The Effect of a Preparation Containing Rhizobial Nod Factors on Pea Morphological Traits and Physiology
Round 1
Reviewer 1 Report
It seems that the authors did not consider my major comment:
The conclusions are a summary of the results. Instead, they should be written in the form of both take-home messages and perspectives/potential for polish/international agriculture.
Author Response
Thank you for the possibility to check once again the text of our manuscript. This time we added some information about the devices used in our experiment and improved English. We added also the new Conclusions with the recommendations concerning the use of NFs preparation in the pea cultivation, as was suggested by a Reviewer. We hope these changes make our manuscript more readable and interesting.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The authors have made significant changes to the manuscript in agreement with the reviewers' suggestions. The replacement of most of the tables by column graphs makes the data particular easy to take in. A final revision of English usage would be very desirable.
Author Response
Thank you for the possibility to check once again the text of our manuscript. This time we added some information about the devices used in our experiment and improved English. We added also the new Conclusions with the recommendations concerning the use of NFs preparation in the pea cultivation, as was suggested by a Reviewer. We hope these changes make our manuscript more readable and interesting.
Author Response File: Author Response.pdf
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
The article “The effect of a preparation containing rhizobial Nod factors on pea morphological traits and physiology” describes the influence of Nod factor treatment on two pea varieties.
The effects of pretreatment of seeds of legumes with Nod factors have been repeatedly investigated. Such studies were carried out for peas, and by the authors of the article (Podleśny et al., The pleiotropic effect of rhizobial lipochitooligosaccharides on growth, development and yielding of pea (Pisum sativum L.). Cent. Eur. J. Biol. 2014, 9(4), 398-409; Podleśny, J., Wielbo, J., Podleśna, A., & Kidaj, D. (2014). The responses of two pea genotypes to Nod factors (LCOs) treatment. J Food Agric Environ, 12, 554-558). The authors have previously compared two pea genotypes in their responses to treatment by Nod factors, and one of the pea varieties was of the afila type, as in this article. Therefore, there is no novelty in this article. In addition, only 38 references were used in the article, of which only a few were published after 2010. No article cited in the past five years.
The article uses strange terminology, some statements are erroneous. For example.
lines 37-39. You wrote: “The unique ability to fixation of this form of nitrogen in a complicated process known as biological nitrogen fixation - BNF [4] is possessed only by physiologically and morphologically diverse diazotrophic bacteria [5–7]. However, to the full course of this process is necessary a legume plant which has to be infected by the proper bacteria strain from the Rhizobium genus”. This is a false statement, since various genera of rhizobia, representing α, β, and γ-proteobacteria, are capable of biological nitrogen fixation with legumes and non-leguminous like Parasponia, and also one should not forget about non-leguminous plants that fix nitrogen when interacting with Frankia.
lines 43-44. You wrote: “The BNF process takes place in specialised nodules, formed from layers of the inner bark of the primary florets of legumes”. This phrase sounds like complete nonsense. Nodules of different types are initiated either in the inner ROOT cortex (not bark!) or outer ROOT cortex.
lines 52-53. This statement must be supported by the appropriate references.
line 83. Nod factor are signalling molecules not “particles”!
In many cases you have used root papillae instead of nodule. This is completely unacceptable.
Author Response
Thank you for meaningful Review and all important comments. In response to comments of Reviewer 1 we would like to explain: 1. Our studies on the effect of a preparation containing of Nod factors were continued during some years. Although our experiments were conducted in the control conditions of greenhouse we used different types of cultivars to prove our earlier results. So, some parameters are publish in the earlier papers. From the scientific point of view it is interesting to know if different types of pea (traditional leafy and afila) show the same response to Nod factors preparation and improve/or not their metabolism and processes which led to achieve higher yields. However, at present the most popular pea cultivars in Poland belong to afila type. 2. We tried to change the “strange terminology” from the text of manuscript. 3. lines 43-44 : we have changed this sentence. 4. lines 52-53: we have changed this sentence and add a reference. 5. line 83: we removed this word from this part of manuscript. 6. We tried to replace the word “papillae” with “nodules”
Author Response File: 
Author Response.docx
Reviewer 2 Report
The Introduction has a somewhat confusingly written description of the nodulation process, best handled as a chronology from early to later stages. May require one or more general references to the known, specific effects of nod factors on plant morphology (ultrastructure) and physiology. These could be used as a basis to discuss possible mechanisms for these effects in the Discussion.
Similar work has apparently done before, again using an undefined mixture of compounds that includes nod factors. Most importantly, the authors can not refer to the material used as "a nod-factor preparation", since it contains all sorts of other things (eg, fatty acids). In all instances, it could be referred to as a "preparation containing nod-factors" or something of that sort.
In the Methods section, the nature of the "nod-factor preparation" should be made explicit, not just by giving a reference to paper 23.
Since the general trends in changes in plant physiology are important, rather than the absolute values, the Tables should in most cases be converted to some sort of graph.
The pea plants were apparently inoculated with rhizobia, but I didn't see any details on this.
The English language usage needs revision.
Other observations:
Line 48, I don't think that the occasional failure to establish a successful symbiosis can be blamed on the "complexity" of the process.
Line 71, is the 1.18 fold greater yield cited between the two cultivars really significant?
Line 91, thinned rather than tinned
Line 93, what does the "by the weight method" refer to?
Line 201, is it 4 pods or 4 pots?
Author Response
Thank you for meaningful Review and all important comments. In response to comments we would like to explain: 1. Our studies on the effect of a preparation containing of Nod factors were continued during some years. Although our experiments were conducted in the control conditions of greenhouse we used different types of cultivars to prove our earlier results. So, some parameters are publish in the earlier papers. 2. We agree with the suggestion that we should use “a preparation containing nod-factors” and we changes this in the text of our manuscript. 3. We tried to add some more information about the method of nod-factor preparation. 4. We would like to show many of our results so we decided to do tables with some groups of them. In the case of figures it is not easy to show all of them and it will be necessary more figures and place in the manuscript. On the other hand we want to show data because the differences are significant but not great and these data are not visible on the figure. 5. Pea plants were not inoculated with rhizobia because the experimental soil originated from the field where pea was cultivated and the abundance of Rhizobium was sufficient. 6. line 48 – you are right. 7. line 71- it is significant difference 8. line 91 – you are right . 9. line 93 – we tried to explain what is “weight method” use to the watering of plants in the pots. 10. line 201 – 4 pots is good
Author Response File: Author Response.docx
Reviewer 3 Report
Major comments:
The conclusions are a summary of the results. Instead, they should be written in the form of both take-home messages and perspectives/potential for polish/international agriculture.
Minor comments and suggested changes:
- 39: replace “bacteria” with “bacterial”
- 40: Rhizobium in italics
- 41: replace “with” with “which”
- 43: add a space after the full stop
- 44: I do not think that “florets” is the correct term
- 74: correct “two pea cultivars were included”
- 82: replace “cultivats” with “cultivars”
- 81-84: the aim of the work is not very clearly written; please, re-write
- 90: correct “into each pot”
- 92: rewrite nutrient values, it is unclear
- 94: what does “ppw” mean?
- 97: correct “two-factors”
- 101: M/dm-3 of water is an unclear concentration. Molarity is independent from volume. Did you mean “mol*dm-3”?
- 104: Correct “Extracts … … were obtained from … …”
- 110-111: this sentence is unclear (I don’t understand when the observation was stopped)
- 114: what is “gdzie”?
- 138: Delete “sampled and”
- 155-166: I think that this part is just a repetition and should be deleted.
- 202: Delete “use” and add a full stop
- 213-214: replace “date” with “data”
- 1: “Model” not in bold
- 216: Modal or Model?
- 2: here also there are incorrect bold type fonts and some commas to be replaced with dots
- 3: SPAD not in bold
- 4,5,6,7: again, incorrect bold fonts need to be corrected for clarity of table content
- 5: What does the values under “LSD 0.05” indicate? Are these the p-values? Then they are all not significant; so why you wrote “ns” for some of them? Please, clarify
- 263-273, 380, 398-400, 414-416: What are “papillae”? Do you mean “nodules”? Please, correct
- 6: the number of nodules is indicated as not significantly different between pea cultivars at BBCH 75 for both water and LCO treatments; is this correct? I ask this because at BBCH 60 the difference between the means are smaller but significant.
- 7: The post-hoc letters are used in a confusing way; sometimes “a” indicate the lower mean and “b” the higher mean and sometimes is the opposite. Moreover, some post-hoc letters are missed (for Number of pods per plant – Wiato – BBCH 75). Please, uniform the use of post-hoc letters for all the tables and correct where necessary
- 339: replace “differ” with “different”
- 370: correct the font of N2 (2 in subscript font)
- 375: “was found” must be moved to the end of the sentence
- 376: Fig. 1, not Fig. 5
- 378: replace “no yield increase” with “no more yield increase"
Author Response
Thank you very much for insightful, helpful Review and all important comments. In response to Reviewer 3 we would like to explain our answers on your comments and questions: 1. Lines 39, 40 and 43 – I did it according to your suggestions, 2. Line 44 – it should be flower, 3. Line 74 – I did the correction I this sentence, 4. Line 82 – I wrote properly a word “ cultivars”, 5. Lines 81-84 - we wrote new aim of our work, 6. Line 90 - We did correction marked place, 7. Line 92 - We wrote once again the amount of NPK which was given to the each pot, 8. Line 94 - ppw is a mistake – it is in Polish and means “field water capacity” 9. Line 97 – “two-factors” is corrected, 10. Line 101 – it is “mol * dm-3”, 11. Line 104 – Extracts … were obtained from” is corrected, 12. Line 110-111 – this sentence is corrected, 13. Line 114 – “gdzie “ is in Polish and means “where”, 14. Line 138 – I deleted “sampled and”, 15. Lines 155-166 – it was a repetition and it was deleted, 16. Lines 213-214 – we have replaced “date” with “data”, Model is wrote without bold, 17. Line 216 – this is Model cultivar, 2) we changed commas with dots, 3) SPAD is not in bold, 4,5,6,7 changed fonts,5) LSD 0.05 indicate: p≥ 0.05, 18. Lines 263-273 and others – papillae is not correct – we changed it on “nodules” , 19. Table 6 – you were right – we corrected these errors 20. Table 7 – we changed the “a” and “b” markers put at the data , 21. Line 339 – now is “different”, 22. Line 370 - now is N2, 23. Line 376 – now is Fig. 1, 24. Line 378 – we changed “no yield increase” on “no more yield increase”
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
In the revised version of the article “The effect of a preparation containing rhizobial Nod factors on pea morphological traits and physiology” some positive changes have been made.
However, I still guess that this article has no significant novelty in comparison with previous work. The reference list is still insufficient.
Author Response
Thank you for the re-review of our manuscript. In the newest version we tried to improve English as well as Introduction and the whole text to eliminate too long sentences and language mistakes. In relation to your last comment on the lack of significant novelity of our article we would like to explain that the submitted manuscript is a part of geater studies performed as a scientific project. Some results from this research were publish earlier and presented data are the last of them. The main aim of these research was to find the answer on the question : is it possible to increase the seed yield of pea by the use of Nod factor preparation and how the plants ( varied types of pea) response to that factor in aspect of different morphological and physiological features. So, some of presented features are repeated in this manuscript and some are the new -like content of chlorophyll and carotenoids. We use availabe in our Institute research equipments like Portable Photosynthesis System or Leaf Area Scanner and all experiments were conducted in the greenhouse to eliminate the other factors. On the end we would like to inform that we added 6 new, important articles to reference list which were published in 2010, 2018 and 2021.
Author Response File: Author Response.pdf
Reviewer 2 Report
I believe that the author's have corrected or at least addressed all of the problems that I indicated in my review of the previous version of the manuscript. However, I don't fully agree with their viewpoint on Point 4 regarding use of figures instead of tables, in particular the claim that "differences are significant but not great", which seems to be contradictory to me, since I don't equate statistical and biological significance. Also, further errors in English usage have been introduced with the modifications made to the text, and these should be corrected if possible. In the footnotes of tables, the word "date" is used instead of "data".
Author Response
Thank you for the re-review of our manuscript. Dear Reviewer we tried to improved our manuscript according your suggestions contained in the first and second review. Although last time we tied to defend the Tables but this time we changed them on Figures. We agree with you that now the differences between objects are more visible ( not less as we tought before !). Next, we tried to improved Introduction and the whole text in aspect of English, too long sentences and mistakes. We hope the new version of our manuscript is more informative and readable.
Author Response File: Author Response.pdf
