Rhizobia: A Promising Source of Plant Growth-Promoting Molecules and Their Non-Legume Interactions: Examining Applications and Mechanisms

Round 1

Reviewer 1 Report

The manuscript entitled  “Rhizobia: A Promising Source of Plant Growth-Promoting Molecules and their Non-Legume Interactions: Examining Applications and Mechanisms” summarised the literature regarding the use of rhizobia for promoting the growth of non-legume plants.

The Manuscripts has a solid structure, and covers the most significant parts of this area.

However, there are some mistakes that should be corrected (mentioned below):

When writing rhizobia: non-italic, lower caps; Rhizobium: italic; all Latin names should be written in italic. This should be done throughout the whole manuscript.

Line 51: add reference.

Lines 85-88: or vice versa?

Line 101: add reference.

Lines 112-113: Find more research where commercial fertilisers are used.

Line 127: Gram-negative, Gram is uppercased.

Lines 152-154: add more references for legume-rhizobia symbiosis.

Line 161: add more references for using rhizobia in abiotic or biotic stress, both for legumes and non-legumes.

Line 596: do not start a sentence with number.

Line 649: italic.

Line 651: replace “et” with “and” in the reference citing.

Lines 789-791: find more research about using liquid bio-inoculants based on rhizobia (both for legumes, such as alfalfa, birdsfoot trfoli and similar, and for non-legumes)

In general, reference list needs to be updated with some recent literature (see: DOI: 10.3390/su13063369, 10.1007/978-981-19-4906-7, 10.1007/s40003-020-00474-3,  10.1016/j.rhisph.2022.100487, 10.1002/9781118917091.ch8… )

Extensive editing of English language is required.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The objective of the review is to describe and discuss the mechanisms by which rhizobia act as PGPR and stimulate the growth of non-legumes.

The authors give no clear justification for this review nor idea of the specific content or knowledge gap to be addressed is given, nor mention of previous reviews on the topic. The 2009 review cited in reference 2 is much better structured and informative. Most of the primary literature in the current manuscript is old, and much of this might have been covered in the reference 2 review.

What is the research experience of the authors that justifies their writing a review on this topic? I find only one citation to one of the 4 authors. This lack of credentials and hence viewpoints on the topic makes the manuscript a bibliographic compilation rather than a critical review.

A selection of corrections or things that need clarification.

1. Rhizobia should not be italicized: it’s not an official taxonomic level.

2. Line 35: “sustainable in the long run” is redundant. Something that’s sustainable is in the long run.

3. Line 36: “its reach”: agricultural systems cannot have a “reach”.

4. Line 42: do you mean “external” rather than “internal”?

5. Line 48/49: should clarify that this applies only to rhizobia that fix nitrogen in free live, not the vast majority of nodule-forming species.

6. Omit colons (:) after subheadings.

7. Lines 86,87: I think these descriptions are reversed (iPGPR live inside the roots). Also, the iPGPR and ePGPR abbreviations are not needed, since they only occur in these lines (so omit).

8. Line 128: gives the impression that rhizobia are dispersed in all of the proteobacterial subclasses, while they exist essentially only in the alpha and beta subclasses.

9. Line 140: they don’t form any nodules, “true” or not.

10. Line 150. Nodules are not “oxygen free”.

11. Line 154: what is meant by “this process may vary….”?

12. Line 179: Nitrogen is an important element: omit as obvious.

13. Line 183: “unavailability in the gaseous form”: on the contrary, N gas is very available in the atmosphere.

14: Line 187. “in our daily life”?

15. Line 189,190. This isn’t a sentence nor does it make any sense.

16: Line 190: “such as e.g.” is redundant

17: Line 193: implies that Frankia are the only symbiotic rhizobia.

18: Don’t capitalize nitrogen, phosphorous, phosphate, in the middle of a sentence (not proper nouns).

19. Line 185 and elsewhere: don’t use contractions.

20: Line 205, nod D = nodD, line 207, nif HDK = nifHDK.

21: Line 208: “and many more”: weak.

22. Line 219: “Given the that the”

23: Line 221: nod factors cannot secrete signals.

24: Line 222 and other places, use of “et” instead of “and”.

25. Line 223, italicize the binomial for white clover.

26: The paragraph beginning line 218 starts talking about nod factors then goes into cell wall degrading enzymes, without linking the two topics. How do they relate?

27 Line 238: Omit “executed/accomplished”.

28. Line 241. It it’s insoluble, it can’t be available.

29: Lines 258 and 272: identical subheadings: the later one should be “Inorganic acid production”.

30. Line 286: “without the ness…” What is “ness”?

31. Line 668: Fluorescent, not fuorescent. Not italicized.

32. Line 681: “menaces the essential organic matter”. Meaning?

33. Line 737: as mentioned, most rhizobia associated with non-legumes or non-host legumes won’t fix nitrogen.

Inconsistent formatting of the references and use of “et al.”

The topic of rhizobial inoculation of non-legumes having a negative effect on their growth, mentioned in the Abstract, is not covered in the review (that I recall).

A bit more should be said in the legends of Fig. 3 and Fig. 4. What kind of nodules for Fig. 3? The figure’s not really very informative without structures. Fig. 4 was taken from something published. I assume the circled atoms represent functional groups of the different siderophores. These details should be described.

English usage is poor: see my comments to authors.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript contains very valuable basic information, uses the information and terminology correctly, the review of the topic could provide insights for the examination of the symbiosis of fixing bacteria in non-legumes from what is known in legumes. However, I detected several aspects in the manuscript that must be corrected and addressed before accepting the manuscript for publication.

All these aspects were indicated in the text of the manuscript in yellow and with commentary text.

My main criticisms are:

1. It is necessary that the authors explicitly state to the reader the objective of their review with an objective.

2. I think that in the topic-by-topic review section on the benefits of fixing bacteria, it would be more important to present a synthesis of what is known in non-legumes as a kind of integration of all these topics.

3. It is very striking that nothing is mentioned about the leguminous/non-leguminous tripartite symbiosis-N-fixing bacteria-arbuscular mycorrhizal fungi, given its functional importance in eco- and agro-ecosystems and its wide use as biofertilizers. I think this is a crucial issue.

4. I believe that pointing out generality about growth-promoting bacteria is much abused, as if all the benefits of this group of bacteria fully apply to N-fixing bacteria in legumes and non-legumes. I suggest taking care of this aspect.

5. Be careful with the correct writing of the References used in the manuscript and with the inclusion of figures that have already been published (unless you have permission to do so). Also be careful with the proper writing of scientific names and with the nomenclature of chemical elements and molecules.

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

The authors made a big effort to get from the very basics of plant physiology to the effect of plant growth-promoting bacteria on plant growth. They extensively consulted literature and tried to structure their review based on metabolites whose concentration might be altered by PGPR. Unfortunately, they tried to touch all aspects of plant physiology and different bacterial phyla including the mechanisms found for legumes, thus losing the focus of the manuscript. At the end of the manuscript the reader is lost and it remains unclear which effects of rhizobia on non-legume plants were observed, which mechanism is behind it and how they could be applied.

A key point is that the authors use Rhizobacteria and rhizobia almost as synonyms. Based on the title the review should be limited to rhizobia, but throughout the text they mention PGPR and rhizobia and it is not always clear who is relevant. Furthermore, the authors describe extensively what happens in legume-rhizobia interactions, but here again, in agreement with the title the authors should focus on non-legume interactions. I strongly recommend to streamline the manuscript and to concentrate on the topics mentioned in the title. In this context please also carefully check if all the very general background information on phytohormones and other metabolites is relevant. For example, the authors mention abscisic acid and that some bacteria were shown to produce it but it remains unclear if it has any effect on plant growth. Similarly, the authors describe the infection process by Rhizobia in detail, but as outlined above the review title indicates that the topic of the manuscript is about the effects of Rhizobia on non-legume plants.

Please also check on page 2 if the definitions for extracellular and intracellular PGPR are used in the correct way, from my perspective it should be vice versa.

The manuscript contains a large number of small mistakes such as missing space between word and reference, wrong use of singular and plural, inconsistent use of capital and small letters, sometimes wrong terms, but as the manuscript has to be substantially rewritten, not all mistakes are listed below but only the ones that are repeated throughout the manuscript. Overall, it would be useful if a native speaker could go through the manuscript.

Please consistently write rhizobia either with a small letter or with a capital letter.

The term rhizobia is plural, use rhizobium if you want to use the singular.

nitrogen, phosphate, soybean, fungi, fluorescent etc. should be written with small letters.

All Latin names should be written in italics.

Line 179-182 - please rewrite, I assume you want to speak about nitrogen fixation, though overall I am not sure if you have to provide that many details on nitrogen. The readers of this manuscript should be aware of the importance of N.

Line 201 - I assume you are talking about conversion

Line 284-289 - please revise and make two sentences for a better understanding.

Line 292  can be achieved

Line 321-332 - highly repetitive content.

Line 350 two pathways ? You mention only one.

Line 366 The general role of CKs is not important in the context of this manuscript, in particular callus formation, but the effect of rhizobia on CKs in plants...

Line 381 what means less identified?

Line 387 showed

Line 402 produced by

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The revised version of the manuscript by Sara Fahde and co-workers has been extensively modified to address the concerns of the reviewers. While the manuscript still suffers from poor English usage, it is obvious that the authors made a significant effort to correct this and other problems with the manuscript, with a good degree of success. Here I point out some mainly minor corrections or requirements for clarification:

For correction or clarification

Lines 23, 24: “the improvement of plant nutrient uptake” is repeated.

Line 46: phrase “other natural resources for a balanced system has access to…” seems out of place in the sentence, doesn’t make sense.

General comment: instead of always using a longer word (eg, line 50, “presents”), use a shorter, simpler word (like “is” instead of “presents”.

Line 52: what are “internal resources”?

Line 92, Rhizobia misspelled.

Line 95, omit “or”

Lines 101 and 102, the ePGPR and iPGRP designations are reversed.

Line 202, diazotrophs (plural)

Line 206, actinorhizal

Starting line 211, fix, nif and nod in italics

Line 230: “Given that Rhizobium infection and symbiotic recognition in the soil is mainly controlled by root hairs and most specifically the Nod factor signals secreted by the bacteria.” is not a sentence.

Line 242: “…the presence of [1]. “The presence of what?

Line 256: omit “however”: the sentence is not stating something that’s an exception to what was said in the text above it.

Line 295, superscript the +.

Line 324: strong? molecules (meaning?).

Several places: tryptophane should be tryptophan.

Line 367, italics for the genes.

Line 405, rephrase “…phytohormones produced by 136 different molecules”

LIne 420: “…mass spectroscopy (GC-MS), HPTLC and HPLC, in order to detect and quantify the gas [20].”  Define the abbreviations. What gas is being referred to?

Line 506: why are there degree symbols (°) in the dissociation constants.

Line 658, cyanide, not Cyanide

See my Comments and Suggestions for Authors

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Thanks to the authors for revising the manuscript and for correcting many of the small mistakes.

Unfortunately, it seems that I did not address my main concern properly - based on the title, the manuscript should be about the role of rhizobia as PGPR in non-legume interactions. However, reading the manuscript it remains unclear which findings are from free-living/root-associated Rhizobia and which findings were obtained from symbiotic interactions. Furthermore, the manuscript still covers a number of results from non-rhizobial species making it difficult for the reader to follow.

In more detail:

Lines 100-132 please delete and finish  line 99 with a sentence such as: in the present review we will focus only on PGPR belonging to the family Rhizobiaceae. (see also my comment below regarding the term "rhizobia".

N-fixation: this chapter mixes up N-fixation by nodulating rhizobia, other N-fixing bacteria and indirect benefits. Please focus on your topic! Clearly state what free-living/root associated rhizobia are doing compared to those living in symbiosis. It is not necessary to provide all the details about N-fixation if it is only done in symbiosis as bacteroids - this aspect definitely has to be mentioned, but currently the text is fuzzy. Furthermore, I do not fully understand why lines 234-242 are mentioned here.

P-solubilization: lines 268-307: it is not necessary to mention all mechanisms of P-solubilization unless earlier studies are cited demonstrating that the different mechanisms have been shown for rhizobia.

Phytohormone production: here again, it would be important to know if the phytohormone biosynthesis was shown for free-living rhizobia or for symbiotic interactions - and if phytohormones were released by the bacteria and directly affected plant growth.

Siderophore production: have all the siderophores shown in Figure 4 been shown to be produced by free-living rhizobia?

Antibiotics synthesis: same comment as above - in lines 572-577 the authors speak about Bacillus and Pseudomonads; then (line 578-585) the authors speak about Rhizobia but to the best of my knowledge, the examples cited are for nodule forming rhizobia interacting with legumes (e.g. Trifolium, soybean).

Induction of systemic resistance: The authors extensively talk about Pseudomonas. Please focus on the main topic of this review!

Production of cell-wall degrading enzymes - please delete. Not a single study with rhizobia is cited.

Throughout the text: please write Rhizobia and Rhizobium in capital letters when you talk about the genus and with small letters when the term covers a broad range of genera belonging to the group of rhizobia.

In this context it might be helpful to better describe which genera/families you want to address with the term "rhizobia" as rhizobial taxonomy has evolved over the last decades - see e.g. Rajkumari et al. 2022

Minor comment: please speak about cytokinins and check for spaces before citations.

The text substantially improved. It still contains a few minor mistakes (see above).

Author Response

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