Harnessing Rhizobial Inoculation for Sustainable Nitrogen Management in Mung Bean (Vigna radiata L.)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors described a study of glass-house screening followed by ¹⁵N field quantification to evaluate cross-legume rhizobial inoculants on mung bean. This paper is highly relevant to sustainable agriculture and to reducing mineral-N inputs. It presents a combined axenic screening with 15N natural-abundance field quantification, comparing the “high-BNF” phenotype (BR 96) with the “high soil-N uptake” phenotype (BR 3302). Nevertheless, several issues must be addressed before publication.

1. Only historical MAPA registration numbers are provided; no fresh authentication has been undertaken. Please supply: (1) 16S rRNA sequences of re-cultured strains (deposited in GenBank), (2) symbiotic gene (nodC/nifH) amplicons for the top two strains, (3) BOX- or ERIC-PCR fingerprints to rule out cross-contamination, and (4) a phylogenetic tree in the Supplementary Information.

2.Lines 191–197: Table 1 uses log(NN + 1) for ANOVA, whereas raw nodule numbers are interpreted in the text; please ensure consistency.

3. Lines 312–320 and 420–423: Figures 1 & 2 lack error bars; add SEM and indicate letter groupings.

4. The claim of “PGPR activity beyond BNF” for BR 3302 remains speculative, as no data on IAA, ACC-deaminase, siderophore or P-solubilisation are presented. Tone this down to a hypothesis and propose concrete assays.

5. Line 597–598: the Sci-Hub URL in Ref 20 must be replaced with the official DOI

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

General comment: The English could be improved to more clearly express the research.

Response: Thank you for your feedback. The current version was corrected using Grammarly (https://www.grammarly.com/).

 

Comment 1: Only historical MAPA registration numbers are provided; no fresh authentication has been undertaken. Please supply: (1) 16S rRNA sequences of re-cultured strains (deposited in GenBank), (2) symbiotic gene (nodC/nifH) amplicons for the top two strains, (3) BOX- or ERIC-PCR fingerprints to rule out cross-contamination, and (4) a phylogenetic tree in the Supplementary Information.

Response: Thank you for pointing this out. We would like to consider some aspects regarding comment 1.

Initially, it is important to clarify that the 11 strains used in this study are commercially used in Brazil and were previously selected for cultivating cowpea (BR3301, BR3302, BR3267, and BR3262), soybean (BR85, BR86, BR96, and BR29), and Phaseolus beans (BR322, BR534, and BR520). Although these sequences are not new, as Brazilian researchers isolated them at least twenty years ago, the soybean isolates, in particular, were obtained in the early 1990s and have since been used in various experiments on phylogenetic characterization and phytotechnical studies.

Brazilian researchers have a long history of developing inoculants with atmospheric N-fixing bacteria. Soybean is a prime example; when it was introduced around 1950, the soils lacked a rhizobial population capable of nodulating its roots, leading to the need for nitrogen fertilizer application. Since then, research efforts to create innovative and effective soybean inoculants have been vigorous, as exemplified by a recent article (see Rossetim et al 2025_Enhancing Soybean Yield Through Inoculation of Multifunctional Microbial Consortia). In 2023, soybean was cultivated on 44.4 million hectares, accounting for 15% of total agricultural land in the country (FAOSTAT; https://www.fao.org/faostat/en/#data, accessed on 2025/11/02). That year, about 158 million tons of soybeans were produced, and it is estimated that seed inoculation was used on around 90% of the planted area.

In this study, we evaluated commercial strains for inoculating mung bean, a crop that has recently gained popularity among Brazilian farmers. Assessing the performance of these strains offers an option to use an inoculant for mung bean without the need to develop new strains, a process that is labor-intensive and time-consuming.

At Embrapa Agrobiologia, we have a Biological Resource Center (BRC), registered with the World Federation for Culture Collections (WFCC, 364), where all strains are deposited and maintained after meeting strict quality control standards. The BRC is responsible for providing the strains to accredited companies for the production of commercial inoculants. Quality control is performed according to the protocols of MAPA (Brazilian Ministry of Agriculture and Livestock).

Technical-scientific publications containing the requested information and the current taxonomy for each strain have been included. The access numbers and locations of the requested gene in GenBank are also provided in the supplementary data.

Regarding the request to include a phylogenetic tree as supplementary data, we understand that this falls outside the scope of the article. Additionally, since none of the co-authors are specialists in this area, it will be necessary to add a new co-author if this requirement is maintained. I have already reached out to a colleague who can assist with this issue.

The following have been included in the text:

• Technical-scientific publications about the strains were included in Table 1 after Line 96.
• Information on the 16S rRNA, nodC, and nifH genes from GenBank, including genome accession numbers, is presented as Supplementary Material (table S1). Please see on Lines 98-99.

• Johanna Döbereiner Biological Resources Center, Embrapa Agrobiologia, Seropédica, Brazil, is registered with the World Federation for Culture Collections (WFCC, 364). Please refer to Lines 92-94.
• MAPA, Brazilian Ministry of Agriculture and Livestock. Please see on Line 97.
• The following strains (see Table 1) registered at MAPA (Normative Instruction No. 13, March 25, 2011; MAPA, 2013) were evaluated. Please see on Lines 90-91.

 

Comment 2: Lines 191–197: Table 1 uses log(NN + 1) for ANOVA, whereas raw nodule numbers are interpreted in the text; please ensure consistency.

Response: We agree with this comment. Therefore, we have changed to higher log (nodule number + 1). See Line 198-200.

Plant roots inoculated with the BR 3302, BR 96, or BR 3301 strains under axenic conditions showed a significantly higher nodule number, as analyzed by log (nodule number + 1), than the other ten strains and the non-inoculated control (Table 2).

 

Comment 3: Lines 312–320 and 420–423: Figures 1 & 2 lack error bars; add SEM and indicate letter groupings.

Response: We agree with this comment. Therefore, we added the standard error of the mean (SEM) and Tukey letter groupings to Figures 1 and 3, where applicable. For Figure 2, which is presented as a boxplot, the variability of the data is already represented by the median, quartiles, and whiskers, therefore, additional error bars were not included. Although this figure displays the data as a boxplot, Tukey's letters were based on comparisons of means and were placed above each box to facilitate interpretation of the statistical differences between treatments.

 

Comment 4: The claim of “PGPR activity beyond BNF” for BR 3302 remains speculative, as no data on IAA, ACC-deaminase, siderophore or P-solubilisation are presented. Tone this down to a hypothesis and propose concrete assays.

Response: Thank you for pointing this out. From your comment, we understand that the research answers a question, which leads to new questions that will be addressed in future works as part of a ongoing series of research and development projects. At this stage, we see the next step as conducting a new experiment. The final text is presented on Lines 503-505.

New experiments involving inoculation with BR 3302 and BR 96 strains, as well as co-inoculation of both strains, could reveal a complementary relationship in the PGPR mechanisms between the two strains.

Comment 5: Line 597–598: the Sci-Hub URL in Ref 20 must be replaced with the official DOI

Response: We agree with this comment and changed it to the oficial DOI. Please see L 630 (reference 32).

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript of dos Santos et al. looks at the contribution of different Bradyrhizobium strains to nodulation, nitrogen fixation, various growth traits, and yield in mung bean. They selected strains using a Leonard jar system and then chose from that set to run a field trial. The field trial showed that increased N from the Bradyrhizobium strains can come from either fixation or improved soil N uptake, suggesting the potential for co-inoculation with strains that specialize in each type of N incorporation. Overall, the manuscript is well-written, and the topic is interesting. I enjoyed reading it.

 

General points:

The introduction seems unnecessarily brief, perhaps to avoid redundancy because the manuscript is part of a special issue on the topic? I think the manuscript could benefit from a brief introduction on what has been done in terms of BNF in mung bean to this point – some references are already in the results and discussion section. This topic could be used to replace the 3^rd paragraph of the introduction as readers of the manuscript and the special issue will presumably be aware broadly of the benefits of inoculation.

Unlike the Introduction, the Results and Discussion section goes on at length, and I had to go back several times to remember which piece of data I was reading about. This is an inherent disadvantage of a combined results and discussion, but the manuscript would be improved by limiting discussion around well-known topics like N fertilizer suppression of nodulation (i.e. see Thornton, H. G. and Nicol, H. (1936). Reduction of nodule numbers and growth produced by the addition of sodium nitrite to lucerne in sand culture. J. Agric. Sci. 1.6: 173-188.) or PGPR effects of rhizobial bacterial.

Specific points:

Abstract:

Lines 28-30: As this paper points out, N fixing bacteria don’t always improve fertility or lower the need for fertilizer, etc. I would re-write to:

 “As a pulse crop, mung beans are associated with nitrogen-fixing bacteria, which can improve soil fertility, lower the need for nitrogen fertilizers, and increase yield and soil quality for subsequent harvests.”

Lines 33-35: The authors didn’t use the 15N natural abundance technique to assess grain yield. Re-write the sentence to clarify that.

Introduction:

Lines 55-57: This sentence doesn’t make sense on a couple levels. (1) Pulses aren’t vegetables they’re pulses, although some pulses can be eaten as vegetables before they dry (peas for example). (2) How does mung bean account for 90% of global production of pulses or vegetables? And (3) what unnamed country has the low grain yield? I would just delete this sentence and start at the next one.

Materials and Methods:

Line 84: add a reference for Leonard jars.

Line 101: Give recipes or references for any media or nutrient solution that are named.

Line 103: How was inoculation done? How many seeds per jar initially? How many CFUs/seed?

Line 105: Norris’s solution (recipe!) was added weekly by placing a 300 mL jar where/how? 300mL in the Leonard jar?

Line 140: How were the inoculants prepared? Reference?

Line 141: How many seeds? Target CFU/seed?

Results and Discussion

Lines 205-207: Missing reference.

Lines 272-274: should read “… probably due to an indigenous non-specific rhizobial bacterial population capable of nodulating mung bean.”

Line 304: Where’s the one hand? Delete “One the other hand” and start the sentence with “Nodulation in plants…”

Line 316: Why is the control called absolute? Why not non-inoculated?

Line 485: Figure 4. Delete “NEW HYPOTHESIS” from figure, as the idea for co-inoculation for different aspects of N incorporation is not new. See:

Pataczek, L., Armas, J.C.B., Petsch, T., Hilger, T., Ahmad, M., Schafleitner, R., Zahir, Z.A. and Cadisch, G., 2024. Single-Strain Inoculation of Bacillus subtilis and Rhizobium phaseoli Affects Nitrogen Acquisition of an Improved Mungbean Cultivar. Journal of Soil Science and Plant Nutrition, 24(4), pp.6746-6759.

The Pataczek et al. paper should be cited.

Author Response

General comment: Figures and tables must be improved

Response: Thank you for your feedback. All figures and tables were redrawn in R software to improve visual clarity and overall graphic quality in the revised version of the manuscript.

 

Comment 1 (general point): The introduction seems unnecessarily brief, perhaps to avoid redundancy because the manuscript is part of a special issue on the topic? I think the manuscript could benefit from a brief introduction on what has been done in terms of BNF in mung bean to this point – some references are already in the results and discussion section. This topic could be used to replace the 3^rd paragraph of the introduction as readers of the manuscript and the special issue will presumably be aware broadly of the benefits of inoculation.

Response: Thank you for pointing this out. As suggested, we have removed the 3rd paragraph and included the text below from Line 64 to 74.

The predominant mung bean symbiont in Brazilian agriculture is Bradyrhizobium. Among the species, B. yuanmingense shows the most significant increase in shoot biomass [6]. This genus significantly improves nodulation, root, and shoot biomass, while strains from the B. japonicum superclade strains perform better than those from the B. elkanii superclade [7]. Bradyrhizobium also nodulates mung beans in Ethiopia, a hotspot for rhizobial diversity [8]. Other rhizobia (Rhizobium, Mesorhizobium, Ensifer) and non-rhizobial endophytes (Leifsonia, Bacillus, Agrobacterium) have been isolated from mung bean nodules [6]. Fast-growing species such as E. aridi, E. meliloti, and R. pusense can also form effective associations with mung beans [9].

Research shows that mung bean nodulation with native rhizobia is generally lower in the Brazilian Cerrado (tropical savanna) compared to the Atlantic Forest [6].

 

Comment 2 (general point): Unlike the Introduction, the Results and Discussion section goes on at length, and I had to go back several times to remember which piece of data I was reading about. This is an inherent disadvantage of a combined results and discussion, but the manuscript would be improved by limiting discussion around well-known topics like N fertilizer suppression of nodulation (i.e. see Thornton, H. G. and Nicol, H. (1936). Reduction of nodule numbers and growth produced by the addition of sodium nitrite to lucerne in sand culture. J. Agric. Sci. 1.6: 173-188.) or PGPR effects of rhizobial bacterial.

Response: We agree with the comment about N fertilizer suppressing nodulation. Therefore, we have removed the following text.

Herridge et al. (2005) [24] evaluated mung bean inoculated with Bradyrhizobium strains under field conditions, observing nodule formation in 34 crops where the presence of high soil nitrate levels (> 100 kg N ha^-1) induced a reduction in nodulation to about 10 nodules per plant. In assessing mung bean nodulation under controlled conditions, Razzaque et al. (2016) [25] observed that doses above 40 kg N ha^-1 reduced nodulation, with the lowest values being observed at a dose of 100 kg N ha^-1. These authors pointed out that BNF is highly dependent on the availability of carbohydrates, which is a high-energy demanding process when compared to the absorption of mineral N and may explain the reduced nodulation in the presence of high N levels. Malik et al. (2014) [26] also observed a reduction in the nodule number in non-inoculated mung bean roots that received optimal doses of N-P-K. Increasing doses of N-fertilizer were able to linearly inhibit the nodule dry and fresh masses in cowpea, resulting in an average reduction of 50% in nodule dry mass at 140 kg N ha^-1 [27].

However, we did not remove the discussion section on plant growth-promoting bacteria, since this topic has become highly relevant in light of the results and was used to interpret them.

 

Abstract

Comment 3 (specific point): Lines 28-30: As this paper points out, N fixing bacteria don’t always improve fertility or lower the need for fertilizer, etc. I would re-write to:

 “As a pulse crop, mung beans are associated with nitrogen-fixing bacteria, which can improve soil fertility, lower the need for nitrogen fertilizers, and increase yield and soil quality for subsequent harvests.”

Response: We agree with your comment, so we have changed the texto to “As a pulse crop, mung beans are associated with nitrogen-fixing bacteria, which can improve soil fertility, lower the need for nitrogen fertilizers, and increase yield and soil quality for subsequent harvests.” Please see Lines 28-30.

 

Comment 4: (specific point): Lines 33-35: The authors didn’t use the 15N natural abundance technique to assess grain yield. Re-write the sentence to clarify that.

Response: Thank you for pointing this out. Promising strains were then tested in the field to assess grain yield and quantify nitrogen fixation using the 15N natural abundance method. Please, see Lines 33-35.

 

Introduction

Comment 5: (specific point): Lines 55-57: This sentence doesn’t make sense on a couple levels. (1) Pulses aren’t vegetables they’re pulses, although some pulses can be eaten as vegetables before they dry (peas for example). (2) How does mung bean account for 90% of global production of pulses or vegetables? And (3) what unnamed country has the low grain yield? I would just delete this sentence and start at the next one.

Response: We agree with this comment. We have deleted 'pulses' from the sentence and corrected the mung bean data to reflect that Asia accounts for 90% of the total production. Please see in Lines 55-58 the revised text.

Native to the India-Burma region, mung beans (Vigna radiata (L.) Wilczek) are widely cultivated throughout Asia, accounting for nearly 90% of the world's supply [3]. While India is the leading producer with an average annual yield of 1.48 million tons, its grain productivity remains relatively low at about 470 kg per hectare [1]. See Lines 54-57.

 

Material and Methods

Comment 6: (specific point): Line 84: add a reference for Leonard jars.

Response: Thank you for the comment. We have added a new reference [10] (Vincent, J.M. 1970) to Line 86. Please, check Line 567.

 

Comment 7: (specific point): Give recipes or references for any media or nutrient solution that are named.

Response: Thank you for pointing this out, we have added two new references regarding YM medium (Line 106 and 111). Please see reference [18] on Line 591; and Norris solution (Line 115), check reference [19] (Line 593).

 

Comment 8: (specific point): How was inoculation done? How many seeds per jar initially? How many CFUs/seed?

Response: Thank you for pointing this out. We have added the following information.

Five seeds were used per jar. Please, check on line 104.

After growth, 1 mL of the bacterial suspension, which results in an approximate concentration of 10⁸ CFU per seedling, was placed at the collar of each seedling using an automatic pipette. Five days after emergence (DAE), one plant was left per jar. See Lines 107-110.

 

Comment 9: (specific point): Norris’s solution (recipe!) was added weekly by placing a 300 mL jar where/how? 300mL in the Leonard jar?

Response: Thank you for pointing this out!

Lines 85-86: An assay was conducted in a greenhouse under axenic conditions at Embrapa Agrobiologia, in the municipality of Seropédica - RJ, using Leonard jars with two compartments [10].

And on Lines 111–114. Norris´s solution (N-free), prepared according to the instructions (S2), was added weekly by adding 300 mL to the lower compartment of the Leonard jar [10, 19]. The upper compartment was filled with a substrate made of sterilized gravel and vermiculite (2:1 v/v), and seeds were then sown.

 

 

Comment 10: (specific point): How were the inoculants prepared? Reference?

Response: Thank you for pointing this out. We don't have a specific publication on how we prepare the rhizobium inoculant; instead, we cited the Embrapa Agrobiologia’s Standard Operating Procedure (SOP) document in the text, which details the procedure.

Please see Lines 140-151. The inoculants were prepared under controlled laboratory conditions to ensure cell viability and uniformity, following the Standard Operating Procedure (code 023.03.02.22.2.001, revision 001) titled “Cell Culture for the Production of Inoculants,” Embrapa Agrobiologia: Seropédica, Brazil, 2004. Each bacterial strain was initially grown in test tubes with 5 mL of YM medium [18], supplemented with 500 μL of a stock suspension stored at −80 °C. The cultures were incubated on a rotary shaker at 150 rpm and 30 °C for 24 hours. Subsequently, the entire content of each tube was transferred to a 500 mL Erlenmeyer flask containing 50 mL of YM medium and incubated again under the same conditions for an additional 24 hours. After incubation, 25 mL of the culture was aseptically mixed with 40 g of autoclaved, sterilized peat that had been previously autoclaved at 121 °C for 30 minutes and stored in sealed containers. The cell concentration of the inoculant was approximately 10⁸ CFU mL^-1.

 

Comment 11: (specific point): How many seeds? Target CFU/seed?

Response: Thank you for pointing this out.  

Lines 161-163: Seeds were pre-inoculated with 10⁴ CFU per seed, and planting was performed manually with 15 seeds per linear meter, resulting in a population density of 300,000 plants per hectare.

Results and Discussion

Comment 12: (specific point): Lines 205-207: Missing reference.

Response: Thank you for pointing this out. You are probably referring to that sentence, “The isolation and characterization of 101 bacterial strains from nodules of two mung bean cultivars grown in different Brazilian soils revealed the prevalence of the Bradyrhizobium genus in inducing nodulation.” The reference for it is in the following sentence, ref 6 (Lines 224-227). We repeated the reference 6 (Line 555) at the end of the cited sequence.

Lines 247-227: The isolation and characterization of 101 bacterial strains from nodules of two mung bean cultivars grown in different Brazilian soils revealed that the Bradyrhizobium genus is involved in nodulation [6]. Biological nitrogen fixation (BNF) efficiency was influenced by the strain’s phylogenetic group and soil type [6].

 

Comment 13: (specific point): Lines 287-288: should read “… probably due to an indigenous non-specific rhizobium bacterial population capable of nodulating mung bean.”

Response: We agree with this comment. See on lines 276-278 the corrected sentence. Since there has been no recent history of legume cultivation in this area, the abundant nodulation in non-inoculated plants was likely caused by an indigenous rhizobial population capable of nodulating mung beans (Table 3).

 

Comment 14: (specific point): Line 304: Where’s the one hand? Delete “One the other hand” and start the sentence with “Nodulation in plants…”

Response: We agree with this comment. Please, check on Line 308, the revised sentence. Nodulation in plants grown in soil-filled pots tends to produce more nodules.

 

Comment 15: (specific point): Line 316: Why is the control called absolute? Why not non-inoculated?

Response: Thank you for pointing this out. The term 'absolute control' in biological nitrogen fixation experiments used to refer to control conditions, distinguished from the nitrogen control. Currently, there is a trend to use the more descriptive term, 'non-inoculated control.' Because of this, 'absolute control' has been replaced throughout the manuscript with 'non-inoculated control,' including in tables and figures.

 

Comment 16: (specific point): Line 485: Figure 4. Delete “NEW HYPOTHESIS” from figure, as the idea for co-inoculation for different aspects of N incorporation is not new. See:

Pataczek, L., Armas, J.C.B., Petsch, T., Hilger, T., Ahmad, M., Schafleitner, R., Zahir, Z.A. and Cadisch, G., 2024. Single-Strain Inoculation of Bacillus subtilis and Rhizobium phaseoli Affects Nitrogen Acquisition of an Improved Mungbean Cultivar. Journal of Soil Science and Plant Nutrition, 24(4), pp.6746-6759.

The Pataczek et al. paper should be cited.

Response: Thank you for pointing this out. We considered it a hypothesis because it was suggested by the results of this study, which, however, needs to be proven through a new experiment. The term 'new hypothesis' was removed as suggested. However, the article Pataczek et al. (2024) was not cited, since a quick search reveals several examples of co-inoculation that promote mung bean growth, which act through different mechanisms. Citing one or more references should be accompanied by a more in-depth discussion, which would be outside the scope of this article. See some examples of mung bean coinoculation.

Cough et al 2022_ The role of nutrients underlying interactions among root-nodule bacteria (Bradyrhizobium sp.), arbuscular mycorrhizal fungi (Funneliformis mosseae) and root-lesion nematodes (Pratylenchus thornei) in nitrogen fixation and growth of mung bean (Vigna radiata).

Htwe et al 2019_ Biofertilizer Produced from Bradyrhizobium and Streptomyces griseoflavus on Plant Growth, Nodulation, Nitrogen Fixation, Nutrient Uptake, and Seed Yield of Mung Bean, Cowpea, and Soybean

Htwe et al 2024_Isolation and characterization of mung bean (Vigna radiata L.) rhizobia in Myanmar

Nraragund et al 2020_ Influence of Crop Establishment Practices and Microbial Inoculants on Nodulation of Summer Green Gram (Vigna radiata) and Soil Quality Parameters.

Salehi et al 2019_ Effects of glomus mosseae and pseudomonas fluorescens on eco-physiological traits and antioxidant production of mung bean under drought condition

Shah et al 2023_Field co-inoculation of Bradyrhizobium sp. and Pseudomonas increases nutrients uptake of Vigna radiata L. from fertilized soil.

Yousefi et al_ Evaluation radiation use efficiency and growth indicators on two mungbean (Vigna radiata L.) genotypes under the influence of biological fertilizers.

Reviewer 3 Report

Comments and Suggestions for Authors

After carefully reading this paper through, I honestly have no comments for the authors. It is an excellent paper. 

Author Response

We sincerely appreciate your kind words and positive review of our manuscript. We are very grateful for your time and careful analysis. Your encouraging feedback reinforces the importance of our study and motivates us to continue improving the quality of our research.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors The authors have addressed the comments or provided reasonable explanations.