Evaluating Microbial Biofertilizers for Root Colonization Potential in Narra (Pterocarpus indicus Willd.) and Their Efficacy in Heavy Metal Remediation

Round 1

Reviewer 1 Report

Overall in the manuscript, the authors have attempted to demonstrate the root colonization potential of mycorrhiza and nitrogen-fixing bacteria (supplemented as defined formulations) on Narra seedlings, in presence of heavy metals like nickel and gold. The exposure to the heavy metals is through the use of mined-out soils; with an aim to explore the possible use of the microbial formulations to augment the ability of Narra in bioremediation. The authors also try to correlate the efficacy of mycorrhiza and NFBs through co-inoculations. Although the study addresses one of the widely explored approach of forest soil rehabilitation, it is restricted only to demonstrating the microbial root/rhizosphere colonization, which seems inadequate to justify the projected potential in bioremediation. 

Several concerns/queries that need to be addressed are as under

1. The soil parameters as in Table 1 were pre-assessed prior to microbial inoculation. However, the same parameters after six months of growth in Ni/Au-mined out soils is missing.

Comparison of soil parameters pre- and post-inoculation becomes a major aspect in determining the efficacy of the AMF and NFB colonization. More so, because both the inoculants used are expected to improve P and N nutrient status, respectively.

2. Plant biomass and/or growth parameters after six months of inoculation and growth in mined-out soils is missing. Again, this becomes crucial to analyze the effect of microbial colonization

3. The compost used for Narra seedling cultivation itself could be source of microbes with diverse potentials, which could have indirect influence on the observed root colonization and the ensuing benefits to the Narra seedling growth. This aspect needs to be included in the discussion. An experimental control without compost may also help understanding its role in microbial root colonization.

4. At several places, the authors mention about the symbiotic relationships of Narra with AMF and NFBs, independently. However, possible interactions between AMF and NFB, in the co-inoculations are underexplored. Given that mycorrhiza improve P availability and P is one of the major factors influencing nodulation by NFBs, this becomes important. The authors may discuss their results on these lines

5. Section 3.3 Title: Does it mean Mycorrhizal root colonization? If yes please specify accordingly.

6. Figure 4: The figure is subdivided into (a) and (b). At the same time the bars are also marked as a, b and c, probably indicating the statistical differences. Please use appropriate symbols to indicate the statistical aspects on the bars in the graph. Plus, the figure legends need to be included describing the meaning of each symbol

7. Line 125: ‘…..as shown in Table 1’. This looks like to referring to results in Materials and Methods, which may be avoided. In fact Lines 125-128 and subsequent details until line 142 appear like results and discussion. They can be included accordingly

8. Line 15 in Abstract ‘Bioremediation technology, the least known strategy…..’ and Line 56 ‘Bioremediation is widely accepted….’, are contrasting each other. The authors need to be careful with this

9. Introduction: Line 46-51 are unclear. This segment should also include some information on how heavy metal accumulation in soil affect the soil enzymes and Narra seedling growth. A line or two about why nickel and gold mined-out sites shall be useful in the introduction. In general, the introduction segment requires revision to improve the flow of the content.

10.  Discussion segment at several places looks like re-mentioning the results. This needs to be avoided. These results need to be discussed in alignment with the previous studies demonstrating effect of the said microbial inoculants on Narra growth, heavy metal accumulation etc.

11. Use of Scanning Electron Microscopy to demonstrate the root colonization by microbial inoculants could enhance the data quality. More so, it could be useful to demonstrate the co-existent AMF colonization and root nodules; and thus justify the impact of microbial co-inoculations on Narra seedlings

12. Line 101 ‘…since the set-up was mimicking the mined-out area Conditions…’ How was this mimicking the mined-out conditions? In what sense?

Comments for author File: Comments.pdf

The manuscript requires significant English language check to improve the readability. Throughout the manuscript, there is no need to write the time points in words and number both. For example, six (6) months is not needed. Either may suffice depending on the formation of the sentence.

Line 76 - ‘In the study of [18].....’ and similar way of citing reference in lines 178, 185 and several other places too, looks inappropriate in terms of the sentence readability.

Some of the other observations are listed below.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The paper presents interesting information but I am offering the following comments for further improvement:

11.Line 294 should not start with a numeric reference.

22. The nutrient mobilization effect of biofertilizers has been described in the paper with reference to the published literature. The authors have only provided the data for soil analysis before the biofertilizer inoculation. Soil analysis after six months of biofertilizer inoculation would provide a better understanding of the effect of biofertilizer on the seedlings. It is also unclear from the data, whether biofertilizers helped in bioremediation.

33. The authors have discussed in the introduction and discussion portion, the phytoremediation potential of Narra inoculated with biofertilizers. This should be supported by the data collected by the authors.

44. The research should provide data on the effect of applied biofertilizers on the Narra seedlings as compared to the control. How do authors verify that the spores that they visualized and the colonies they observed on nitrogen-free Dobereiners agar media were of the same biofertilizer they inoculated? Compost can be a source of these spores/cells.

55. The number of cells/spores of AMF or nitrogen-fixing bacteria inoculated should be mentioned. This is also important to verify how many cells infect the root, colonize and multiply.

66. It is unclear that the microbial root colonization term that the authors used in line 233, point 3.3 in the result and discussion refers to colonization by nitrogen-fixing bacteria or AMF or both?

77.  Figure 1, 2 and 3 are missing

The language quality is good. 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Overall, the authors have made sufficient effort to incorporate the suggestions. The readability of manuscript and correlation of results has also improved. However, several concerns that need to be addressed are as under:

1. As explained by the authors, the observations on plant biomass and/or growth parameters after six months of inoculation and growth in mined-out soils, have been published as a part of another paper. Hence, major inclination of the manuscript is towards root colonization potential and associated depletion of heavy metals. Therefore, the title should be changed accordingly.

2. Line 203: Convert into one sentence.

3. In Section 3.1, the soil parameters after results are compared with Table 1 that describes soil properties before planation of Narra seeding. For the parameters after 6 months, a similar comparison between the control and the treated soils, is required, as that would highlight the potential of the biofertilizer. It should be clearly mentioned, which biofertilizer performs relatively better, in each case.

4. In figures 1 and 2, in some cases, the error bar on the graphs are apparently extending on the negative axis. The authors need to explain this.

5. Line 311-313: Rephrase, its unclear.

6. Line 430: Sentence is abrupt. Several such abrupt sentences are noted across the manuscript

7. Figure 4: The authors have now sufficiently differentiated between the statistical difference and the figure subdivisions. Hence, use of alternative conventional symbols like *, ‡ etc (as implied in previous comment) to indicate statistical differences, is ruled out.

However, the figure legends should define the meaning of the alphabets and their combinations like a, b, c, d, e, ab, bc etc. In fact, this is required for all the figures.

8. Tracking the changes in the re-submission was difficult.

As submitted by the authors, the English language has been corrected using grammar checker; however, professional help in English language may help further improvement.

Author Response

I greatly appreciate your comments. Please see the attachment for the action done.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors have revised the manuscript according to the comments.

Author Response

Please see attachment.

Author Response File: Author Response.pdf