Isolation of Diverse Phosphate- and Zinc-Solubilizing Microorganisms from Different Environments

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Although to conclude this manuscript needs improvement. The manuscript is of great importance to the scientific community working in the field of environment, and I recommend publishing this article because.This research demonstrates the importance of isolating diverse microorganisms that solubilize phosphate and zinc from different environments.

Author Response

Response to Reviewer 1:

Comment 1: Although to conclude this manuscript needs improvement. The manuscript is of great importance to the scientific community working in the field of environment, and I recommend publishing this article because. This research demonstrates the importance of isolating diverse microorganisms that solubilize phosphate and zinc from different environments.

Response 1:  We appreciate the reviewer's recognition of the importance of our research and their recommendation for publication. We have improved the manuscript as highlighted.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is of particular interest for the development of organic farming, however, the novelty of the study is not entirely clear? Please add the name of the soils according to the WRB classification. What kind of forest soils are these? Why were they chosen for the study? Were the plowed soils also forested before use? Please describe what methods you used to determine the parameters of soils and soil amendments. The ultimate goal of the study is not entirely clear: do you want to obtain strains of microorganisms with the necessary parameters or give recommendations for improving soil quality? The conversion of phosphorus into a form accessible to plants is a very complex process, which is influenced not only by pH, but also by the iron content, which must be taken into account when choosing a substrate for isolating microorganisms. I understand that these are primary studies, but it is advisable to expand the sections of the Discussion manuscript and Conclusions by providing more detailed explanations and links (if necessary) to similar works. I hope the questions and comments will help improve the manuscript.

Author Response

Response to Reviewer 2:

Comment 1: The manuscript is of particular interest for the development of organic farming, however, the novelty of the study is not entirely clear?

Response 1: We have revised lines 86-91 to clearly delineate the novel aspects of our research: below the highlight in the manuscript.

“The present study introduces a novel approach for the isolation of PSMs and ZnSMs. To achieve this, our objectives were to: 1) isolate PSMs and ZnSMs from different environments, including those with different land uses, crop rotation systems, and soil amendments; 2) quantitatively and qualitatively screen PSMs and ZnSMs capable of solubilizing different sparingly soluble P sources (i.e., TCP, CaP, and RP) and Zn sources (i.e., ZnC, ZnO, and ZnP). These beneficial microbes hold potential use in agricultural biofertilization.

 

Comment 2: Please add the name of the soils according to the WRB classification.

Response: We have added the names of the soils according to the World Reference Base (WRB) classification in lines 98-101. This addition will aid readers in better understanding the specific soil contexts of our study:

“According to the World Reference Base for Soil Resources (WRB), the forest soil (FS) is classified as Luvisols, the agricultural soils, including the wheat-barley soil (WBS), corn-sorghum soil (CSS), and pinto beans-cowpea soil (PCS), are classified as Chernozems.”

Comment 3: What kind of forest soils are these? Why were they chosen for the study? Were the plowed soils also forested before use. Please describe what methods you used to determine the parameters of soils and soil amendments,

Response 3: Thank you for your comment. the forest soil (FS) is classified as Luvisols. We have addressed the reason for choosing natural forest soil in the introduction section (lines 75-76):

“Land use also plays a role, with uncultivated soils (e.g., forest soils) harboring a richer diversity of microbes compared to agricultural, grassland, and mined soils.”

 The agricultural soils have been under continuous agricultural production for over 100 years and were not forested prior to this use (see Materials & Methods section, see lines 112-117):

The physicochemical characterization of both soil samples and soil amendments (Table 1) was conducted using the following methods: 1:1 soil-to-water ratio for pH measurement; DTPA-extractable Zn method for zinc analysis; loss-on-ignition method for organic matter determination; and Menlich 3 method for testing phosphorus. These analyses were performed by Ward Laboratories, Inc. (Kearney, NE).

Comment 4: The ultimate goal of the study is not entirely clear: do you want to obtain strains of microorganisms with the necessary parameters or give recommendations for improving soil quality?

Response 4Thank you for your comment. We have revised our objectives for greater clarity. Please see the updated objectives on line 444-449:

“This study also underscores the potential of using microbial consortia to solubilize/mineralize diverse insoluble soil sources, which could significantly enhance crop yields and quality. The inclusion of specific fungi, such as Fusarium circinatum and Fusarium longifundum, along with Mucor circinelloides and Aspergillus awamori, enriches this potential. These findings highlight the importance of diverse microbial species in improving nutrient bioavailability in soil and, consequently, agricultural productivity.”

Comment 5: The conversion of phosphorus into a form accessible to plants is a very complex process, which is influenced not only by pH, but also by the iron content, which must be taken into account when choosing a substrate for isolating microorganisms.

Response 5: We thank the reviewer for this comment. In this study, we did not evaluate Fe sources for isolating microorganisms because previous studies indicated that in acid soils, phosphorus associates with aluminum and iron. While in alkaline soils, calcium is the main fixing constituent. However, we have provided a brief explanation in the materials and methods section see line (117-125). We hope this explanation and the corresponding modifications meet the reviewer's approval:

“The conversion of phosphorus into a form accessible to plants is significantly influenced by soil properties, notably pH and iron content. These factors affect the availability of phosphorus by forming soluble or insoluble complexes. For the isolation of effective phosphorus-solubilizing microorganisms (PSMs), it is critical to choose substrates that closely mimic these natural soil conditions. This approach ensures that the microorganisms isolated are not only effective under laboratory conditions but are also capable of enhancing phosphorus availability in a variety of agricultural settings. Thus, our substrate selection was guided by these considerations to optimize the practical applicability of the isolated PSMs.”

Comment 6: I understand that these are primary studies, but it is advisable to expand the sections of the Discussion manuscript and Conclusions by providing more detailed explanations and links (if necessary) to similar works. I hope the questions and comments will help improve the manuscript.

Response 6: We are grateful for the reviewer's constructive feedback. In response, we have carefully revised and expanded the conclusion sections, as indicated on lines 455-459 and 469-474 (highlighted in yellow) of the manuscript:

“In conclusion, adopting a more efficient approach to the identification of efficient PSMs and ZnSMs involves isolating strains from different soils or soil management practices, such as crop rotation systems or soil amendments. Our results revealed that some fungal strains did not produce halo zones on agar plates but exhibited solubilization in liquid media, whereas bacterial strains produced halo zones but showed lower solubilization in liquid media compared to the fungal strains. This highlights that the solubilization of P and Zn varies depending on the insoluble sources and the inoculated strains. Thus, while tests in media with various insoluble P and Zn sources in tandem serve as a valuable prescreening method to narrow down strains, additional tests in broths are advisable to provide an acceptable indication of P and Zn solubilizing ability, as well as to identify efficient PSMs and ZnSMs. Out of the 25 isolates, Aspergillus awamori, Fusarium circinatum, Fusarium longifundum, and Mucor circinelloides were selected as the most efficient strains to solubilize P and Zn. This study also showed that strains of the Mucor and Fusarium genera can solubilize Zn. Despite their capability to release P from both organic and inorganic sources, as well as Zn from different sources, given the complexity of soil conditions compared to in vitro settings, further studies on their interactions should be carried out before considering application. Further studies should also explore the combined effects of these PSMs and ZnSMs from different environments, aiming to develop a composite biofertilizer with higher-quality and multifunctional properties. This might enhance P and Zn bioavailability and improve their effectiveness under field conditions, despite challenging factors such as soil properties, environmental conditions, and competition with native soil microorganisms.”

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

 

The manuscript titled. "Isolation of Diverse Phosphate and Zinc Solubilizing Microorganisms from Different Environments" is very interesting. The topics of the article will certainly be of interest to readers. The methodology of the study is given in detail. The results . discussion are described correctly. Despite this, the paper needs some changes, namely:

§  Please expand the "Introduction" section.

§  The innovativeness of the research should be more highlighted.

§  In the section " Materials and Methods" subsection "Soil sample collection and soil amendments", please supplement the soil physicochemical properties with total nitrogen content, soil hydrolytic acidity, sum of base exchangeable cations. Please also state what was the granulometric composition of the soils and their classification according to WRB.

 

§  Please provide in your work what methods were used to determine the physicochemical properties of soil and soil additives.

§  The sequences of the identified bacteria and fungi should be entered into the Gen Bank database and a phylogenetic tree should be created based on them.

§  Some figures are not very clear. Please improve it.

§  The subsection numbers are incorrect. Please check it and correct it.

§  Please check the accuracy of the cited bibliography.

 

 

Author Response

Reviewer 3:

The manuscript titled. "Isolation of Diverse Phosphate and Zinc Solubilizing Microorganisms from Different Environments" is very interesting. The topics of the article will certainly be of interest to readers. The methodology of the study is given in detail. The results discussion are described correctly. Despite this, the paper needs some changes, namely:

Comment 1:   Please expand the "Introduction" section.

Respond 1: Thank you for your constructive feedback and the positive comments about our manuscript. We appreciate your suggestion to expand the Introduction section. We have expanded the introduction (see sections highlighted in yellow).

Comment 2:   The innovativeness of the research should be more highlighted.

Respond 2: Thank you for your feedback. We have emphasized the innovative aspects of our research in the introduction and discussion sections of our manuscript (see lines 86-91 and lines 444-449).

Comment 3: In the section " Materials and Methods" subsection "Soil sample collection and soil amendments", please supplement the soil physicochemical properties with total nitrogen content, soil hydrolytic acidity, sum of base exchangeable cations. Please also state what was the granulometric composition of the soils and their classification according to WRB.

 Respond 3:  Thank you for your insightful comments regarding the additional soil physicochemical properties. In this study, we did not measure the total nitrogen content, soil hydrolytic acidity, granulometric composition, or the sum of base exchangeable cations. These parameters were not included in our initial study design as our focus was primarily on parameters related to P and Zinc availability ( 1:1 soil-to-water ratio for pH measurement; DTPA-extractable Zn method for zinc analysis; loss-on-ignition method for organic matter determination; and Menlich 3 method for testing phosphorus) that directly influence the isolation and efficacy of phosphate-solubilizing microorganisms and zinc-solubilizing microorganisms under varying soil management practices.

For the classification according to the World Reference Base for Soil Resources (WRB), our classification was limited to identifying the soils as Luvisols for forest soils and Chernozems for agricultural soils, as per the details mentioned in the "Soil sample collection and soil amendments" section. Further detailed granulometric composition was not analyzed in this study. We appreciate your suggestions and will take them into account to enhance the robustness of future studies and their findings.

Comment 4:  Please provide in your work what methods were used to determine the physicochemical properties of soil and soil additives.

Respond 4:  Thank you for your comment. The methods used to determine the physicochemical properties of the soil and soil additives are detailed in the "Materials and Methods" section, lines 113 to 118. This includes measurements of soil pH, organic matter, available phosphorus, and zinc using standard protocols. These analyses were performed by Ward Laboratories, Inc. (Kearney, NE. USA)

Comment 5:  The sequences of the identified bacteria and fungi should be entered into the GenBank database and a phylogenetic tree should be created based on them.

 Respond 5:  We appreciate your feedback. Figure 1 displays a circular phylogenetic tree. The fasta sequences for the identified bacteria and fungi were submitted to GenBank with submission IDs SUB14576178 (Prokaryotic 16S rRNA) and SUB14575763 (Eukaryotic Nuclear rRNA/ITS). 

 Comment 6:   Some figures are not very clear. Please improve it.

 Respond 6: Thank you for pointing out the clarity issues with some figures in our manuscript. We have addressed this comment.

Comment 7:  The subsection numbers are incorrect. Please check it and correct it.

Respond 7: Thank you for pointing out the issue with the subsection numbers. We have reviewed and corrected them to ensure they are accurate. Your attention to detail is greatly appreciated.

Comment 8:  Please check the accuracy of the cited bibliography.

Respond 8: We appreciate your feedback on the bibliography. We have double-checked and

verified the accuracy of all cited sources. Thank you for bringing this to our attention.

We hope that the revisions and clarifications provided address the concerns of the reviewers adequately. We believe that these changes have significantly strengthened our manuscript, making it a valuable contribution to the field. We are thankful for the opportunity to improve our work based on such constructive feedback. We look forward to the possibility of our work being published in [Applied Microbiology] and believe it will make a significant impact in the field.