Deciphering the Symbiotic Plant Microbiome: Translating the Most Recent Discoveries on Rhizobia for the Improvement of Agricultural Practices in Metal-Contaminated and High Saline Lands

Round 1

Reviewer 1 Report

General comments

In the manuscript Bellabarba et co-workers, reviewed the knowledge on the impact of rhizobia inoculant in agricultural practices. In particular, the authors focus on conditions of metalloid and high saline lands.

The review summarizes the different aspects of the interaction of rhizobia with plants and point out the effective role on plant fitness.

 Overall, the manuscript is well written and gives a global overview of the knowledge of rhizobia influence on plant growth in harsh soil conditions for possible applications to crops of agricultural interest. The manuscript is easy to follow and the reviewed data could be of interest for the scientific community working on the better understanding the role of rhizobia inoculants.

 

The specific comment concerns:

 Fig1: the legend should include all the abbreviations utilized in the figure.

Table 1: it should be reviewed since the layout is not correct (ex: in the main line it is indicated Metal concentration that is not provided in the table..). The section concerning co-inoculation could be presented as a table apart.

Author Response

We would like to thank Reviewer 1 for the suggestions. We modified the Figure 1 without abbreviations and the relative legend. Furthermore, we corrected the main line of Table 1 from “Metal concentration” in “Metal contamination”.

We thank the referee for the suggestion, but we preferred to not modify the section concerning the co-inoculation: considering that only one case of co-inoculation is well described, it was left in the same Table 1 to avoid displacement in the table and main text symmetry .

Reviewer 2 Report

The review "Deepening the symbiotic plant microbiome: translating the most recent discoveries on rhizobia for the improvement of agricultural practices in metalloid and high saline lands" by  Bellabarba et al. covers  the recent studies on plant growth promoting rhizobia as effective inoculants for agricultural practices in saline and heavy metal contaminated soils. This review covers important related matters ranging from molecular to genetic mechanisms and from rhizobial inoculants to genomic strategies for improving crop yield.

The manuscript is well written and summarizes the main ideas related to the importance of rhizobia for improve legume productivity and includes general molecular mechanisms adopted by legume and rhizobia that allow them to overcome such harsh conditions.

However, in the section "Plant Growth Promoting Rhizobia in heavy metal contaminated soils" is not discussed if rhizobia and legumes are or not metal tolerant to metal toxicity. Moreover, what are the main constraints to the legume-rhizobia symbioses under metal toxicity ? The mechanisms involved in plant tolerance described herein are mainly from non-legumes. The same mechanisms were detected in legumes? What about the prospecting rhizobial tolerance to metals? All rhizobial genera are tolerant to metals? Are there specific genera more tolerant than others? These aspects should be discussed to improve the quality of this section.

Author Response

We thank the reviewer for the suggestions. Indeed, we improve the paragraph adding few sentences and the relevant references highlighting the importance of the rhizobial symbiotic relationship in metal contaminated soils. In particular, referring to their role in legume’s role in bioremediation, we improved the main text in lines 225-227:“Legumes may be ideal species for bioremediation as surveys of plant species surviving in long-term metal-contaminated environments showed legumes to account for a dominant portion of these populations [122]”.

Referring to the rhizobial mechanisms for phytostabilization and phytoimmobilization (in rhizosphere and in symbiosis), we improved the main text in lines 241-247: ”The main contribution of rhizobia towards phytostabilization and phytoimmobilization is plant growth enhancement [132]. Bacteria that nodulate their hosts may increase metal accumulation in root nodules, while those that remain in the rhizosphere would reduce metal toxicity locally by precipitation, chelation, immobilization and biosorption. The role of the nodule itself has an important role in metal-resistance: once the symbiosis is established, nodules could serve as storage areas that provide plants an extra place to stock metals and reduce the risk of direct exposure [133].”

and lines 259-265: “Although not all rhizobia are intrinsically tolerant to metals, metal-tolerant strains of taxonomically diverse rhizobia have been isolated from various plants in heavy metal contaminated environments [37, 142]. Metal resistance determinants provide protection for rhizobia to survive and maintain effective nodulation of legumes, allowing them to play a role in promoting plant growth. In addition, the existence of a symbiotic relationship may provide protection for the survival of rhizobia in soils with elevated metal concentrations [143,144].”

Reviewer 3 Report

Review of Bellabarba et al. MDPI

This will be a useful paper for people not familiar with the field because it summarizes some of the recent discoveries for improving saline and metal-contaminated soils.

 

The reviewer recommends that you change the title from “Deepening” to “Deciphering, etc.” “Deepening”, which can suggest digging a hole or diving into the ocean. It seems to me that you are trying to do the exact opposite of that and rather are trying to understand the complexity of the symbiotic microbiome.

Also, change "metalloid" in the title to "metal-contaminated" or something similar.  Definition of metalloid:  an element (e.g. arsenic, antimony, or tin) whose properties are intermediate between those of metals and solid nonmetals or semiconductors.

 

Overall, this is an interesting paper , but there are omissions and at times confusing statements that can be easily corrected, which will make this a much more useful contribution.  

 

Specific Comments.

p.2, line 8.  The concept of the nodulating beta-rhizobia has expanded to consist of Paraburkholderia,Cupriavidus, and Trinickia (see Estrada-de los Santoset al. 2018. Genes.  9(8), 389; https://doi.org/10.3390/genes9080389and Estrada de los Santos et al., 2019. Trinickia dabaoshanensissp. nov., a new name for a lost species.  Arch. Microbiol.  https://doi.org/10.1007/s00203-019-01703-2.)

 

p.3, line 4, siderophore (singular) production.  The phrase mineral phosphate seems out of place.  Do you mean to say phosphate and zinc solubilization?

 

p.3, line 114. …to improve yield of legumes grown in arid or metal-contaminated soils.  

 

p. 3, line 120.  You have written a sentence about salinity and in the next paragraph you make an abrupt jump to arid soils.  The transition should be improved.

 

p. 4, line 128.  “Withered” is the wrong word.  Synonyms:  dry, desiccated, dehydrated.

 

Line 132. “Continued” instead of “The progressive”.

 

Line 134.  Delete “the “.  Start sentence with “Salt tolerance…”.

 

Line 145.  No doubt multiple cytosolic proteins are affected.   Use the plural.

 

Line 150.  Delete:  “This is highlighted by the work of”, and start the sentence with “Sakrouchi and” and delete “who” before isolated.

 

Line 152.   …organic solvents, osmo-protectants,” and in the next line insert after [54] “such”.

 

Line 156.  Close up space after “poly-“.

 

Line 166.  Depletion is awkward. Write:   soil “nutrient loss”.

p. 5, line 194.  Azospirillum is a “diazotroph”.  You can say non-rhizobial diazotroph but “not rhizobia” by itself.  This is not the correct definition.

 

p. 5, line 2013 “to improve”

 

p. 5, “Anthropogenic” is the correct word; “human-made”.

 

p. 6, line 244.   EPS is often an abbreviation for exopolysaccharide.   Please check to see if you are correct with this abbreviation for extracellular polymeric substances or can something else be used (e.g., no abbreviation at all).

 

p. 6, line 270.  Complex sentence.  Perhaps a comma after carbon source, which….

 

p. 10, line 368.  What do you mean by “conjunct”?

 

 

Please check references.  Reference 210 is missing. 

 

Author Response

Ref 3

The reviewer recommends that you change the title from “Deepening” to “Deciphering, etc.” “Deepening”, which can suggest digging a hole or diving into the ocean. It seems to me that you are trying to do the exact opposite of that and rather are trying to understand the complexity of the symbiotic microbiome.

Also, change "metalloid" in the title to "metal-contaminated" or something similar.  Definition of metalloid:  an element (e.g. arsenic, antimony, or tin) whose properties are intermediate between those of metals and solid nonmetals or semiconductors.

 

We thank the reviewer for the suggestions: we accordingly modified the title and the main text.

 

Overall, this is an interesting paper , but there are omissions and at times confusing statements that can be easily corrected, which will make this a much more useful contribution. 

 

We are grateful to the referee for this comment. We read and correct the manuscript making it more clear and accurate.

 

Specific Comments.

p.2, line 8.  The concept of the nodulating beta-rhizobia has expanded to consist of Paraburkholderia,Cupriavidus, and Trinickia (see Estrada-de los Santoset al. 2018. Genes.  9(8), 389; https://doi.org/10.3390/genes9080389 and Estrada de los Santos et al., 2019. Trinickia dabaoshanensissp. nov., a new name for a lost species.  Arch. Microbiol.  https://doi.org/10.1007/s00203-019-01703-2.)

 We thank the referee for the bibliography suggestions. We add them in the main text (line 59, references 20-21).

 

p.3, line 4, siderophore (singular) production.  The phrase mineral phosphate seems out of place.  Do you mean to say phosphate and zinc solubilization? 

Correction line 97: “ This is due to their PGP features such as indoleacetic acid, siderophore production, phosphate and zinc solubilization.”.

 

p.3, line 114. …to improve yield of legumes grown in arid or metal-contaminated soils.  

We correct as suggested by the reviewer (line 114).

 

3, line 120.  You have written a sentence about salinity and in the next paragraph you make an abrupt jump to arid soils.  The transition should be improved.

Correction line 121: “Salinity due to water deficiency conditions is one of the largest environmental constraints for plant growth and productivity in stricken regions”

 

4, line 128.  “Withered” is the wrong word.  Synonyms:  dry, desiccated, dehydrated.

Correction line 129 “Desiccated soils can lead to alterations in plant homeostasis due to a reduction in the osmotic potential and inappropriate ionic distribution.”

 

Line 132. “Continued” instead of “The progressive”.

We corrected the main text according to the referee suggestion in line 133.

 

Line 134.  Delete “the “.  Start sentence with “Salt tolerance…”.

We corrected the main text according to the referee suggestion in line 135.

 

Line 145.  No doubt multiple cytosolic proteins are affected.   Use the plural.

We corrected the main text according to the referee suggestion in line 146.

 

Line 150.  Delete:  “This is highlighted by the work of”, and start the sentence with “Sakrouchi and” and delete “who” before isolated.

We corrected the main text according to the referee suggestion, starting with “For example” to mantain connection with the previous sentence, in line 151.

 

Line 152.   …organic solvents, osmo-protectants,” and in the next line insert after [54] “such”.

We corrected the main text in line 153 e 156.

 

Line 156.  Close up space after “poly-“.

We corrected the main text according to the referee suggestion in line 157.

 

Line 166.  Depletion is awkward. Write:   soil “nutrient loss”.

We corrected the main text according to the referee suggestion in line 167.

 

5, line 194.  Azospirillum is a “diazotroph”.  You can say non-rhizobial diazotroph but “not rhizobia” by itself.  This is not the correct definition.

We corrected the main text according to the referee suggestion in line 195.

 

5, line 2013 “to improve”

We corrected the main text modyfing the sentence in line 203. We used “thus improving” and not “to improve” considering that the final aim of bacteria in producing ACC deaminase is not plant growth promotion, but of course the process facilitate it.

 

5, “Anthropogenic” is the correct word; “human-made”.

We corrected the main text according to the referee suggestion in line 215.

 

6, line 244.   EPS is often an abbreviation for exopolysaccharide.   Please check to see if you are correct with this abbreviation for extracellular polymeric substances or can something else be used (e.g., no abbreviation at all).

We corrected the main text according to the referee suggestion removing the abbreviation and using the complete definition in lines 255,256

 

6, line 270.  Complex sentence.  Perhaps a comma after carbon source, which….

We corrected the main text according to the referee suggestion in line 287.

 

10, line 368.  What do you mean by “conjunct”?

We corrected the main text with the term “inclusion” in line 385.

 

Please check references.  Reference 210 is missing. 

 We thank the referee for the suggestion. We corrected and improved the references list.