Preservation and Recovery of Metal-Tolerant Fungi from Industrial Soil and Their Application to Improve Germination and Growth of Wheat

Round 1

Reviewer 1 Report

This is an interesting paper, which can be revised and accepted.

 

1. The introduction is too jumbled. According to your research content, suggest yourintroduction mainly divides into four parts to write, first, the present situation and the harm of heavy metal polluted soil, and second, the main heavy metal contaminated soil repair methods and the advantages and disadvantages, and third, the introduction of bioremediation, are mainly bioremediation and phytoremediation of research as well as repair mechanism, finally, a brief introduction to your research content.

 

2. Please indicate the meaning of each abbreviation in the table below the table.

 

3. In Table 3, if "STD" stands for "prescribed limit", then the soil is also obviously contaminated by Zn and Pb. Please give a reasonable explanation.

 

4. The author pointed out in 3.7 "Tolerance index revealed best tolerance of Cd by MS1... (Fig. 4)." However, in Fig. 4 "MS1" was not found to be the best tolerance to Cd. Please give a reasonable explanation.

 

5. Section 3.9, on the metal adsorption capacity of fungi, suggest you write detailed data and give relevant analysis. In fact, metal concentration, temperature and pH belong to external conditions, and your study provides a tolerance degree of the fungus to relevant external conditions. Therefore, it is suggested that you analyze how related conditions affect the adsorption capacity of the fungus, and the influence degree of different conditions and other contents.

 

6. The author pointed out "These mor-phological changes describe the detoxification mechanisms." in "4. Discussion", please further discuss the mechanism between the morphological changes and detoxification mechanisms of the fungi screened in this paper in the discussion.

 

7. The author pointed out in "4. Discussion" that "Results of this study depicted that the increasing metal concentration also improves biosorption capacity of fungus.", please further discuss the relationship between the adsorption capacity of the fungi screened in this paper and the concentration of heavy metals in the discussion.

 

8. Please discuss the effect between the fungi screened in this study and the germination rate of wheat seeds in "4. Discussion".

 

Author Response

Answers to Reviewer 1

1. The introduction is too jumbled. According to your research content, suggest your introduction mainly divides into four parts to write, first, the present situation and the harm of heavy metal polluted soil, and second, the main heavy metal contaminated soil repair methods and the advantages and disadvantages, and third, the introduction of bioremediation, are mainly bioremediation and phytoremediation of research as well as repair mechanism, finally, a brief introduction to your research content.

Answer: As per instruction, introduction has been re-written and divided into four suggested parts. 

2. Please indicate the meaning of each abbreviation in the table below the table.

Answer: As per instruction, meaning of each abbreviation has been mentioned below the table. 

3. In Table 3, if "STD" stands for "prescribed limit", then the soil is also obviously contaminated by Zn and Pb. Please give a reasonable explanation.

Answer: In table 3, "STD" stands for standard deviation, and it is not related to any prescribed limit. As replied in query 3, meaning of STD has been written below table 3. 

4. The author pointed out in 3.7 "Tolerance index revealed best tolerance of Cd by MS1... (Fig. 4)." However, in Fig. 4 "MS1" was not found to be the best tolerance to Cd. Please give a reasonable explanation.

Answer: Thank you much sir/madam for pointing out this mistake. It is actually MS5 and it has been corrected in the text and highlighted. 

5. Section 3.9, on the metal adsorption capacity of fungi, suggest you write detailed data and give relevant analysis. In fact, metal concentration, temperature and pH belong to external conditions, and your study provides a tolerance degree of the fungus to relevant external conditions. Therefore, it is suggested that you analyze how related conditions affect the adsorption capacity of the fungus, and the influence degree of different conditions and other contents.

Answer: As per instruction, more information has been incorporated. The detailed influence of all factors has been described and highlighted.

6. The author pointed out "These mor-phological changes describe the detoxification mechanisms." in "4. Discussion", please further discuss the mechanism between the morphological changes and detoxification mechanisms of the fungi screened in this paper in the discussion.

Answer: We have observed two morphological changes (colour of mycelia and pattern of mycelia). We have explained it in a better way, in last two lines of paragraph and highlighted it.

7. The author pointed out in "4. Discussion" that "Results of this study depicted that the increasing metal concentration also improves biosorption capacity of fungus.", please further discuss the relationship between the adsorption capacity of the fungi screened in this paper and the concentration of heavy metals in the discussion.

Answer: Information has been incorporated and and highlighted.   

8. Please discuss the effect between the fungi screened in this study and the germination rate of wheat seeds in "4. Discussion".

Answer: Information has been incorporated and and highlighted.  

Reviewer 2 Report

The authors have focused their study on the Preservation and Recovery of Metal Tolerant Fungi from Industrial Soil and their Application to Improve Germination and Growth of Wheat. The authors have not respected the Instructions for authors, so the paper has serious shortcomings in writing, structure and content. At least one of the 17!!! authors had to do that. 

Please see few of my suggestions bellow.

Lines of the manuscript  are not numbered so it is more difficult to exactly indicate the place for changes. Please number them.

Keywords must reflect the main characteristic words of the paper (usually reflected also by the title) in the best way to increase the paper's relevance and chances to be find when searching it after key words. So, for the actual title, I suggest the following  keywords: preservation; recovery; metal tolerant fungi; industrial soil; application; wheat germination; wheat growth.

1. Introduction 

First paragraph. "very important is mentioned twice, in 2 consecutive sentence. Please reshape. Mentioning that something is very important is not and academic statement. The importance must be highlighted by a clear statement.

Text is very repetitive: i.e. 2nd paragraph: Soil pollution....Pollution....Soil pollution...Soil pollutants.... English must be extensively revised, avoiding these annoying repetitions.

After ref. [2]. The sentence beginning with "Soil pollutants..... " Exemplify them in parenthesis. Reference is also needed to be added. Sentence beginning with "Different chemical pesticides..." also needs reference. I suggest adding Bungau et al. Expatiating the impact of anthropogenic aspects and climatic factors on long term soil monitoring and management. Environ Sci. Pollut. Res. 2021, 202, 30528-30550. https://doi.org/10.1007/s11356-021-14127-7 and Samuel, A.D., et al. Effects of Long Term Application of Organic and Mineral Fertilizers on Soil Enzymes. Revista de Chimie, 2018, 69(10), 2608-1612. https://doi.org/10.37358/RC.18.10.6590. 

Please make the actual aim of study (presented in the SEPARATE, LAST paragraph of Introduction) relevant, to increase its visibility. Responding to the following questions would be helpful: What makes special this study? Which is its novelty character or its special aspects? Why have the author chosen this topic? What differentiate this paper from others in the same/similar topic? Actual text is not relevant/attractive at all (there are tenths of papers in similar topic), mentioning the site of the study not being enough to justify the relevance and necessity of publishing your study.

2. Material and methods

As the unit of measure for volume in your research, please replace ml with mL (as Litter being the international unit of measure for volume). Please check/revise the entire manuscript in this regard.

I suggest a new section 2.1. Reagents and apparatus. Please provide here the Model, Producer/manufacturer, City and Country for each apparatus used in the research, and the Producer and Country for each reagent/chemical used.  OR, I suggest adding all the mandatory information after each reagent/apparatus. The Authors will decide.

I suggest restructuring this section (avoiding so many very short sections/subsections) - the text will be more fluid, as follows:

2.1. Reagents and apparatus, if the authors decide to make it as a separate subsection;

2.2. Soil sample collection and processing

2.3. Physiochemical properties of soil samples, removing the actual subtitles 2.2.1. and 2.2.2. The text is very clear, with no other subtitles.

etc...

In the actual 2.5., as well is not needed of 3! subsections for providing 3 equations. 

To not detail every subsection, I suggest that authors to merge some of them. They are much too many, not relevant some of them (2 lines cannot be considered a subsection). Please revise the structure of the entire section.

2.14. In the Statistical analysis subsection please also check that all computers programs used for analysis to be provided together with their variants.

3.1. "... helped us.. " is not a scientific way to write. I think the authors knew and understood "Different physiochemical properties of soil " before this study. Please reshape the sentence.

According to the instructions for authors: https://www.mdpi.com/journal/sustainability/instructions (I suggest checking and applying them, as the Instructions for authors are not optional but must be followed):

• All Figures, Schemes and Tables should be inserted into the main text close to their first citation and must be numbered following their number of appearance (Figure 1, Scheme I, Figure 2, Scheme II, Table 1, etc.).
• Adapt the size of the figures to be similar to the characters used in the main text (10 size)
• Figure 5 is blurred. Please replace it with a better quality one.
• Acronyms/Abbreviations/Initialisms should be defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table. When defined for the first time, the acronym/abbreviation/initialism should be added in parentheses after the written-out form.

On the Sustainability draft please see the requested format for Tables (type and size of characters, lines for the table, etc.

4. Discussion 

This section is very poor. Please develop it. A table (having as the last column Ref.) making comparison with other already published data would be relevant.

Moreover, as the Introduction section is very long, the authors can move some information from Introduction to Discussion.

Additionally, considering that the soil enzymology is one of the main characteristics when it is about soil proprieties [Samuel A.D.,et al. Enzymological and physicochemical evaluation of the effects of soil management practices. Rev. Chim. 2017, 68(10), 2243-2247. https://doi.org/10.37358/RC.17.10.5864 ; Samuel A.D., et al. Enzymatic indicators of soil quality.  J. Environ. Prot. Ecol. 2017, 18(3), 871-878], what about the connection between this enzymology and industrial polluted soil?

What about nanotechnology usage in the soil management practices related to soil pollution [Behl, T.; Kaur, I.; Singh, S.; Sharma, N.; Bhatia, S.; Al-Harrasi, A.; Bungau S. The dichotomy of nanotechnology as the cutting edge of agriculture: Nano-farming as an asset versus nanotoxicity, Chemosphere2022, 288 Part 2, 132533. https://doi.org/10.1016/j.chemosphere.2021.132533 ]. Develop these ideas.

Please complete which are the strengths and weakness of your study (if there is any) - it should be mentioned as the last paragraph of 4. Discussion section.

5. Conclusion. Please reshape "helped us"!!! Underline better the achievement of the aim of the study, considering also the novelty/special aspects of your study.

References

• Please check the Instructions for authors,
• provide all the requested information for each reference and
• use the MDPI style,
• number them in the requested way
• update some of them as they are older than 2) years. 

Author Response

Review Report 2

The authors have focused their study on the Preservation and Recovery of Metal Tolerant Fungi from Industrial Soil and their Application to Improve Germination and Growth of Wheat. The authors have not respected the Instructions for authors, so the paper has serious shortcomings in writing, structure, and content. At least one of the 17!!! authors had to do that. 

Please see few of my suggestions bellow.

Query: Lines of the manuscript are not numbered so it is more difficult to exactly indicate the place for changes. Please number them.

Answer: Lines of the manuscript has been numbered.

Keywords must reflect the main characteristic words of the paper (usually reflected also by the title) in the best way to increase the paper's relevance and chances to be find when searching it after key words. So, for the actual title, I suggest the following keywords: preservation; recovery; metal tolerant fungi; industrial soil; application; wheat germination; wheat growth.

Answer: Thanks for very kind suggestion. Described keywords have been incorporated.

1. Introduction

First paragraph. "very important is mentioned twice, in 2 consecutive sentence. Please reshape. Mentioning that something is very important is not and academic statement. The importance must be highlighted by a clear statement.

Answer: As per suggestion, sentence has been replaced as “contamination free soils are imperative for the community” and highlighted in the revised manuscript file.

Text is very repetitive: i.e. 2nd paragraph: Soil pollution....Pollution....Soil pollution...Soil pollutants.... English must be extensively revised, avoiding these annoying repetitions.

Answer: As per suggestion, English has been revised.

After ref. [2]. The sentence beginning with "Soil pollutants..... " Exemplify them in parenthesis. Reference is also needed to be added.

Answer: Changes have incorporated, and reference has been added and highlighted.

Sentence beginning with "Different chemical pesticides..." also needs reference. I suggest adding Bungau et al. Expatiating the impact of anthropogenic aspects and climatic factors on long term soil monitoring and management. Environ Sci. Pollut. Res. 2021, 202, 30528-30550. https://doi.org/10.1007/s11356-021-14127-7 and Samuel, A.D., et al. Effects of Long Term Application of Organic and Mineral Fertilizers on Soil Enzymes. Revista de Chimie, 2018, 69(10), 2608-1612. https://doi.org/10.37358/RC.18.10.6590. 

Answer: Suggested references have been incorporated and highlighted.

Please make the actual aim of study (presented in the SEPARATE, LAST paragraph of Introduction) relevant, to increase its visibility. Responding to the following questions would be helpful: What makes special this study? Which is its novelty character or its special aspects? Why have the author chosen this topic? What differentiate this paper from others in the same/similar topic? Actual text is not relevant/attractive at all (there are tenths of papers in similar topic), mentioning the site of the study not being enough to justify the relevance and necessity of publishing your study.

Answer: All suggested information has been incorporated and highlighted.

2. Material and methods

As the unit of measure for volume in your research, please replace ml with mL (as Litter being the international unit of measure for volume). Please check/revise the entire manuscript in this regard.

Answer: Suggested information has been incorporated and highlighted.

I suggest a new section 2.1. Reagents and apparatus. Please provide here the Model, Producer/manufacturer, City and Country for each apparatus used in the research, and the Producer and Country for each reagent/chemical used.  OR, I suggest adding all the mandatory information after each reagent/apparatus. The Authors will decide.

I suggest restructuring this section (avoiding so many very short sections/subsections) - the text will be more fluid, as follows:

2.1. Reagents and apparatus, if the authors decide to make it as a separate subsection.

2.2. Soil sample collection and processing

2.3. Physiochemical properties of soil samples, removing the actual subtitles 2.2.1. and 2.2.2. The text is very clear, with no other subtitles.

etc...

Answer: As per suggestion, new section has been added and highlighted. Names of  

In the actual 2.5., as well is not needed of 3! subsections for providing 3 equations. 

To not detail every subsection, I suggest that authors to merge some of them. They are much too many, not relevant some of them (2 lines cannot be considered a subsection). Please revise the structure of the entire section.

Answer: As per suggestion, new section has been added and highlighted. The Model, Producer/manufacturer, City and Country for each apparatus used in the research has been described in detail and highlighted.

2.14. In the Statistical analysis subsection please also check that all computers programs used for analysis to be provided together with their variants.

Answer: As per suggestion, missing information has been incorporated and highlighted in lines 208-210.

3.1. "... helped us.. " is not a scientific way to write. I think the authors knew and understood "Different physiochemical properties of soil " before this study. Please reshape the sentence.

Answer: As per suggestion, information has been incorporated and highlighted as “Different physiochemical properties revealed the nature and composition of collected soil samples.” in line 213.

According to the instructions for authors:

 https://www.mdpi.com/journal/sustainability/instructions (I suggest checking and applying them, as the Instructions for authors are not optional but must be followed):

• All Figures, Schemes and Tables should be inserted into the main text close to their first citation and must be numbered following their number of appearance (Figure 1, Scheme I, Figure 2, Scheme II, Table 1, etc.).
• Adapt the size of the figures to be similar to the characters used in the main text (10 size)
• Figure 5 is blurred. Please replace it with a better quality one.
• Acronyms/Abbreviations/Initialisms should be defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table. When defined for the first time, the acronym/abbreviation/initialism should be added in parentheses after the written-out form.

On the Sustainability draft please see the requested format for Tables (type and size of characters, lines for the table, etc.

Answer: All the format changes has been settled according to the format of “sustainability”.

4. Discussion 

This section is very poor. Please develop it. A table (having as the last column Ref.) making comparison with other already published data would be relevant.

Moreover, as the Introduction section is very long, the authors can move some information from Introduction to Discussion.

Answer: All the suggestions have been incorporated and highlighted.

Additionally, considering that the soil enzymology is one of the main characteristics when it is about soil proprieties [Samuel A.D.,et al. Enzymological and physicochemical evaluation of the effects of soil management practices. Rev. Chim. 2017, 68(10), 2243-2247. https://doi.org/10.37358/RC.17.10.5864 ; Samuel A.D., et al. Enzymatic indicators of soil quality.  J. Environ. Prot. Ecol. 2017, 18(3), 871-878], what about the connection between this enzymology and industrial polluted soil?

What about nanotechnology usage in the soil management practices related to soil pollution [Behl, T.; Kaur, I.; Singh, S.; Sharma, N.; Bhatia, S.; Al-Harrasi, A.; Bungau S. The dichotomy of nanotechnology as the cutting edge of agriculture: Nano-farming as an asset versus nanotoxicity, Chemosphere2022, 288 Part 2, 132533. https://doi.org/10.1016/j.chemosphere.2021.132533 ]. Develop these ideas.

Please complete which are the strengths and weakness of your study (if there is any) - it should be mentioned as the last paragraph of 4. Discussion section.

Answer: Instruction has been followed and both references has been incorporated and discussion has been modified.

5. Conclusion. Please reshape "helped us"!!! Underline better the achievement of the aim of the study, considering also the novelty/special aspects of your study.

Answer: Instruction has been followed and information has been incorporated.

References

• Please check the Instructions for authors,
• provide all the requested information for each reference and
• use the MDPI style,
• number them in the requested way
• update some of them as they are older than 2) years. 

Answer: All the instruction has been followed and references been settled modified according to MDPI style.

Reviewer 3 Report

The manuscript ,,Preservation and Recovery of Metal Tolerant Fungi from Industrial Soil and their Application to Improve Germination and Growth of Wheat,, addresses an interesting topic that could be of interest to the scientific community. 

I have the following comments on the manuscript:

The abstract should be factual, but should include background, results and conclusions. 

I would welcome a more extensive section on results and discussion than the experimental section.

The significance of this study should be more emphasize in the introduction.

Page 2: ,,Many scientists have started working on the remediation of heavy
metals which are being released from different industries like leather, steel, tanning and pulp processing,,

This issue has been addressed in great detail by this very important paper and therefore it should be mentioned here.

https://www.sciencedirect.com/science/article/pii/S0304389421031757

Page 2 ,,The heavy metals remain in soil for many years, it has become imperative to find different remediation methods to control soil contamination. Many people are working on devising new methods and strategies to solve the problems of soil pollution. Generally, soil remediation is brought about in situ and ex situ. In in situ remediation, soil is subjected to remediation treatment in the original place, while in ex situ remediation, soil is treated
in laboratory or some other place. Scientists use both methods according to the requirement of site,,. 

Tha same here. https://www.sciencedirect.com/science/article/pii/S0048969722011354

Please add line numbering to a future version of the manuscript.

A list of the most important abbreviations could be added at the beginning of the manuscript.

At the end of the introduction, emphasize the novelty and importance of your work.

Page 3: ,,Following physiochemical properties of collected soil samples were examined:,, This sentence should be deleted.

,,2.2.1. Soil organic matter.,, State the purity and molar concentration of all chemicals used in the experiments.

,,2.5.3. Pollution index (PI),, Improve the quality of this equation. It is blurred and probably added as an image.

,,3. Results,, The results should be discussed in more detail. In the current situation, this would not be enough.

,,4. Discussion,, The discussion should also be more extensive.

,,Conclusion,, Extend the conclusions with your most important findings. Indicate the possible risks of such research. Add your recommendations for future research.

,,References,, Make sure the references are added correctly according to the journal's instructions.

,,Table 1 Nutrient analysis of industrial soil samples,, This should be Table 2.

Add measurement deviations to the individual tables.

Distinguish the graphs in the figures in color.

   

 

Author Response

Reviewer 3

The manuscript “Preservation and Recovery of Metal Tolerant Fungi from Industrial Soil and their Application to Improve Germination and Growth of Wheat,, addresses an interesting topic that could be of interest to the scientific community. 

I have the following comments on the manuscript:

The abstract should be factual, but should include background, results and conclusions.

Answer: As per instruction, desired information has been incorporated. 

I would welcome a more extensive section on results and discussion than the experimental section.

The significance of this study should be more emphasize in the introduction.

Answer: As per instruction, introduction has been extensively modified and highlighted for your information. Following the instruction of all reviewers, results and discussion sections have been revised, extensively. 

Page 2: Many scientists have started working on the remediation of heavy
metals which are being released from different industries like leather, steel, tanning and pulp processing,,

This issue has been addressed in great detail by this very important paper and therefore it should be mentioned here.

https://www.sciencedirect.com/science/article/pii/S0304389421031757

Answer: As per instruction, desired reference has been incorporated. 

Page 2 ,,The heavy metals remain in soil for many years, it has become imperative to find different remediation methods to control soil contamination. Many people are working on devising new methods and strategies to solve the problems of soil pollution. Generally, soil remediation is brought about in situ and ex situ. In in situ remediation, soil is subjected to remediation treatment in the original place, while in ex situ remediation, soil is treated
in laboratory or some other place. Scientists use both methods according to the requirement of site,,. 

Tha same here. https://www.sciencedirect.com/science/article/pii/S0048969722011354

Answer: As per instruction, desired reference has been incorporated. 

Please add line numbering to a future version of the manuscript.

Answer: As per instruction, line numbers have been added in the revised manuscript. 

A list of the most important abbreviations could be added at the beginning of the manuscript.

Answer: According to your suggestion and following the journal format, all the abbreviation have been carefully described in the text. 

At the end of the introduction, emphasize the novelty and importance of your work.

Answer: Desired information has been incorporated and highlighted.

Page 3: ,,Following physiochemical properties of collected soil samples were examined:,, This sentence should be deleted.

Answer: As per instruction, sentence has been deleted.

,,2.2.1. Soil organic matter.,, State the purity and molar concentration of all chemicals used in the experiments.

Answer: As per instruction, desired information has been incorporated and highlighted.

,,2.5.3. Pollution index (PI),, Improve the quality of this equation. It is blurred and probably added as an image.

Answer: As per instruction, the quality of equation has been improved.

,,3. Results,, The results should be discussed in more detail. In the current situation, this would not be enough.

Answer: As per instruction of all reviewers, results have been described better and highlighted.

,,4. Discussion,, The discussion should also be more extensive.

Answer: As per instruction, discussion has been improved.

,,Conclusion,, Extend the conclusions with your most important findings. Indicate the possible risks of such research. Add your recommendations for future research.

Answer: As per instruction, discussion has been described in more detail and highlighted.

,,References,, Make sure the references are added correctly according to the journal's instructions.

Answer: As per instruction, all the references has been rephrased according to MDPI format.

,,Table 1 Nutrient analysis of industrial soil samples,, This should be Table 2.

Answer: In the revised manuscript, Nutrient analysis of industrial soil samples is Table 2.

Add measurement deviations to the individual tables.

Answer: As per instruction, measurement deviations have been incorporated.

Distinguish the graphs in the figures in color.

Answer: As per instruction, graphs have been modified for better understanding.

Reviewer 4 Report

Dear Authors,

I would like to thank you for all your sincere efforts in bringing out this manuscript. The work seems fine with regard to the isolation and characterization of fungal spp. capable of mycoremediation. The species F. solani and P. oxalicum demonstrated the highest biosorption capacity of Cu and Cd, respectively. However, the practical utility doesn't seem to be much promising. The isolated species performed best at 28 0C and pH 6.0. But under field conditions, this ideal situation may not be met and their performance may be severely affected. Besides that, you have studied the performance of the wheat crop for a very brief period of time (10 days) but again how long this will work remains a question. The general wheat crop cycle varies from 120-150 days. Whether these fungal stains would be able to retain the heavy metals Cd/Cu within themselves for such a long period still remains a question. Also, the farmers judge any technology based on the benefit-cost ratio. How much yield advantage a farmer can get using this technology or how much production cost can be saved by him needs to answer for getting these findings to gain a practical utility. 

  Anyway, your work does have some merit. However, I could find some very basic mistakes say as the unit of electrical conductivity mS/m. if the unit is true then the values are too less and such values we normally don't encounter in soil. It seems that the unit must have been mS/cm and that corresponds to dS/m. Similarly, you have mentioned that you have estimated N using AAS. I have never heard of it. Please do check it and correct it. Normally for estimating soil pH or EC, we use 1:2 or 1:2.5 soil: water ratio, but you have used 1:1 for pH or 1:9 for EC. Why is it So? Any specific reason? Besides, some spelling and typing mistakes have been highlighted.

Author Response

Reviewer 4

Query 1:

I would like to thank you for all your sincere efforts in bringing out this manuscript. The work seems fine with regard to the isolation and characterization of fungal spp. capable of mycoremediation. The species F. solani and P. oxalicum demonstrated the highest biosorption capacity of Cu and Cd, respectively. However, the practical utility doesn't seem to be much promising. The isolated species performed best at 28 0C and pH 6.0. But under field conditions, this ideal situation may not be met and their performance may be severely affected.

Answer:

In this study, the isolated pathogen performed well at 28 °C and pH 6.0. We agree that maintaining pH 6.0 is not easy, in the field but 28 °C is the most common in Pakistan and these fungi can perform well in the months of autumn, winter and spring. At this temperature, about 80% of sub-continent crops are grown and except 2-3 months of summer (when temperature raises above 40 °C), these bioinoculants can be used. Soil pH can definitely affect their efficiency, but it can work better than other physical and chemical bioremediation agents.

Query 2:

Besides that, you have studied the performance of the wheat crop for a very brief period of time (10 days) but again how long this will work remains a question. The general wheat crop cycle varies from 120-150 days. Whether these fungal stains would be able to retain the heavy metals Cd/Cu within themselves for such a long period still remains a question. Also, the farmers judge any technology based on the benefit-cost ratio. How much yield advantage a farmer can get using this technology or how much production cost can be saved by him needs to answer for getting these findings to gain a practical utility. 

Answer:

Sir, regarding your another query “Whether these fungal stains would be able to retain the heavy metals Cd/Cu within themselves for such a long period still remains a question.” It is stated that the studied fungi are soil-borne, and they can retain heavy metals for very long time due to their biosorption ability. Moreover, we did germination experiment of wheat because it is the most important phenomenon to guarantee the successful growth of ant plant. These fungi can help plant to germinate and mitigate toxic environment of heavy metals. Previous findings have also proved that once the fungi absorb metals, they keep it attached with their body (Vacar et al. 2021).

V ˘acar, C.L.; Covaci, E.; Chakraborty, S.; Li, B.; Weindorf, D.C.; Frent,iu, T.; Pârvu, M.; Podar, D. Heavy Metal-Resistant Filamentous Fungi as Potential Mercury Bioremediators. J. Fungi 2021, 7, 386. https://doi.org/10.3390/jof7050386

Sir we are extremely thankful to your thought-provoking ideas and suggestions. We will keep these factors in consideration while going to the field application of these mycoremediation agents. Sir, it is a lab study and gave us some good results and we are looking for its field application after the approval of national biosafety committee (NBC). In our country we need approval form NBC before their application in the field.

Query 3:

Anyway, your work does have some merit. However, I could find some very basic mistakes say as the unit of electrical conductivity mS/m. if the unit is true then the values are too less and such values we normally don't encounter in soil. It seems that the unit must have been mS/cm and that corresponds to dS/m.

Answer: It was a mistake and the units have been corrected as dS/m.

Similarly, you have mentioned that you have estimated N using AAS. I have never heard of it. Please do check it and correct it.

Answer: The sentence has been corrected and justified with the reference.

Normally for estimating soil pH or EC, we use 1:2 or 1:2.5 soil: water ratio, but you have used 1:1 for pH or 1:9 for EC. Why is it So? Any specific reason? Besides, some spelling and typing mistakes have been highlighted.

Answer: We followed the protocol of Mclean et al. 1983 and writing 1:1 and 1:9 ratios was a mistake. We have corrected it in the manuscript and highlighted. Spelling and tying mistakes have been corrected. 

Round 2

Reviewer 2 Report

The authors responded to my requests.

Author Response

thanks sir/madam for your kind suggestions.

Reviewer 3 Report

Manuscript can be accepted.

Author Response

Thanks for your kind review and final suggestions,

Reviewer 4 Report

Dear Authors,

You have made corrections. However, some minor corrections remain. In section 2.3 2^nd para second last line change the unit to deciSiemens per metre. Similarly in section 3.1 third line also change the unit to dS/m. You have mentioned two tables as Table No. 1. Please change the to Table No. 2 in the second table. Further, there is no homogeneity in the no. of digits after decimal places in data, and I had made those corrections in the original document. Please do the needful. Again I am adding that file for your ready reference. 

 

Comments for author File: Comments.pdf

Author Response

All suggested minor changes have been incorporated as track changes of the final manuscript. 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.