Promotion of Peanut (Arachis hypogaea L.) Growth by Plant Growth-Promoting Microorganisms
and Everlon Cid Rigobelo *
Round 1
Reviewer 1 Report
In this work, the authors have reported the effects of various bacterial inoculation on the growth of peanut plants. Authors have systematically performed experiments with various microbial inoculants to study how the inoculant changes plant height, dry matter, the concentration of nitrogen and phosphorus, etc. While I commend the authors for undertaking this challenging project, I have a few concerns that I would like the authors to provide an explanation, especially regarding the design of this study.
- Line 148 (Methods section) authors report that B. japonicum was used only in the seed treatment, showing the best results compared to all other bacterial inoculation. This begs the question of whether the authors’ results are because of different application strategies between different bacteria or bacterial species. Why was the application of bacterial inoculation not consistent across all bacterial strains?
- It is unclear why the concentration of bacterial inoculant was kept at 1 x 108 colony-forming units. What would happen if a different concentration was used? Is inoculation with 1 x 108 CFU a standard in the field? Please cite appropriate references on why these values were used,
- In methods, please provide more details on how the bacteria were applied to the soil.
- Did authors verify if the inoculated bacteria survive in the soil over time? How do bacteria count in soil change over time? If the bacterial species die over time at different rates?
- Why was the combination of bacteria tested, and how was the combination selected?
- In the introduction, Lines 82-83, it is unclear how PGPM is a promising alternative. The authors haven’t provided sufficient motivation. Authors claim that PGPM is a promising alternative but haven’t clarified what PGPM will replace. Urease, Ammonia? Fertilizers? Please clarify.
- The discussion currently reads as a repetition of the results, I would encourage authors to dive deeper and provide some insights and reasoning into why they are observing what they have reported. List of questions that they could begin answering:
o Why is one kind of bacteria performing better than? What has literature reported on it? What is the reasoning behind one bacterial species performing better than another for plant growth?
o Are what authors reporting is it consistent with what has been reported in the literature? Why or why not?
Other minor comments
- In the graphs, what do the floating letter indicate – ‘a, ‘b’? Please explain in the figure caption.
- Please provide a separate section on statistics in the methods section. How was the analysis performed, and what was considered significant?
Author Response
Reviewer1 - In this work, the authors have reported the effects of various bacterial inoculation on the growth of peanut plants. Authors have systematically performed experiments with various microbial inoculants to study how the inoculant changes plant height, dry matter, the concentration of nitrogen and phosphorus, etc. While I commend the authors for undertaking this challenging project, I have a few concerns that I would like the authors to provide an explanation, especially regarding the design of this study.
All the changes were written in red color
Answer: We thank you, reviewer, for the opportunity given to us to improve our manuscript. We really appreciate your work.
Reviewer1- Line 148 (Methods section) authors report that B. japonicum was used only in the seed treatment, showing the best results compared to all other bacterial inoculation. This begs the question of whether the authors’ results are because of different application strategies between different bacteria or bacterial species. Why was the application of bacterial inoculation not consistent across all bacterial strains?
Answer: It is a very good question. Usually, B. japonicum is applied through the seeds and B. subtilis and T. harzianum do not, and we made it in the greenhouse to follow as the farmers do. In the field trial, B. japonicum was applied through leaves. We compared different microorganisms following the usual way of application of each one.
Reviewer1- It is unclear why the concentration of bacterial inoculant was kept at 1 x 108 colony-forming units. What would happen if a different concentration was used? Is inoculation with 1 x 108 CFU a standard in the field? Please cite appropriate references on why these values were used,
Answer: We kept the same microbial concentration found in the chamber for 72 hours after microbial growth. The microbial concentration used in the studies varies from 108 and 109. We evaluated the effect of microbial concentration and found the same results for 104 and 1010 in the cotton crop. Please see Escobar Diaz, P. A., Dos Santos, R. M., Baron, N. C., Gil, O. J. A., & Rigobelo, E. C. (2021). Effect of Aspergillus and Bacillus concentration on cotton growth promotion. Frontiers in Microbiology, 12, 737385.
Reviewer1- In methods, please provide more details on how the bacteria were applied to the soil.
Answer: In the greenhouse conditions, the microorganisms were applied directly to the soil surface near the plant with the aid of a pipette. In the field condition, the microorganisms were applied with the aid of an automatic sprayer. It has been added in the text.
- Did authors verify if the inoculated bacteria survive in the soil over time? How do bacteria count in soil change over time? If the bacterial species die over time at different rates?
Answer: In this study, it was not carried out. Usually, the applied bacteria keep in the soil for 2-3 weeks. We made it in the past. The appropriate concentration of bacterial inoculum permits bacterial colonization in the soil, and low concentration may not. When the concentration of microbial inoculum permits microbial colonization; higher concentrations of the same inoculum have no effect. Please see Nascimento, F. C., Santos, C. H. B., Kandasamy, S., & Rigobelo, E. C. (2019). Efficacy of alginate-and clay-encapsulated microorganisms on the growth of Araçá-Boi seedlings (Eugenia stipitata). Acta Scientiarum. Biological Sciences, 41, 43936.
- Why was the combination of bacteria tested, and how was the combination selected?
Answer: The combination was carried out according to the abilities of each microorganism. B. japonicum has a high affinity with legumes. Bacillus subtilis has the ability to solubilize phosphorus, and T. harzianum has the ability to synthase indole acetic acid. Our research group has worked with these microorganisms previously, and some combinations have been carried out. Please see the references below.
LOBO, Laiana Lana Bentes; DOS SANTOS, Roberta Mendes; RIGOBELO, Everlon Cid. Promotion of maize growth using endophytic bacteria under greenhouse and field conditions. Australian Journal of Crop Science, v. 13, n. 12, p. 2067-2074, 2019.
Diaz, P. A. E., Baron, N. C., & Rigobelo, E. C. (2019). 'Bacillus' spp. as plant growth-promoting bacteria in cotton under greenhouse conditions. Australian Journal of Crop Science, 13(12), 2003-2014.
Lobo, L. L. B., da Silva, M. S. R. D. A., Carvalho, R. F., & Rigobelo, E. C. (2022). The negative effect of coinoculation of plant growth-promoting bacteria is not related to indole-3-acetic acid synthesis. Journal of Plant Growth Regulation, 1-10.
- In the introduction, Lines 82-83, it is unclear how PGPM is a promising alternative. The authors haven’t provided sufficient motivation. Authors claim that PGPM is a promising alternative but haven’t clarified what PGPM will replace. Urease, Ammonia? Fertilizers? Please clarify.
Answer: Plant growth-promoting microorganisms is a group of microorganisms with several abilities related to plant growth. These abilities are phosphorus solubilizing, phytohormones production, induced systemic resistance, nitrogen fixation, biocontrol and etc. These abilities are offered to plants promoting the increase of growth and yields. In this way, these microorganisms can be used to reduce the amount of fertilizers and pesticides in crop production. This text has been added.
Reviewer1- The discussion currently reads as a repetition of the results, I would encourage authors to dive deeper and provide some insights and reasoning into why they are observing what they have reported. List of questions that they could begin answering:
Answer: The results of some studies have been added. There are few studies with B. japonicum and Trichoderma in peanuts. But we compared some results.
Reviewer1 - Why is one kind of bacteria performing better than? What has literature reported on it? What is the reasoning behind one bacterial species performing better than another for plant growth?
Answer: Although these microorganisms be carriers of many abilities related to plant growth, this effect depends on several factors such as plant genotype, type of soil, nutritional status, and plant development. Therefore, maybe there are no better microorganisms than others. But in this specific condition, for this plant genotype, B. japonicum promoted higher plant growth than A. brasilense, B. subtilis, and T. harzianum.
Reviewer1 - Are what authors reporting is it consistent with what has been reported in the literature? Why or why not?
Answer: We expected better results for B. subtilis because our group has worked with this strain in many crops, and this strain has shown interesting results. But the results are consistent with the literature because B. japonicum has a great affinity with legumes, mainly soybean and peanut is a legume.
Other minor comments
- In the graphs, what do the floating letter indicate – ‘a, ‘b’? Please explain in the figure caption.
Answer: Means followed by the same letters did not differ by Duncan's test at 10% probability.
- Please provide a separate section on statistics in the methods section. How was the analysis performed, and what was considered significant?
Answer: It has been done.
Analysis of results
The data obtained were subjected to variance analysis, with the F test application using the program AgroEstat [46]. When there was significance, a comparison of means was performed using the Scott Knott test at 10% probability.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript entitled "Promotion of peanut growth by microorganisms" presents the evaluation the effects of different Plant growth-promoting microogranisms on peanuts.
The manuscript is interesting and easy to understand. I have only few minor suggestions and questions as follows:
1. How and why you choose those microorganisms in table 1 in this study?
2. What's the strain information of T. harzianum and B. japonicum?
3. As B. japonicum was the most effective, I suggest the authors to introduce more about the effect/mechanisms of B. japonicum on the other plants in the introduction part.
4. For the crop fertilization, why with a 20% reduction and what's the result without reduction? Please address.
5. B. japonicum was only used in the seed treatment and other microorganisms are not, please discuss the possible effects on different treatment and why use different treatments.
6. The result shows inoculation of B. japonicum alone is better than mixing microorganisms, please compare with previous studies and discuss more about this phenomenon.
Author Response
Reviewer2 - The manuscript entitled "Promotion of peanut growth by microorganisms" presents the evaluation the effects of different Plant growth-promoting microogranisms on peanuts.
Reviewer2 - The manuscript is interesting and easy to understand. I have only few minor suggestions and questions as follows:
Answer: We thank the reviewer by the opportunity given to us to improve our manuscript. We really appreciate your work.
Reviewer2 - 1. How and why you choose those microorganisms in table 1 in this study?
Answer: Our group has worked with these microorganisms and had interesting results with them. Then we decided to evaluate them in peanut because there is few studies with these microorganisms in peanut. Please see the studies from our group.
LOBO, Laiana Lana Bentes; DOS SANTOS, Roberta Mendes; RIGOBELO, Everlon Cid. Promotion of maize growth using endophytic bacteria under greenhouse and field conditions. Australian Journal of Crop Science, v. 13, n. 12, p. 2067-2074, 2019.
Diaz, P. A. E., Baron, N. C., & Rigobelo, E. C. (2019). 'Bacillus' spp. as plant growth-promoting bacteria in cotton under greenhouse conditions. Australian Journal of Crop Science, 13(12), 2003-2014.
Lobo, L. L. B., da Silva, M. S. R. D. A., Carvalho, R. F., & Rigobelo, E. C. (2022). The negative effect of coinoculation of plant growth-promoting bacteria is not related to indole-3-acetic acid synthesis. Journal of Plant Growth Regulation, 1-10.
Bueno, C. B., Dos Santos, R. M., de Souza Buzo, F., & Rigobelo, E. C. (2022). Effects of chemical fertilization and microbial inoculum on Bacillus subtilis colonization in soybean and maize plants. Frontiers in Microbiology, 13.
Reviewer2 - 2. What's the strain information of T. harzianum and B. japonicum?
Answer: These information have been added. B. japonicum and T. harzianum were acquired from Embrapa, classification NC 230 (SEMIA 6439), and CEN199, respectively.
Reviewer2 - 3. As B. japonicum was the most effective, I suggest the authors to introduce more about the effect/mechanisms of B. japonicum on the other plants in the introduction part.
Answer: Much information have added in Introduction and Discussion section.
Reviewer2 - 4. For the crop fertilization, why with a 20% reduction, and what's the result without reduction? Please address.
Answer: Interesting question. Some studies show that nutritional necessity improves the interaction between plants and microbes. Then the amount of fertilizer was reduced by 20%. I think without reduction, the interaction could be decreased.
Reviewer2 - 5. B. japonicum was only used in the seed treatment and other microorganisms are not, please discuss the possible effects on different treatment and why use different treatments.
Answer: According to Agriculture Brazilian Agency B. japonicum must be applied through seeds. We decided to compare other microorganisms with the standard recommended by the Agency. I think now we should evaluate other ways to apply the B. japonicum.
Reviewer2 - 6. The result shows inoculation of B. japonicum alone is better than mixing microorganisms, please compare with previous studies and discuss more about this phenomenon.
Answer: It is a good discussion. In fact, the use of formulated preparations consisting of a single microbial species or isolates as inoculants (i.e., a single antagonist against a single pathogen) has often resulted in inconsistent performance in agriculture and, consequently, in low representation in the world inoculant market. One of the reasons for such failure may be that a single microbial agent is unlikely to be active in all soil environments (in the presence of different biotic and abiotic stressors) or against all pathogens attacking the host plant. One way to overcome this problem is to include different species or strains of beneficial microbes in the same microbial formulation. The application of binary or multiple mixtures would mimic the natural situation more closely and could broaden the spectrum of biocontrol activity (Raupach and Kloepper, 1998). However, co-inoculation may not promote effects when the strain interacts with the plant genotype.
Author Response File: Author Response.docx
Reviewer 3 Report
The study has some interesting aspects which would be of interest to the international scientific community. However, this manuscript needs significant improvements, both in terms of language and scientific content.
Title (Line 2): „Promotion of peanut growth by microorganisms“ – too vague. I suggest „Promotion of peanut (Arachis hypogaea L.) growth by plant growth-promoting microorganisms“
Abstract (Line 9): „In addition, the use of microorganisms that promote plant growth„ – include abbreviation – microorganisms that promote plant growth (PGPM)
Abstract (Line 12): „is the use of microorganisms that promote plant growth“ – the use of PGPM
Abstract (Line 12-15): „The objective of this study was to evaluate the effects on growth promotion and productivity of peanuts inoculated with the microorganisms Azospirillum brasilense, Bacillus subtilis, Bradyrhizobium japonicum and Trichoderma harzianum“ – include more information about experimental design, inoculation method and experimental conditions
Abstract (Line 15-18): „The results show that, in general, all the microorganisms evaluated in the present study promoted increases in the various parameters analyzed, root dry mass, shoot dry mass, phosphorus concentrations and plant height, under both conditions, in a greenhouse and under field conditions compared to the control treatments that did not receive microbial inoculation“ – A lot of unnecessary words, delete: The results show that, in general; various parameters analyzed; under both conditions; treatments that did not receive microbial inoculation.
Abstract (Line 19-21): „These results show that it is possible to obtain increases in the peanut crop with the inoculation of plant growth-promoting microorganisms without major changes in the management and conduct of the crop“ – delete entire sentence.
Abstract (Line 24-25):These results show that mixing microorganisms with different abilities does not necessarily guarantee an advantage for the culture - – delete entire sentence.
Abstract (Line 25-26): Of all the treatments performed, the inoculation of the bacterium B. japonicum was the most promising for the peanut crop - the inoculation of the bacterium or the peanut?
Abstract: Proper conclusions based on the new findings made in the study are missing in the Abstract.
Keywords (Line 27): „hypogaea“ – should be italicized
Introduction generally has a bad flow; the section on peanuts should be followed by the issues related to chemical fertilizers (mentioned in the abstract but not in the introduction); biotic and/or abiotic stresses; biological nitrogen fixation and nitrogen fixing microorganisms; other plant growth promoting microorganisms and their role/mechanisms; and finally the hypothesis and objective. Too much detail about urease and phosphatase which would be suitable for the discussion.
Introduction (Line 47-49): „Because it is a legume species, the peanut presents the characteristic of association with nitrogen-fixing bacteria, which allows it efficiency in the process of absorption of this nutrient“ – its efficiency?
Introduction (Line 56): They – Ureases
Introduction (Line 63-65): As much of the nitrogen applied in the field is lost, some chemical compounds have been mixed with urea with the objective of inhibiting urease activity, reducing the hydrolysis rate and, consequently, the losses by volatilization [15]. However – delete this part
Introduction (Line 67-68): Nitrogen is the element that most limits plant growth, followed by phosphorus [17] – move this sentence to the beginning of the paragraph
Introduction (Line 69-81): Explain the difference between P-solubilization and P-mineralization, and the mechanisms behind both.
Introduction (Line 82): microorganismos – microorganisms
Introduction (Line 85-86): „Among the genera with potential already known as growth promoters in some plant species are Azospirillum, Bacillus, Bradyrhizobium and Trichoderma“ – unclear, rewrite
Introduction (Line 90-91): „and in association with nonlegumes such as rice, maize and pine [32] – is 32 an appropriate reference to cite here?
Introduction (Line 96): „the biosynthesis of compounds“ – what kind of compound?
Introduction (Line 106-110): rewrite the objectives by combining them into one meaningful sentence
In Materials and Methods more information about each strain including isolation source/origin should be included. On the basis of which criteria they were selected for the study?
Materials and Methods (Line 112): UNESP –Write full name.
Materials and Methods (Line 114, 123): include more information about cultivars
Materials and Methods (Line 114): a variety with a low growth habit – rewrite
Materials and Methods (Line 127, 136): a previously performed soil chemical analysis – include the results of soil chemical analysis in the main manuscript
Materials and Methods (Line 128-130, 135-138): „with a 20% reduction in the fertilization required for the crop, the macronutrients calcium, sulfur, phosphorus, magnesium and nitrogen were supplied, as well as the micronutrients boron, copper, molybdenum and zinc“ – unclear; specify amounts
Materials and Methods (Line 141): FCAV – write full name.
Materials and Methods (Line 141-142): The bacterium B. subtilis was isolated from maize plant and identified through automatic sequencing and the deposit of GenBank Access Number MZ133755 - which part of the plant?
Materials and Methods (Line 142-143): The bacterium A. brasilense is from Embrapa A. brasiliensis strain F111 (Access Number MZ133758) [42] –unclear, rewrite
Materials and Methods (Line 141-143): What about Bradyrhizobium and Trichoderma?
Materials and Methods (Line 145-146): The inoculant B. subtilis was prepared in nutrient broth, the fungus T. harzianum in malt extract, A. brasilense in DIG medium and B. japonicum in YM medium – explain abbreviations for growth media.
Materials and Methods (Line 148-152): „With the exception of B. japonicum, which was used only in the seed treatment, the other microorganisms were applied directly to the soil in a total of four applications in the greenhouse and a single application in the field. In the greenhouse, the first application was performed on the seventh day after sowing and then every ten days; in the field, the application was performed on the thirtieth day after sowing“ – include the amounts of inocula and include more information abot preparation of single and co-inoculants.
Materials and Methods (Line 159-161): Based on the data obtained in the greenhouse regarding shoot and root dry matter and plant height, five of the treatments mentioned above were selected and evaluated in the field conditions. The plots were divided into randomized blocks, with 4 replicates – include this part in the field experiment section of Materials and Methods
Materials and Methods (Line 177): both analyses according to – add Malavolta et al.
Materials and Methods (Line 179-180): The enzymatic activity of acid phosphatase was determined according to [44]. To de- 179 termine the enzymatic activity of urease, the method of [45] – to, of?
Materials and Methods (Line 180): Both experiments were performed using soil samples collected from the experiment in a greenhouse – why the field soil samples were not analyzed?
Results: separate the data obtained in greenhouse from field data
Results (Line 192-200): repetition of methodology
Results (Line 203-204): Plants treated with T. harzianum (0.15 g) and A. brasilense (0.152 g) presented lower root development than treatment 7 (A. brasilense + B. Japonicum) - like all other treatments
Results (Line 210-214): use italic for scientific names
Results (All figures): What do the bars on the charts represent?
Results (Figures 6, 8): include letters of significance (although there was no statistical difference among the treatments)
Discussion (Line 384): B. japonicum japonicum – delete one japonicum
Discussion (Line 418-422): The present study evaluated mixtures of microorganisms, such as A. brasilense + B. subtilis, A. brasilense + B. japonicum, A. brasilense + T. harzianum, B. subtilis + B. japonicum, B. subtilis + T. harzianum, and B. japonicum + T. harzianum, and the consortium of microorganisms was a mixture of A. brasilense + B. subtilis + B. japonicum and T. harzianum – repetition of methodology, delete entire sentence
Discussion (Line 424-426): Specifically, in the field, the highest productivity was found in the inoculation of B. japonicum alone, and the mixture of microorganisms did not have same effect. These results show that mixing microorganisms with different abilities does not necessarily guarantee an advantage for the peanut crop - whether the mutual compatibility of the strains was examined before their mixing?
Discussion (Line 429-432): Interestingly, this species is highly adapted to soybean and less 429 adapted to common bean, both legumes, showing that the success of the B. japonicum plant 430 interaction is dependent on specificity and even like that this bacterium promoted in- 431 creases in the peanut crop - the origin of B. japonicum strain is not stated, nor is it capable of nodulating peanuts
Conclusions (Line 434-437): too short and too vague; proper conclusions based on the new findings made in the study are missing, rewrite this section.
Author Response
Reviewer3
Reviewer3 - The study has some interesting aspects which would be of interest to the international scientific community. However, this manuscript needs significant improvements, both in terms of language and scientific content.
Answer: We thank the reviewer for the opportunity given to us to improve our manuscript.
Reviewer3 - Title (Line 2): „Promotion of peanut growth by microorganisms“ – too vague. I suggest „Promotion of peanut (Arachis hypogaea L.) growth by plant growth-promoting microorganisms“
Answer: We accepted your suggestion, and the new title was adopted.
Reviewer3 - Abstract (Line 9): „In addition, the use of microorganisms that promote plant growth„ – include abbreviation – microorganisms that promote plant growth (PGPM)
Answer: It has been included.
Reviewer3 - Abstract (Line 12): „is the use of microorganisms that promote plant growth“ – the use of PGPM
Answer: It has been used.
Reviewer3 - Abstract (Line 12-15): „The objective of this study was to evaluate the effects on growth promotion and productivity of peanuts inoculated with the microorganisms Azospirillum brasilense, Bacillus subtilis, Bradyrhizobium japonicum and Trichoderma harzianum“ – include more information about experimental design, inoculation method, and experimental conditions
Answer: in greenhouse and field conditions. In the greenhouse, the experiment was conducted with 12 treatments with six repetitions. In field conditions, the experiment was conducted with five treatments with four repetitions. Both were conducted in randomized blocks.
Reviewer3 - Abstract (Line 15-18): „The results show that, in general, all the microorganisms evaluated in the present study promoted increases in the various parameters analyzed, root dry mass, shoot dry mass, phosphorus concentrations and plant height, under both conditions, in a greenhouse and under field conditions compared to the control treatments that did not receive microbial inoculation“ –
A lot of unnecessary words, delete: The results show that, in general; various parameters analyzed; under both conditions; treatments that did not receive microbial inoculation.
Answer: These words have been deleted.
Reviewer3 - Abstract (Line 19-21): „These results show that it is possible to obtain increases in the peanut crop with the inoculation of plant growth-promoting microorganisms without major changes in the management and conduct of the crop“ – delete entire sentence.
Answer: It has been deleted.
Reviewer3 - Abstract (Line 24-25):These results show that mixing microorganisms with different abilities does not necessarily guarantee an advantage for the culture - – delete entire sentence.
Answer: It has been deleted.
Reviewer3 - Abstract (Line 25-26): Of all the treatments performed, the inoculation of the bacterium B. japonicum was the most promising for the peanut crop - the inoculation of the bacterium or the peanut?
Answer: Inoculation of the bacterium.
Reviewer3 - Abstract: Proper conclusions based on the new findings made in the study are missing in the Abstract.
Answer: It has been added
Keywords (Line 27): „hypogaea“ – should be italicized
Answer: It has been italicized.
Reviewer3 - Introduction generally has a bad flow; the section on peanuts should be followed by the issues related to chemical fertilizers (mentioned in the abstract but not in the introduction); biotic and/or abiotic stresses; biological nitrogen fixation and nitrogen fixing microorganisms; other plant growth promoting microorganisms and their role/mechanisms; and finally the hypothesis and objective. Too much detail about urease and phosphatase which would be suitable for the discussion.
Answer: These enzymes were removed from the introduction section.
Reviewer3 - Introduction (Line 47-49): „Because it is a legume species, the peanut presents the characteristic of association with nitrogen-fixing bacteria, which allows its efficiency in the process of absorption of this nutrient“ – its efficiency?
Answer: Yes the correct is “its”
Reviewer3 - Introduction (Line 56): They – Ureases
Answer: It has been removed from introduction section.
Introduction (Line 63-65): As much of the nitrogen applied in the field is lost, some chemical compounds have been mixed with urea with the objective of inhibiting urease activity, reducing the hydrolysis rate and, consequently, the losses by volatilization [15]. However – delete this part
Answer: This part has been deleted.
Reviewer3 - Introduction (Line 67-68): Nitrogen is the element that most limits plant growth, followed by phosphorus [17] – move this sentence to the beginning of the paragraph
Answer: It has been changed.
Reviewer3 - Introduction (Line 69-81): Explain the difference between P-solubilization and P-mineralization, and the mechanisms behind both.
Answer: Solubilization is a process that transforms unavailable forms of phosphorus in available ones. Mineralization is a process that degrades organic matter, releasing the immobilized phosphorus content.
Reviewer3 - Introduction (Line 82): microorganismos – microorganisms
Answer: It has been corrected.
Reviewer3 - Introduction (Line 85-86): „Among the genera with potential already known as growth promoters in some plant species are Azospirillum, Bacillus, Bradyrhizobium and Trichoderma“ – unclear, rewrite
Answer: The genera Azospirillum, Bacillus, Bradyrhizobium and Trichoderma are carriers of several abilities related to plant growth.
Reviewer3 - Introduction (Line 90-91): „and in association with nonlegumes such as rice, maize and pine [32] – is 32 an appropriate reference to cite here?
Answer: Better citations have been added.
Reviewer3 - Introduction (Line 96): „the biosynthesis of compounds“ – what kind of compound?
Answer: Phytohormones like indole acetic acids.
Reviewer3 - Introduction (Line 106-110): rewrite the objectives by combining them into one meaningful sentence
Answer: The objective of this study was to evaluate the effects on growth promotion and productivity of peanuts inoculated with plant growth-promoting microorganisms.
Reviewer3 - In Materials and Methods more information about each strain including isolation source/origin should be included. On the basis of which criteria they were selected for the study?
Answer: : These information have been added. B. japonicum and T. harzianum were acquired from Embrapa, classification NC 230 (SEMIA 6439), and CEN199, respectively.
For more information about these strains, please see the papers below.
ARAÚJO, Fábio Fernando de; HUNGRIA, Mariangela. Soybean nodulation and yield when co-inoculated with Bacillus subtilis and Bradyrhizobium japonicum/Bradyrhizobium elkanii. Pesquisa Agropecuária Brasileira, v. 34, p. 1633-1643, 1999.
CARVALHO, Daniel Diego Costa et al. Biocontrol of seed pathogens and growth promotion of common bean seedlings by Trichoderma harzianum. Pesquisa Agropecuária Brasileira, v. 46, p. 822-828, 2011.
LOBO, Laiana Lana Bentes; DOS SANTOS, Roberta Mendes; RIGOBELO, Everlon Cid. Promotion of maize growth using endophytic bacteria under greenhouse and field conditions. Australian Journal of Crop Science, v. 13, n. 12, p. 2067-2074, 2019.
LOBO, Laiana Lana Bentes et al. The negative effect of coinoculation of plant growth-promoting bacteria is not related to indole-3-acetic acid synthesis. Journal of Plant Growth Regulation, p. 1-10, 2022.
Reviewer3 - Materials and Methods (Line 112): UNESP –Write full name.
Answer: Universidade Estadual Paulista - UNESP
Reviewer3 - Materials and Methods (Line 114, 123): include more information about cultivars
Answer: Cultivar IAC-OL3 was developed by Instituto Agronômico de Campinas. This cultivar's cycle is 130 days; its potential to yield varies from 3 to 4-ton ha-1 The plant is small creeping and bushy leaves.
Reviewer3 - Materials and Methods (Line 114): a variety with a low growth habit – rewrite
Answer: It has been removed.
Reviewer3 - Materials and Methods (Line 127, 136): a previously performed soil chemical analysis – include the results of soil chemical analysis in the main manuscript
Answer: It has been included. The soil composition was pH 6.5; organic matter 11 g dm3; phosphorus 20 mg dm3; soil 12 mg dm3; potassium 0.7 mmolc dm3; magnesium 17 mmolc dm3; and the sum of bases 24.4 mmolc dm3.
Reviewer3 - Materials and Methods (Line 128-130, 135-138): „with a 20% reduction in the fertilization required for the crop, the macronutrients calcium, sulfur, phosphorus, magnesium and nitrogen were supplied, as well as the micronutrients boron, copper, molybdenum and zinc“ – unclear; specify amounts
Answer: The amounts were (Ca= 60g, Mg=15g and S=15 g)
Reviewer3 - Materials and Methods (Line 141): FCAV – write full name.
Answer: FCAV – Faculdade de Ciências Agrárias e Veterinárias
Reviewer3 - Materials and Methods (Line 141-142): The bacterium B. subtilis was isolated from maize plant and identified through automatic sequencing and the deposit of GenBank Access Number MZ133755 - which part of the plant?
Answer: It was isolated from Root .
Reviewer3 - Materials and Methods (Line 142-143): The bacterium A. brasilense is from Embrapa A. brasiliensis strain F111 (Access Number MZ133758) [42] –unclear, rewrite
Answer: Embrapa is a Research Brazilian Agency that sells microbial strains for research. This isolate came from Embrapa.
Reviewer3 - Materials and Methods (Line 141-143): What about Bradyrhizobium and Trichoderma?
Answer:
Reviewer3 - Materials and Methods (Line 145-146): The inoculant B. subtilis was prepared in nutrient broth, the fungus T. harzianum in malt extract, A. brasilense in DIG medium and B. japonicum in YM medium – explain abbreviations for growth media.
Answer: B. japonicum and T. harzianum were acquired from Embrapa, classification NC 230 (SEMIA 6439), and CEN199, respectively [40, 41] .
Reviewer3 - Materials and Methods (Line 148-152): „With the exception of B. japonicum, which was used only in the seed treatment, the other microorganisms were applied directly to the soil in a total of four applications in the greenhouse and a single application in the field. In the greenhouse, the first application was performed on the seventh day after sowing and then every ten days; in the field, the application was performed on the thirtieth day after sowing“ – include the amounts of inocula and include more information abot preparation of single and co-inoculants.
Answer: All the microorganisms were prepared separately. Each vase received 10 mL from each microorganism. The vase that received two microorganisms received 10 mL of each microorganism, grown separately and inoculated simultaneously.
Reviewer3 - Materials and Methods (Line 159-161): Based on the data obtained in the greenhouse regarding shoot and root dry matter and plant height, five of the treatments mentioned above were selected and evaluated in the field conditions. The plots were divided into randomized blocks, with 4 replicates – include this part in the field experiment section of Materials and Methods
Answer: It has been included.
Reviewer3 - Materials and Methods (Line 177): both analyses according to – add Malavolta et al.
Answer: It has been added. Please see the reference section.
Reviewer3 - Materials and Methods (Line 179-180): The enzymatic activity of acid phosphatase was determined according to [44]. To de- 179 termine the enzymatic activity of urease, the method of [45] – to, of?
Answer:
Reviewer3 - Materials and Methods (Line 180): Both experiments were performed using soil samples collected from the experiment in a greenhouse – why the field soil samples were not analyzed?
Answer: The soil was. But it belongs to a farmer, and we did not have access to the results.
Reviewer3 - Results: separate the data obtained in greenhouse from field data
Answer: Dear Reviewer. All suggestions made by you were excellent. And we accepted all of them. Regarding separating the results from two conditions, we authors decided to keep the results together. Because if the results are separated, we will have results with all parameters doubled.
Reviewer3 - Results (Line 192-200): repetition of methodology
Answer: It has been removed.
Reviewer3 - Results (Line 203-204): Plants treated with T. harzianum (0.15 g) and A. brasilense (0.152 g) presented lower root development than treatment 7 (A. brasilense + B. Japonicum) - like all other treatments
Answer: It has been changed.
Reviewer3 - Results (Line 210-214): use italic for scientific names
Answer: Interesting. All the scientific names were written in italic. But the Journal’s system changed it. In the original paper, all of them are written in italic.
Reviewer3 - Results (All figures): What do the bars on the charts represent?
Answer: The bars represent the standard deviation. It was included in all figures.
Reviewer3 - Results (Figures 6, 8): include letters of significance (although there was no statistical difference among the treatments)
Answer: It has been done
Reviewer3 - Discussion (Line 384): B. japonicum japonicum – delete one japonicum
Answer: It has been removed.
Reviewer3 - Discussion (Line 418-422): The present study evaluated mixtures of microorganisms, such as A. brasilense + B. subtilis, A. brasilense + B. japonicum, A. brasilense + T. harzianum, B. subtilis + B. japonicum, B. subtilis + T. harzianum, and B. japonicum + T. harzianum, and the consortium of microorganisms was a mixture of A. brasilense + B. subtilis + B. japonicum and T. harzianum – repetition of methodology, delete entire sentence
Answer: It has been removed.
Reviewer3 - Discussion (Line 424-426): Specifically, in the field, the highest productivity was found in the inoculation of B. japonicum alone, and the mixture of microorganisms did not have same effect. These results show that mixing microorganisms with different abilities does not necessarily guarantee an advantage for the peanut crop - whether the mutual compatibility of the strains was examined before their mixing?
Answer: It is a very good question. It was evaluated in another study.
Please see this paper
LOBO, Laiana Lana Bentes et al. The negative effect of coinoculation of plant growth-promoting bacteria is not related to indole-3-acetic acid synthesis. Journal of Plant Growth Regulation, p. 1-10, 2022.
Reviewer3 - Discussion (Line 429-432): Interestingly, this species is highly adapted to soybean and less 429 adapted to common bean, both legumes, showing that the success of the B. japonicum plant 430 interaction is dependent on specificity and even like that this bacterium promoted in- 431 creases in the peanut crop - the origin of B. japonicum strain is not stated, nor is it capable of nodulating peanuts
Answer: This strain B. japonicum was isolated from soil. This strain can nodulate peanuts too. It varies according to the efficiency of the interaction between the microbes and the plant host.
Reviewer3 - Conclusions (Line 434-437): too short and too vague; proper conclusions based on the new findings made in the study are missing, rewrite this section.
Answer: It has been rewritten.
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
Line 13-18: „The objective of this study was to evaluate the effects on the growth promotion and productivity of peanuts inoculated with the microorganisms Azospirillum brasilense, Bacillus subtilis, Bradyrhizobium japonicum, and Trichoderma harzianum. The experiments were carried out in greenhouse and field conditions. In the greenhouse, the experiment was conducted with treatments with six repetitions. In field conditions, the experiment was conducted with five treatments with four repetitions. Both were conducted in randomized blocks“ – rewrite, I suggest: The objective of this study was to evaluate the effects of Azospirillum brasilense, Bacillus subtilis, Bradyrhizobium japonicum, and Trichoderma harzianum, as a single inoculant and co-inoculant on the growth promotion and productivity of peanuts in greenhouse and field conditions.
Line 25-26: „However, under field conditions, inoculation with the bacterium B. japonicum was the most promising, increasing peanut productivity“ - repetition of „Specifically, in the field, the highest productivity was found in the inoculation of B. japonicum alone“.
Line 93: Rhizobium is not a family
Line 95-96: At the root-hair tips of soybean plants, Rhizobium bacteria colonize symbiosomes and are then located within the plant membrane – to redundant, delete.
Line 98: Soybean inoculation with B. japonicum frequently boosts seed yield [29]. – already mentioned in line 90; there is no need to highlight soybean, delete.
Line 170-171: The vase that received two microorganisms received 10 mL of each microorganism, grown individually and inoculated simultaneously – unclear; 10 ml for single inoculants, and 2 or 4 × 10 ml for mixed inoculants? for each treatment the volume of the inoculum must be the same – 10 ml, and the ratio of microorganisms must be the same 2 × 5 ml for mixture of two microorganisms, or 4 × 2,5 ml for mixture of four microorganisms.
Line 394-404: The B. japonicum synthesizes a wide range of carbohydrates, including lipopolysaccharides, capsular polysaccharides, exopolysaccharides (EPS), nodule polysaccharides, lipochitin oligosaccharides, and cyclic glucans. This bacterium produces polysaccharide-degrading enzymes, such as polygalacturonase and carboxymethylcellulose, which cleave glycosidic bonds in the epidermal layer of the roots, creating pits in the root's epidermis where the bacterium can gain entry [29]. Rhizobia penetration into the root and cortex and spreading inside the nodules occurs without intracellular infection thread formation and involves intercellular penetration (crack entry). The infection site is at the junction of a hair cell with epidermal and cortical cells, and rhizobia penetrate the root by breaching the epidermal barrier instead of entering through curled root hairs. After entry, rhizobia occupy the space between epidermal and cortical cells and further spread through the root cortex [53] - to redundant, delete.
Author Response
Answer: We want to thank the reviewer for his patience and the opportunity given to us to improve our manuscript.
Reviewer3 - Line 13-18: „The objective of this study was to evaluate the effects on the growth promotion and productivity of peanuts inoculated with the microorganisms Azospirillum brasilense, Bacillus subtilis, Bradyrhizobium japonicum, and Trichoderma harzianum. The experiments were carried out in greenhouse and field conditions. In the greenhouse, the experiment was conducted with treatments with six repetitions. In field conditions, the experiment was conducted with five treatments with four repetitions. Both were conducted in randomized blocks“ – rewrite, I suggest: The objective of this study was to evaluate the effects of Azospirillum brasilense, Bacillus subtilis, Bradyrhizobium japonicum, and Trichoderma harzianum, as a single inoculant and co-inoculant on the growth promotion and productivity of peanuts in greenhouse and field conditions.
Answer: The objective was changed accordingly.
Reviewer3 - Line 25-26: „However, under field conditions, inoculation with the bacterium B. japonicum was the most promising, increasing peanut productivity“ - repetition of „Specifically, in the field, the highest productivity was found in the inoculation of B. japonicum alone“.
Answer: It has been removed.
Reviewer3 - Line 93: Rhizobium is not a family
Answer: It has been removed.
Reviewer3 - Line 95-96: At the root-hair tips of soybean plants, Rhizobium bacteria colonize symbiosomes and are then located within the plant membrane – to redundant, delete.
Answer: It has been deleted.
Reviewer3 - Line 98: Soybean inoculation with B. japonicum frequently boosts seed yield [29]. – already mentioned in line 90; there is no need to highlight soybean, delete.
Answer: It has been deleted.
Reviewer3 - Line 170-171: The vase that received two microorganisms received 10 mL of each microorganism, grown individually and inoculated simultaneously – unclear; 10 ml for single inoculants, and 2 or 4 × 10 ml for mixed inoculants? for each treatment the volume of the inoculum must be the same – 10 ml, and the ratio of microorganisms must be the same 2 × 5 ml for mixture of two microorganisms, or 4 × 2,5 ml for mixture of four microorganisms.
Reviewer3 - Line 394-404: The B. japonicum synthesizes a wide range of carbohydrates, including lipopolysaccharides, capsular polysaccharides, exopolysaccharides (EPS), nodule polysaccharides, lipochitin oligosaccharides, and cyclic glucans. This bacterium produces polysaccharide-degrading enzymes, such as polygalacturonase and carboxymethylcellulose, which cleave glycosidic bonds in the epidermal layer of the roots, creating pits in the root's epidermis where the bacterium can gain entry [29]. Rhizobia penetration into the root and cortex and spreading inside the nodules occurs without intracellular infection thread formation and involves intercellular penetration (crack entry). The infection site is at the junction of a hair cell with epidermal and cortical cells, and rhizobia penetrate the root by breaching the epidermal barrier instead of entering through curled root hairs. After entry, rhizobia occupy the space between epidermal and cortical cells and further spread through the root cortex [53] - to redundant, delete.
Answer: This text has been deleted.
Author Response File: Author Response.docx
