The Impact of Co-Inoculation with Bradyrhizobium japonicum and Azospirillum brasilense on Cowpea Symbiosis and Growth

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The symbiotic nitrogen fixation system of rhizobia in leguminous plants is of great significance in the natural nitrogen cycle and sustainable agricultural development. Developing microbial agents that promote nitrogen fixation in leguminous plants can help farmers reduce their use of nitrogen fertilizers. The author used Bradyrhizobium and Azospirillum to inoculate cowpeas and obtained the optimal combination to promote plant nodulation and growth. However:
Firstly, yield is the most important in cowpeas producitons, so the author needs to measure the yield indicators of the plants.
Secondly, why did you use Bradyrhizobium  instead of symbiotic rhizobia isolated from cowpea? Is there a more suitable symbiotic rhizobium in cowpeas?
Thirdly, Figs, There are no error lines in Fig 2, 3, and 4. It is necessary to clearly indicate how many plants were used for data analysis and to have methods and results for statistical analysis.

Author Response

Dear reviewer,

Thank you very much for your valuable contributions to improving the article.

Indeed, yield is the most important variable when working with agricultural crops. Yield variables and yield were measured in the experiment, however, along with other variables, they will be part of another article that is being prepared for publication.

We used a commercial Bradyrhizobium-based product (Gelfix 5®) because it is widely used in other legumes, such as soybeans. There is no symbiotic rhizobium that is more suitable for cowpeas.

The error lines were inserted in Figures 2, 3, and 4. Line 123 describes that twenty plants were collected from each plot for analysis of the field variables.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript studied the impact of coinoculation of rhizobium and plant growth-promoting rhizobacteria on Cowpea growth and symbiosis process. Considering the importance of biofertilizer application, this study provides effective results and supports the practical potential of biofertilizer application in agricultural fields. The following comments improve the manuscript:

Topic:

Line 1: Please change the topic to “ The impact of coinoculation with Bradyrhizobium japonicum and Azospirillum brasilense on Cowpea symbiosis and growth”

Abstract:

Lines 18-19: The abstract introduction is relatively more extended, and please summarize and focus more on the results.

Line 20-21: … Azospirillum enhances the symbiosis process and root exudates production in 

Line 22: control (is it a negative control?).

Line 22: isolated? Did you isolate these bacterial strains?

Line 29: Please support this sentence by adding numerical values and a statistical significance level.

Keywords: you can write your keywords as Vigna unguiculata; Coinoculation; Bradyrhizobium Japonicum; Azospirillum brasilense ; nodulation; isoflavones

Introduction:

Line 33: remove the second prentice of the scientific name.  

Line 51: Bradyrhizobium was not found in this reference. Please check all your references again.

Line 77-79: Please write your objectives in detail.

Method and materials:

Line 89: In Figure 1, only the average of precipitation and Temperature is enough and not necessary for minimum and maximum and relative air humidity. Also, instead of the name of the month, the number of the month is enough. Additionally, please revise the caption and incorporate a reference.

Line 93-99: Please make a table.

Line 94: In the chemical formula, please use the subscript.

Line 107: What is BRS?

Line 111: please check again writing, T1 (control: no inoculation), ….,

Line 112: How many cells/ mL were there?

Line 114-115: T5 is missing in the writing.

Line 117: Standak Top® and Protreat®, did these chemicals not affect the PGPB?

Line 125: for making the abbreviation, only the parameter is enough. For example, NN: number of nodules is enough, no need to add per plant (plant^-1).

Line 156: Please recheck the symbol (no underline under the degree symbol).

Line 172: Adding subtopics will better present the manuscript results.

Line 179. In the caption, the word “nodulation” is not appropriate, as well as in fig. 3 (nitrogen and dry mass); delete all.

Discussion

Line 341: The family name should be italicized.

Line 366: How do you explain the lower doses based on the mL unit and recommend it for other researchers and producers?

Line 384-385: Please rewrite this sentence and make it more visible as one of your main findings.

 

 

 

 

 

 

 

Author Response

Dear reviewer,

Thank you very much for your valuable contributions to improving the article.

Line 1: The title of the article has been changed to ‘The impact of coinoculation with Bradyrhizobium japonicum and Azospirillum brasilense on Cowpea symbiosis and growth.’

Lines 18 and 19: The introduction to the abstract has been summarised and more information has been added to the results.

Line 22: The control treatment is the control, i.e. the treatment to which nothing was applied.

Line 22: Isolated application means without coinoculation, only one bacterium.

Line 29: We understand that it is not feasible to insert these values in the conclusion because we worked with two varieties and the highest genistin values, for example, are over 4000 for Tumucumaque, but less than 1000 for NovaEra. The conclusion would be too long.

Keywords: Scientific writing recommends that we should not include words that appear in the title of the article in the keywords.

Line 33: the scientific name is correct

Line 51: the reference has been corrected

Lines 77-79: the objectives have been described in detail

Line 89: In Figure 1, the reviewer is mistaken because there is no data on minimum, maximum, and average relative humidity. There is data on precipitation, minimum and maximum temperature, and relative humidity, which are extremely important for understanding the behaviour of cowpea crops throughout the experiment's evaluation cycle. The name of the month is automatically generated by the program that created the graph. We understand that there is no reference to climate data from the Institute of Meteorology (INMET).

Lines 93-99: Table 1 was inserted as suggested by the reviewer.

Line 94: corrected as suggested by the reviewer

Line 107: BRS stands for Embrapa (Brazilian Agricultural Research Corporation), followed by the name of the cultivar. It is a nomenclature system used to identify plant varieties developed or recommended by Embrapa, a Brazilian public agricultural research company.

Line 111: corrected according to the reviewer's suggestion

Line 112: This information is in the line above, i.e., 5x109 CFU mL-1. CFU stands for colony-forming units

Lines 114-115: corrected according to the reviewer's suggestion

Line 117: we can verify from the results obtained that Standak Top® and Protreat® products did not affect PGPB. These are registered products recommended for legumes.

Line 125: corrected according to the reviewer's suggestion

Line 156: corrected according to the reviewer's suggestion

Line 172: corrected according to the reviewer's suggestion

Line 179: corrected according to the reviewer's suggestion

Line 341: according to scientific nomenclature, the name of the family is not italicised, only the genus and species

Line 366: the benefit of low doses of co-inoculation with bacteria is explained by the competition between the two species when together. At high doses, competition increases, leading to higher mortality, which reduces the benefit to plants.

Lines 384-385: corrected according to the reviewer's suggestion.

Reviewer 3 Report

Comments and Suggestions for Authors

In the article entitled “Coinoculation with Bradyrhizobium and Azospirillum in 2 Cowpea Seeds,” Luiz et al present a well-executed field study on the co-inoculation of Bradyrhizobium japonicum and Azospirillum brasilense in cowpea (Vigna unguiculata) and its impact on nodulation, nitrogen content, and isoflavone production. The work addresses a relevant topic in sustainable agriculture, linking biological nitrogen fixation to reduced fertilizer use and potential nutraceutical value. The inclusion of both morphological and biochemical traits, along with multivariate analysis (PCA), adds depth to the findings.

 

The manuscript makes a meaningful contribution to the field of legume microbiology and sustainable agriculture.

Author Response

Dear reviewer,

Thank you very much for your comments.

Reviewer 4 Report

Comments and Suggestions for Authors

The main objectives of the research are (i) to evaluate whether co-inoculation of Bradyrhizobium japonicum and Azospirillum brasilense in cowpea seeds results in improved nodulation, nitrogen content, and isoflavone production compared to (ii) single inoculations or the control, focusing on two cultivated varieties (BRS Tumucumaque and BRS Novaera).

 

The paper, in the opinion of the present Reviewer, is affected by the following scientific issues:

- Introduction: there is an incomplete description of the "state of the art." Indeed, the theoretical framework, while mentioning some key references, does not offer a critical discussion of the multiple agronomic and physiological variables involved in co-inoculation and often presents an overly descriptive summary of the literature, without exploring the limitations of previous research or the specificities of the varieties used.

 

- Materials and Methods: there is a lack of clarity in the experimental description. For example, when describing treatments and doses, the scientific rationale for the choice of volumes, especially the intervals between treatments, is not explained. Additionally, there is no clarity on how the effective establishment of the inoculated strains in the plant roots was ensured, a crucial factor given microbial competition in the soil.

 

- M&M: also, statistics and management of replications lack scientific robustness. Indeed, although block randomization with four replicates is indicated, the size of the individual plots and the achieved statistical significance are not specified. Moreover, details on handling missing data and verifying the assumptions underlying the tests used, such as normality and homoscedasticity, are entirely lacking.

 

- Results: there is a very limited characterization of soil and environmental conditions. Indeed, while pre-plant chemical analyses and climate data are reported, there is no critical discussion of the impact of soil characteristics, such as residual fertility and the natural population of endogenous rhizobia; a factor that is not negligible when attributing effects to inoculation alone.

 

- Discussion: in commenting main obtained outcomes, is the opinion of this Reviewer that the authors overinterpret some data. For example, the direct attribution to an increase in isoflavones following co-inoculation is not supported by a thorough explanation of the variations between varieties. It fails to consider other possible limiting or confounding factors. The analysis of the causal relationships between inoculation, nodulation, and secondary metabolite synthesis appears excessively linear and lacking in caution.

 

- Discussion: there is a great weakness in managing multivariate analysis. A PCA is presented, but a deep, scientifically based discussion of (i) the robustness of the selected components, (ii) the explained variance, and (iii) any negative correlations that may emerge is lacking. Overall, all these aspects leave the present Reviewer without adequate tools to assess the actual informative contribution.

 

- Discussion: the author's choice was to present data primarily through figures, without fully available tables showing mean values and standard Dev or (even better) SE. This could limit the reproducibility and interpretability of the results. The use of letters to indicate statistical differences between treatments is confusing, making it difficult for this Reviewer to evaluate the obtained results independently.

 

- Conclusions: the conclusions often seem dogmatic, suggesting the adoption of co-inoculation in the field based solely on a single trial, without considering (i) environmental variability, (ii) varietal differences, and (iii) interactions between factors that may emerge in other sites or seasons.

 

Considering all the previously reported aspects, rejection of the paper should be considered.

Author Response

Dear reviewer,

We understand that the introduction addresses the topic in a manner that is relevant to the understanding and importance of the manuscript. The reviewer suggests a critical review of the subject, however, the authors understand that this is a scientific article and not a review article on the subject in question.

The justification for choosing the co-inoculation volumes of Bradyrhizobium and Azospirillum was based on the recommended dose (150 mg kg-1) for legumes such as soybeans, since there is no recommendation for cowpeas, half the dose (75 mg kg-1), one and a half doses (225 mg kg-1) and double the dose (300 mg kg-1).

The size of the plots and the statistical significance used are described in the methodology section of the study. There were no missing data, and the assumptions underlying the tests used, such as normality and homoscedasticity, were verified. However, as the authors consider this information to be basic principles of experimentation, we understand that there is no statistical analysis if these assumptions are not met, and therefore there is no need to describe that they were verified.

No analyses of residual fertility and natural population of endogenous rhizobia were performed. However, we understand that the control, in which nothing was applied, was significantly inferior to the other treatments in all variables analysed. Therefore, if these items pointed out by the reviewer had interfered with the treatments, we would not have found any difference between the control and the treatments tested.

The authors took the utmost experimental care to isolate all factors that could interfere with the results. Thus, the differences in the data are indeed due to the treatments and not to uncontrolled factors, as the reviewer suggests, so that we can indeed attribute the increase in isoflavones and other variables to the effects of the treatments.

The authors believe that the Principal Component Analysis figures are well explained in the discussion section of the paper.

Error bars were inserted in Figures 2, 3, and 4 to facilitate the reviewer's understanding. The use of letters to indicate statistical differences between treatments is the simplest way to demonstrate the data obtained and is widely used in the scientific community.

The conclusions presented respond to the objective of the study in a practical way.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Please consider the following comments:

Line 30: Please write your keywords as:

Azospirillum brasilense; Bradyrhizobium japonicum; Cowpea; Coinoculation; Flavonoids

Keywords are the words that are frequently used in the manuscript, and their mention is essential to find your article and cite it.  

Line 33: please write as: (Vigna unguiculata L. Walp)

Line 91: What is the letter “c“at the end of the sentence?

Line 153: The symbol for degrees Celsius differs from the one used in line 130.

Fig. 2, start the Y axis from 0, not -20.

Line 184: add the “Determination of shoot dry weight and nitrogen content” instead of your subtitle.

Line 198: Quantification of … isoflavones

Author Response

We thank the reviewer for their contributions to improving the manuscript. We would like to inform you that all of the reviewer's suggestions have been accepted and corrected in the text.

Reviewer 4 Report

Comments and Suggestions for Authors

After carefully examining the revised version of the manuscript and the authors’ replies, I regret to inform you that the major concerns raised in my initial review remain substantially unresolved. While some minor improvements were attempted (such as clarifying the rationale for doses and adding error bars), the most critical scientific and methodological issues were not addressed in a constructive or satisfactory way.

The introduction continues to lack a critical framing of the state of the art and the specific agronomic or varietal gaps that justify the study. In the materials and methods, the authors still decline to provide transparency regarding statistical assumptions and handling of potential confounding factors, considering them "basic principles" that do not need to be reported. This position underscores a limited understanding of the importance of methodological reproducibility. Similarly, the lack of evaluation of background soil fertility and endogenous rhizobia remains unaddressed, which represents a major limitation when attributing observed effects solely to inoculation.

In the results and discussion sections, causal interpretations are overstated, and the possibility of other limiting or intervening factors is dismissed rather than discussed. The discussion of the PCA analysis remains superficial and does not meet scientific expectations of robustness. Finally, the conclusions are still expressed in a categorical and prescriptive way, despite being derived from a single trial under one site-year condition.

The overall tone of the response to the review also suggests that the authors perceive the process as adversarial rather than collaborative. Instead of engaging with the feedback to strengthen the manuscript, they frequently justify why no change was necessary. Such an approach indicates a lack of understanding of the role of peer review in improving the quality, reproducibility, and interpretative caution of scientific publications.

In my judgment, the manuscript does not meet the scientific standards required for publication in its present form. I therefore recommend rejection. Furthermore, given the nature of the authors’ replies and their unwillingness to address key scientific concerns, I will no longer be available to continue in the review process for this manuscript. 

 

 

 

 

Author Response

We regret to learn that our justifications were not accepted by the reviewer and that he will no longer be available to review our work. However, we would like to point out that three other reviewers also suggested changes to our article, which were accepted, thereby improving the manuscript.