Co-Inoculation of Bacillus spp. for Growth Promotion and Iron Fortification in Sorghum

Round 1

Reviewer 1 Report

The authors have addressed a topic that is relevant today, which has been thoroughly substantiated and proven by their studies.  

After reading the article, I would like to make the following comments and suggestions:

<Introduction>

In the introduction chapter, the importance of iron in the metabolism of microorganisms and the forms it can take up and make soluble should be better highlighted. 
In relation to PGPR bacteria, it is important to highlight which PGPR bacteria are important and why the Bacillus spp. species are important. 
It would be worth highlighting how PGPR species help plants to absorb iron and how these processes occur in sorghum. 
What other microbiological tests are suitable for sorghum?

In addition, the authors should make the purpose of the study clearer and more understandable.  

<Materials and methods> 

The chapter on materials and methods is well explained and the methods and tools used are presented in detail. 

However, it should be made clear whether PGPR and PGP are the same thing.  

In chapter 2.5, the authors mention two Bacillus spp. selected on the basis of the 16s rRNA sequence. Why were these two isolates chosen?  

In chapter 2.6, the authors mention that two types of soil were mixed with compost. According to the WRB, what categories of soils are used? 
Which raw materials were used for the compost? Which composting system was used?  

Please clarify the PGPB used in chapter 2.7?  

For statistical analysis, the authors use only 5%. Does this 5% indicate the level of significance? 
It would be worthwhile for the authors to indicate p<0.05 or 5% as the significance level.  

<Results> 

In chapter 3.1, what caused the difference in the growth promoting of the selected Bacillus spp. plants? 

Have the authors examined the relationships between the parameters studied? (Table 1.) 

Chapter 3.2 should not be a separate sub-chapter, it can be written in Chapter 3.1 as an explanation of Table 1. 

The authors mention the extracellular enzymes studied in Chapter 3.3. 
Why were these enzymes studied? Why are these three enzymes important? 
What causes the differences between the isolates and the enzymes examined in Table 2?  

In chapter 3.6, what exactly does SEM mean? 

What can you see in the electron micrographs in Figure 3? What differences should readers notice?  

<Discussion>

The discussion chapter is well explained.  

Author Response

Reviewer 1

The authors have addressed a topic that is relevant today, which has been thoroughly substantiated and proven by their studies.  

After reading the article, I would like to make the following comments and suggestions:

We thank the reviewers for their valuable suggestions and comments to improve the manuscript. We have attended to the suggestions given and incorporated changes in the manuscript.

Introduction

1. In the introduction chapter, the importance of iron in the metabolism of microorganisms and the forms it can take up and make soluble should be better highlighted. 
   In relation to PGPR bacteria, it is important to highlight which PGPR bacteria are important and why the Bacillus spp. species are important. It would be worth highlighting how PGPR species help plants to absorb iron and how these processes occur in sorghum. 

Authors’ response: Now the importance of iron in metabolism of microorganisms is well explained and added in the introduction. Simultaneously, importance of Bacillus sp., their role in aiding plants for iron absorption.

2. In addition, the authors should make the purpose of the study clearer and more understandable.  

Authors’ response: Now, the purpose of study is well explained in introduction, results and discussion. The results obtained are discussed properly with previous citations for clear understanding of the work.

Materials and methods 

The chapter on materials and methods is well explained and the methods and tools used are presented in detail. However, it should be made clear whether PGPR and PGP are the same thing.

R: Now, both the abbreviations are explained for clarity.  

3. In chapter 2.5, the authors mention two Bacillus spp. selected on the basis of the 16s rRNA sequence. Why were these two isolates chosen?  

Authors’ response: The purpose of selecting two Bacillus spp. (RHPR20 AND MMRH22) was they exhibited copious amounts of Plant growth promoting traits.

4. In chapter 2.6, the authors mention that two types of soil were mixed with compost. According to the WRB, what categories of soils are used? 

Authors’ response: Black soil (Vertisol), sand and compost in a ratio of 3:2:1 was used. Only one type of soil was used in the study.

5. Which raw materials were used for the compost? Which composting system was used?  

Authors’ response: Compost made of farm waste was used in the study.

6. Please clarify the PGPB used in chapter 2.7?  

Authors’ response: now mentioned in 2.7.

7. For statistical analysis, the authors use only 5%. Does this 5% indicate the level of significance? It would be worthwhile for the authors to indicate p<0.05 or 5% as the significance level.  

Authors’ response: Yes. 5% indicates the level of significance. Tables are rewritten as per the reviewer’s suggestions.

Results 

8. In chapter 3.1, what caused the difference in the growth promoting of the selected Bacillus spp. plants? 

Authors’ response: Based on the variation in the solubilization of nutrients, production of indole, ammonia, siderophore and ACC deaminase enzyme could be attributed for growth promotion of sorghum by selected Bacillus spp.

9. Have the authors examined the relationships between the parameters studied? (Table 1.) 

Authors’ response: Only qualitative and quantitative estimations of PGP parameters were done. Based on significant results invitro, the greenhouse study was done.

10. Chapter 3.2 should not be a separate sub-chapter, it can be written in Chapter 3.1 as an explanation of Table 1. 

Authors’ response: Now, it is written in chapter 3.1 as suggested.

11. The authors mention the extracellular enzymes studied in Chapter 3.3. 
    Why were these enzymes studied? Why are these three enzymes important? 
    What causes the differences between the isolates and the enzymes examined in Table 2?  

Authors’ response: Most of the Plant growth promoting bacteria produce hydrolytic enzymes such as cellulase, amylase, protease. Extracellular enzymes are known to inhibit phytopathogens and play a key role in maintaining plant health. Variation in the isolates used in the study could be the reason for difference in enzyme production.

12. In chapter 3.6, what exactly does SEM mean? 

Authors’ response: SEM means Scanning electron microscope. The abbreviation is mentioned in methods.

13. What can you see in the electron micrographs in Figure 3? What differences should readers notice? 

Authors’ response: Co-inoculation of the two Bacillus strains used in this study has aided in effective root colonization (as evident in fig 3) of sorghum when compared to inoculation with individual strains (RHPR20 or MMRH22). Under invitro studies, we observed both the strains, RHPR20 and MMRH22 were compatible to each other under invitro conditions. However, we did not observe any significant colonization in uninoculated control.

Discussion

14. The discussion chapter is well explained. 

 Authors’ response: The authors are thankful to the Reviewer for the comment

Reviewer 2 Report

Dear Authors,

Please check all the comments and please acknowledge them in your revised version. Good luck!

Comments for author File: Comments.pdf

Author Response

Reviewer 2

Abstract

1. PGP traits

Author’s response: changed to PGP traits.

Introduction

2. For all the citations in between sentences, please use the following order:

Author 1, Author 2.... [2]

Author’s response: Changed as suggested

3. rhizobacteria

Author’s response: Text changed as rhizobacteria.

Materials and methods

4. in italics

Author’s response: Text Italicized

5. You mentioned their source as Marine but can you please explain in detail from which marine part did you isolate them?

Author’s response: Marine water source

6. in italics

Author’s response: Text Italicized

7. What about the other remaining 5 isolates? Did they belong to the same two potential Bacillus spp?

Author’s response: Among all the Bacillus spp screened in this study for PGP traits, two (RHPR20, MMRH22) exhibited copious PGP traits and used for greenhouse studies. Hence, only these two Bacillus spp were identified.

7. Why did you use only 2 Bacillus spp for the study? Why didn't you use the other 5?

Author’s response: Among all the Bacillus spp screened in this study for PGP traits, two (RHPR20, MMRH22) exhibited copious PGP traits and hence used for greenhouse studies.

8. You used the acronym PGPR in lines 55-56. Please stick to only one acronym

Full form of PGPR is made clear in line 55-56 and here the title is changed to root colonization by Bacillus spp.

9. Perhaps it would be better if you say

"Estimation of Fe content in sorghum grains" also please make the same changes in the result section

Author’s response: Title modified as suggested.

10. citation and reference?

Author’s response: Citation and reference incorporated.

Results

11. traits or activites?

Author’s response: Traits

12. In that case why didn't you tried the remaining bacterial isolates and their consortia?

Author’s response: The present study is aimed at evaluation of co-inoculation of Bacillus spp. isolated from marine water and rhizosphere for growth promotion, yield and iron fortification of sorghum. Among the Bacillus spp screened in this study, the two strains RHPR20, MMRH22 showed superior PGP traits. Hence, only these two were selected.

13. Which mean and SE values are these? Please rewrite this table

Author’s response: Table is rewritten as per suggestion.

14. Again where is the mean values and where is the SE?

Please rewrite this table

Author’s response: Table is rewritten

15. Please indicate the colonization using an arrow

Author’s response: Image showing colonisation indicated with arrow mark is inserted.

16. Perhaps this section comes before the studies. Please re-arrange it

Author’s response: Sections are rearranged as suggested.

17. The subsections from material and methods do not match with the subsections of results. Please rearrange the subsections accordingly in both.

Author’s response: Subsections are rearranged.

18. Perhaps you can present this data in graphical form

Author’s response: Data is changed to graphical form.

Discussion

19. [33,34,35]

Author’s response: Reference changed as suggested.

20. Lines 316-320 strikes through

Author’s response: Now the appropriate text of results is moved to the results section.

21. All the text that were cancelled belong to Results.

Please Discuss your results with other similar studies rather than explaining the results in word form.

Author’s response: Now the appropriate text of results is moved to the results section.

Conclusion

22. .....and improve uptake of iron content in sorghum

Author’s response: Text changed as suggested.

References:

23. Please re-write the references as per the journal specifications

Author’s response: References edited as per journal specifications

Round 2

Reviewer 2 Report

Dear Authors,

Some minor comments are mentioned which I believe after correcting is ready to get published.

Comments for author File: Comments.pdf

Author Response

All the minor comments annotated by the Reviewer in the pdf file have been corrected