We have revised the Introduction section of the manuscript. (Line 84~85).

Point 3: I also have a major issue with MI affecting soil properties. The data presented are observations and cannot be termed as "cause and effect". My apprehensions are with how can MI directly influence soil properties when for a major part of their lifecycle, they are restricted inside the plant roots. If the authors want to continue with this argument, I would recommend to add other studies or references where direct effects of RKNs or other nematodes have been studies with respect to soil physicochemical & biochemical properties. Or you can include these data as observations.

Response 3: Thank you for your comments. It is acknowledged that the data presented in this study are observational in nature and do not establish causality. Our hypothesis posits that the parasitic activity and consequential damage inflicted by RKNs on plant roots have an impact on the exudation of plant roots, thereby influencing the composition of rhizosphere microorganisms and ultimately leading to alterations in soil characteristics.

It has been shown that root-knot nematodes parasitizing the root system of crown chrysanthemum secrete and produce lauric acid to regulate the chemotaxis of nematodes and other microorganisms, thereby reducing nematode populations and mitigating nematode damage [1].

Rhizosphere soil properties are influenced by plant root activities such as the exudation of reactive carbon compounds and the uptake of mobile nutrients and water [2,3]. The plant-rhizosphere system also affects the biomass and activity of soil microorganisms that is generally enhanced due to root exudates [4]. Different soil types harbor particular indigenous microorganisms that control the influence of plant root activity on rhizosphere microbial communities [5]. Plant species releasing root exudates are thought to select for rhizosphere microbial populations that respond with chemotaxis and fast growth [6].

We are more than happy to make any further modification as necessary.

• Dong L, Li X, Huang L, Gao Y, Zhong L, Zheng Y, Zuo Y. Lauric acid in crown daisy root exudate potently regulates root-knot nematode chemotaxis and disrupts Mi-flp-18 expression to block infection. J Exp Bot. 2014 Jan;65(1):131-41. doi: 10.1093/jxb/ert356. Epub 2013 Oct 29. PMID: 24170741; PMCID: PMC3883285.
• George, T. S., Turner, B. L., Gregory, P. J., Cade-Menun, B. J., and Richardson, A. E. (2006). Depletion of organic phosphorus from Oxisols in relation to phosphatase activities in the rhizosphere. Eur. J. Soil Sci. 57, 47– doi: 10.1111/j.1365-2389.2006.00767.x
• Hartmann, A., Rothballer, M., and Schmid, M. (2008). Lorenz Hiltner, a pioneer in rhizosphere microbial ecology and soil bacteriology research. Plant Soil 312, 7-14. doi: 10.1007/s11104-007-9514-z
• Raaijmakers, J. M., Paulitz, T. C., Steinberg, C., Alabouvette, C., and MoenneLoccoz, Y. (2009). The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms. Plant Soil 321, 341– doi: 10.1007/s11104-008-9568-6
• Berg, G., and Smalla, K. (2009). Plant species and soil type cooperatively shape the structure and function of microbial communities in the rhizosphere. FEMS Microbiol. Ecol. 68, 1– doi: 10.1111/j.1574-6941.2009.00654.x
• Hartmann, A., Schmid, M., van Tuinen, D., and Berg, G. (2009). Plant-driven selection of microbes. Plant Soil 321, 235– doi: 10.1007/s11104-008-9814-y

Point 4: The legends of the panels in figure 3 are too small and difficult to read. I would recommend the authors to make them larger in size or present them as separate figures in supplementary.

Response 4: We are very sorry for the unqualified image work. We have replaced the image quality of Figure 3 and also made modifications to the description of the legends.

Point 5: In section 3.4, the authors have compared fungal and bacterial taxa. I would like to know the reason for this comparison as I am unable to find its rationale in the discussion.

Response 5: Thank you for your comments. We have modified this section again in the discussion. Following your suggestion, we have reorganized Discussion 4.2 in the revised manuscript to add descriptions of important microorganisms at the bacterial and fungal genus levels and their interactions with RKNs. We are more than happy to make any further modification as necessary. (Line 356~403)

Point 6: Section 4.1. I am having the same apprehensions as discussed above in point 3. The observations are interesting given that all the greenhouses had the same soil type. However, arguments on RKNs directly affecting soil properties, that too to such an extend is far fetched. The important questions that I have is 'why & how?'. Also, the supporting citations are for Fusarium, a microbial plant parasite which is not pathologically or metabolically similar to MI.

Response 6: We appreciate your comment, thank you for pointing it out. It is acknowledged that the data presented in this study are observational in nature and do not establish causality. Our hypothesis posits that the parasitic activity and consequential damage inflicted by RKNs on plant roots have an impact on the exudation of plant roots, thereby influencing the composition of rhizosphere microorganisms and ultimately leading to alterations in soil characteristics. The relevant references are listed in point 3. In addition, according to your suggestion, we have removed the inappropriate literature citations. We are glad to make any further modification as necessary. (Line 343~346)

Point 7: Some of the references are not appropriate. For example reference number 37 (line 328) does not even have the word pathogen in the publication. Similarly, reference number 42 (line 344) discusses about induced systemic resistance (ISR) and not the argument that Bacillus species can keep away pathogens. I understand there are a huge number of publications available on this regard and recommend the authors to use appropriate references.

Response 7: We apologize for this embarrassing mistake and have cited appropriate literature in the revised manuscript. Thanks to the reviewer for pointing it out.

Point 8: Section 4.2. The first sentence (line 326-327) is too vague. I suggest the authors to be more specific or add appropriate reference to the argument.

Response 8: Thank you for your comments. We have improved this section in the discussion and added relevant references in the revised manuscript. (Line 357~367)

Comments on the Quality of English Language

Point 1. Lines 51 & 52 I would recommend using the term 'nematicides'  as different terms such as insecticides and pesticides have been used for the same set of chemicals to control nematodes. Or please use a consistent term throughout the manuscript (in the Discussion section 'pesticide' has been used)

Response 1: Thank you for your advice. We agree with your comments, and we have improved this issue in the revised manuscript.

Point 2. Line 302 it is of great value 'to research' can be changed to something like 'to study'

Response 2: Thank you for your advice. We agree with your comments, and we have improved this issue in the revised manuscript.

Point 3. Section 4.2 has several instances where authors cite only one reference but mention the term 'studies' in their statements. Lines 327, 328, 342

Response 3: Thank you for pointing out these errors, and we have modified each of them in the revised manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

In the introduction you should consider work on the microbiota and its effect on crop pathogens.

 

 

They could consider in the part of the discussion works where the genus Pseudomonas has nematicidal activity.

 

 

Also in fungi the genus Trichoderma if there are reports with nematicidal activity

 

Emphasize the bacterial and fungal genera that are present in each treatment, as well as what biological activity they have to inhibit nematodes.

 

 

The work is very interesting and provides information on the microbiota and its possible function in the control of nematodes, however, they should take advantage of all the metagenomic results for discussion and the possible effect between bacterial communities and against the nematode.

Minor editing of English language required

Author Response

Comments and Suggestions for Authors

Point 1: In the introduction you should consider work on the microbiota and its effect on crop pathogens.

Response 1: Thanks for the constructive suggestions to improve our manuscript. According to your comments, we have made the changes. (Line 69~80)

Point 2: They could consider in the part of the discussion works where the genus Pseudomonas has nematicidal activity.

Response 2: Thanks for the comment. We have added some new studies related to the ability of Pseudomonas to suppress RKNs in section 4.2 of the discussion. (Line 378~390)

Point 3: Also in fungi the genus Trichoderma if there are reports with nematicidal activity

Response 3: Thanks for the comment. We have added some new studies related to the ability of Trichoderma to suppress RKNs in section 4.2 of the discussion. (Line 398~404)

Point 4: Emphasize the bacterial and fungal genera that are present in each treatment, as well as what biological activity they have to inhibit nematodes.

Response 4: Thank you for your advice. Following your suggestion, we have reorganized Discussion 4.2 in the revised manuscript to add descriptions of important microorganisms at the bacterial and fungal genus levels and their interactions with RKNs. We are more than happy to make any further modification as necessary. (Line 357~404)

Point 5: The work is very interesting and provides information on the microbiota and its possible function in the control of nematodes, however, they should take advantage of all the metagenomic results for discussion and the possible effect between bacterial communities and against the nematode.

Response 5: Thank you, we agree with your comment. The different molecular methods used (amplicon sequencing, qPCR, and metagenomics) each have their strengths and weaknesses and were used to complement each other. For example, amplicon sequencing provides an excellent taxonomic overview for many samples and allows detecting even very rare members of the community. Metagenomics, on the other hand, enables studying community functions in an untargeted way. However, both methods deliver only compositional data with many limitations. qPCR, therefore, complements these methods by providing absolute gene quantity values.

Among the methods currently used in the study, amplicon sequencing is very well-established and common. However, there are still certain problems. In fact, our collaborating team also performed metagenomics sequencing and qPCR assays based on this study and made some progress, but still need a lot of experiments to confirm our speculations. Next, we will explore the effect of inter-root microorganisms on RKNs in more depth.

 

Comments on the Quality of English Language

Point 1. Minor editing of English language required

Response 1: Thanks for the constructive suggestions to improve our manuscript. And sorry for the language problem, we had improved the language using a language editing service. We are glad to make any further modification as necessary.

Author Response File: Author Response.docx