Comparative Analysis of Rhizosphere Microbiomes in Different Blueberry Cultivars

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

The manuscript horticulture 3630769 entitle Comparative Analysis of Rhizosphere Microbiomes in Different Blueberry Cultivars” include the metagenomics analyses of microorganism associated to six different blueberries cultivars, however, before that was accept it the authors needs considered the following comments.

Specific comments

1. The expression high-through sequencing its, correct?
2. Please correct the names of the phyllum throughout the text as they are misspelled in the case of Actomycetota and should be updated in the case of Proteobacetria.
3. In the introduction paragraph the authors mentioned the functional microbes, But the present work does not show evidence of how this point can be addressed, since only metagenomics was performed and the functionality of the microorganisms was not observed.
4. I was unable to access the supplementary material.

Major points

• In the section 2.3 Bioinformatic analyses the authors needs add the references used to determinate the alpha diversity.
• The authors need improve the PCA analysis explication.
• The authors need to improve the quality of figure 6.
• The present work not give enough results to conclude “However, differences in rhizosphere microbial communities among blueberry cultivars arise from the combined effects of genetic traits, root exudate composition, and environmental factors”, please correct.
• The authors need to improve the discussion paragraph.

Minor points

• Line 129 y 130, please change “ml” by “mL”
• In table 1, please change sub-treatment by cultivars.
• In the figure 7, the bacteria names must be writing in italics.
• In line 386 please write “Enterobacter” in italics.
• Please delate the line 412-413.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors utilised 16S rRNA and ITS sequencing to evaluate variations in rhizosphere microbial communities across eight blueberry cultivars, representing two groups: highbush and rabbiteye. While previous studies have characterised the microbiomes of both groups, the authors position their work as novel in assessing variation among multiple cultivars within each group. However, this has been addressed to some extent in earlier research, notably in the study ‘Comparative Analysis of Rhizosphere Microbiomes of Southern Highbush Blueberry (Vaccinium corymbosum L.), Darrow's Blueberry (V. darrowiiCamp), and Rabbiteye Blueberry (V. virgatum Aiton)’, which included comparative analysis of multiple Vaccinium types, including different cultivars.

 

1. A relevant citation is missing from the introduction. Che et al. (2022) investigated the microbiome of Vaccinium virgatum (formally Vaccinium ashei, rabbiteye blueberry) across plantations of varying ages, identifying long-term cultivation as a key driver of microbial community dynamics. This study should be cited to acknowledge prior work on  virgatum microbiome dynamics.

Che, J. et al. Long-term cultivation drives dynamic changes in the rhizosphere microbial community of blueberry. Front. Plant Sci. 2022, 13, 96275.

 

2. In the methods section, it is stated that only healthy plants were selected for microbiome analysis. However, in the results, there are two instances where comparisons between healthy and diseased rhizosphere samples are described.

 

• Line 190: ‘The number of unique bacterial (Figure 1C) and fungal (Figure 1D) ASVs of the diseased rhizosphere of highbush blueberry are similar to those of the rabbit-eye blueberry’
• Line 256: ‘The relative abundance of 5 fungal 255 phyla differed significantly between healthy and diseased rhizospheres (Figure 5A).

 

This appears to contradict the earlier description of sampling only healthy plants. My understanding up until this point, has been that the study compares healthy rhizosphere samples between V. virgatum and V. corymbosum cultivars. Please clarify whether diseased samples were also included in the analysis.

 

3. The discussion section could be strengthened by explicitly comparing your findings with existing studies such as:

• Sun et al. (2021) Comparison and interpretation of characteristics of Rhizosphere microbiomes of three blueberry varieties. BMC Microbiol. 21:92
• Li et al. (2020). Comparative Analysis of Rhizosphere Microbiomes of Southern Highbush, Darrow's and Rabbiteye Blueberry. Front Microbiol. 11:370.

 

These studies examined both species- and cultivar-level variation in blueberry rhizosphere microbial communities. A more thorough comparison of your findings and those of these prior studies would help to clarify the novel contributions of your work.

 

4. Line 186: it appears there may be a naming error, when you refer to ‘Premier’, I believe you intended to reference ‘Brightwell’. Please confirm and correct if necessary.

 

5. Line 319: I believe it should read ‘fungal OTUs’ rather than ‘bacterial OTUs’.

 

6. Figure text readability: In general, the text within the figures is quite small. This if particularly true for Figure 6, which remains difficult to read even when zoomed in. Please consider increasing font size.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Comparative Analysis of Rhizosphere Microbiomes in Different Blueberry Cultivars” presents high-throughput DNA sequencing data regarding bacterial and fungal community analyses of the rhizosphere of multiple cultivars of both highbush and rabbiteye blueberry groups. Although the research results have merit and the manuscript is, for the most part, well written, there are several points that should be addressed before it is suitable for publication in Horticulturae. Although I don’t see any major faults in the work, I have many minor comments below for the authors to consider and address.

 

General Comments:

 

There are several mostly minor grammatical errors throughout the manuscript that, as a whole, should be corrected. Therefore, this manuscript would benefit from careful English language editing by someone well versed in the language. I would not recommend the use of AI for this, as I suspect this has already been done, and the program did not catch several oddities.

 

Several taxa discussed in the manuscript have recently been updated and renamed, yet the authors are not using the newest naming system in their manuscript. For example, the bacterial phyla Proteobacteria and Firmicutes have now been reclassified as Pseudomonadota and Bacillota, respectively. I recommend the authors consult www.bacterio.net to find the most updated names of bacterial taxa and incorporate these names into this manuscript.

 

The font sizes used in every figure are very small to the point that they are unreadable in printed form. The authors should increase the font sizes of the textual components in the figures so that readers are not forced to read the paper digitally and magnify the figures to read them.

 

Scientific taxa in the text and in each figure should be set in italics. Underscores should not be included in scientific names. In every case, the genus should be capitalized, and the species epithet should be lower case, with both in italics.

 

For all figures with multiple parts (i.e., “A,” “B,” etc.), put descriptions in the figure legends for each part to help the reader navigate the figure. A number of the figure legends seem to be too sparse in their descriptions, Figures 1, 2, 3, and 6, especially.

 

Large run-on sentences occur in Section 3.4. Please edit the writing here.

 

The Conclusions section has some issues. First, I suggest moving up the Conclusions heading to be in front of the text beginning at Line 406, which begins, “In summary...” Also, delete the sentence starting at line 431. I suggest also deleting Lines 432-444 and moving the text in Lines 445-448 up to close out the previous paragraph (and the Conclusion itself).

 

 

Specific Comments:

 

Abstract, lines 23-31: It’s unclear to me if this refers to results of the present study (I assume so) or another report. Please clarify the writing here, as this reads more like an introduction than an abstract.

 

Lines 54-60: It seems like a general reference (or references) is necessary here. A highly regarded textbook or perhaps a review article would suffice. Please check and add if necessary.

 

Line 56: “20-30%” not “20%-30%”

 

Lines 60-61: Please clarify how the compatible solutes synthesized by halotolerant microbes can alleviate salinity stress in plants. These solutes are produced and maintained in the cytoplasm of the bacteria, so how do they aid the plant?

 

Line 128: What is meant by “super-clean bench”?

 

Line 178: Delete “respectively,” as it is not used correctly.

 

Line 185: Conversely, the word “respectively” should be added after “…bacterial and fungal ASVs” for clarity. 

 

Lines 186-187: Presumably, “Premier” would be “Pr” instead of “Br.”

 

Lines 233-241 and elsewhere: Where taxa are "unclassified," do not italicize this word, as it is not part of the taxon designation. It should be written “unclassified Archaea,” “unclassified Vicinamibacterales,” “unclassified Fungi,” etc. Also, in cases of a candidatus designation, it should be formatted as “Candidatus Solibacter” (Line 279), for example. Italicize the word “Candidatus” but not the proposed taxon name. Cases such as “uncultured forest soil bacterium” (Line 241) should not be italicized at all. In addition, all underscores should be removed in these names. Those were presumably added by the software used, but they have no place in scientific nomenclature.

 

Figure 4B: The proper designation would be “Candidatus Patescibacteriota.” Also, update Proteobacteria to Pseudomonadota and Firmicutes to Bacillota. Also in this figure, “uncultured forest soil bacterium” is not a genus. I suggest simply grouping those genera that could not precisely be determined into a group called “Unclassified.”

 

Line 281: “genus Pseudomonas” not “genera Pseudomonas”

 

Lines 282-283: Burkholderiales and Latescibacteraceae are not genus names.

 

Line 284: What is this organism “mle1_7”? Can the authors determine the genus (or family) designation of this recovered sequence and call it that instead? This “mle1_7” name is certainly no genus.

 

Line 294: “are shown” not “were showed”

 

Figures 7 and 8: These figures are far too small to be useful. Can they be expanded?

 

Line 335: “taxa” not “taxon”

 

Lines 362-364: It seems like references are needed here.

 

Line 386: “Enterobacter” should be “Enterobacter” (in italics).

Comments on the Quality of English Language

The English is not bad but does have some issues. See my comments on the manuscript for these.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed all previously raised concerns, resulting in a clearer and more coherent manuscript. However, please note that in Figure 8A, some of the labels appear to overlap, which may affect readability. 

Author Response

We attach great importance to your comments and make the following replies:

Comments: The authors have satisfactorily addressed all previously raised concerns, resulting in a clearer and more coherent manuscript. However, please note that in Figure 8A, some of the labels appear to overlap, which may affect readability.  

Response: Thank you for your attention. The evolutionary branching diagram derived from LEfSe analysis illustrates statistically significant biomarkers by facilitating comparisons both between and within subgroups. In Figure 8, the circles extending from the center to the periphery of the diagram denote taxonomic levels ranging from phylum to species, with each small circle at a given taxonomic level. In instances where there are numerous distinct biomarker entries, they may be reclassified or obscured within the diagram. Consequently, the details of these distinct biomarker entries are displayed adjacent to their respective classifications.

Reviewer 3 Report

Comments and Suggestions for Authors

The revised manuscript is improved, but some issues remain. For example, the authors say the font sizes in the figures are larger, and in some cases, this appears to be true, but several figures still have fonts so small that the paper must be read in an expandable, digital format to read them. There are also some places where the wording is odd, such as the last half of Line 245 and Line 257. There are some punctuation errors, as well: 1) add commas to separate items in Line 248; 2) replace the comma with a period and begin a new sentence in Line 399; 3) remove the unnecessary underscore in Line 317; 4) add the missing italics in Line 307; 5) remove the hyphen in Line 135; and 6) remove the comma in Line 185. I also recommend adding an "unpublished results" reference to Line 61. The revised Conclusion section is redundant with Lines 336-361 in the Discussion. I recommend modifying the Conclusion how I recommended in my original review of this manuscript.

Comments on the Quality of English Language

The English language is, for the most part, fine, but there are still areas where the wording is odd. See my "suggestions for the authors" for those cases where the wording could be improved.

Author Response

We attach great importance to your suggestions and make the following revision and replies:

Comments: The revised manuscript is improved, but some issues remain. For example, the authors say the font sizes in the figures are larger, and in some cases, this appears to be true, but several figures still have fonts so small that the paper must be read in an expandable, digital format to read them. There are also some places where the wording is odd, such as the last half of Line 245 and Line 257. There are some punctuation errors, as well: 1) add commas to separate items in Line 248; 2) replace the comma with a period and begin a new sentence in Line 399; 3) remove the unnecessary underscore in Line 317; 4) add the missing italics in Line 307; 5) remove the hyphen in Line 135; and 6) remove the comma in Line 185. I also recommend adding an "unpublished results" reference to Line 61. The revised Conclusion section is redundant with Lines 336-361 in the Discussion. I recommend modifying the Conclusion how I recommended in my original review of this manuscript.

Response: Line 245 and Line 257 have been partially modified as “A comprehensive analysis of the bacterial communities revealed the presence of 40 phyla, 108 classes, 312 orders, 671 families, and 1413 genera (Supplementary Table 1). We examined the differences in taxonomic composition between the rhizosphere microbiomes of V. virgatum (VV) and V. corymbosum (VC) samples. Notably, the relative abundance of more than 10 bacterial phyla exhibited significant variation between VV and VC (Figure 4A). Specifically, the relative abundances of Campylobacterota, Halobacterota, and Thermoplasmatota were significantly elevated in VV, whereas the relative abundance of unclassified Archaea was markedly decreased. The predominant bacterial phyla, including Pseudomonadota, Acidobacteriota, Actinobacteriota, Bacteroidota, and unclassified Bacteria, collectively constituted a relative abundance of ≥80% in each group (Figure 4B). At the genus level, the bacterial amplicon sequence variants (ASVs) in the blueberry rhizospheres were predominantly classified into Acinetobacter, Pseudomonas, Enterobacter, Burkholderia-Caballeronia-Paraburkholderia, Occallatibacter, and Acidibacter (Figure 4B).”

We have add commas to separate items in Line 248.

We have replaced the comma with a period and began a new sentence in Line 399. The sentences have been partially modified as “Prolonged cultivation of blueberries fosters a distinctive rhizosphere microenvironment. The exudates from blueberry roots selectively promote or suppress specific microbial taxa.”

We have removed the unnecessary underscore in Line 327 in the revised version.

We have added the missing italics in Line 317 in the revised version.

We have removed the hyphen in Line 135.

We have removed the comma in Line 190 in the revised version.

We have added an "unpublished results" reference to Line 61.

We have revised the conclusion section in accordance with your suggestions, which involved eliminating redundant phrases and refining the language to enhance the conclusion's clarity and precision. The conclusion section was shown as “This study used high-throughput sequencing to examine the rhizosphere microbial diversity in eight blueberry cultivars, revealing significant bacterial and fungal diversity differences. Ten major bacterial phyla, including dominant Acidobacteriota, Actinobacteriota, and Pseudomonadota, made up 92.32%-97.08% of bacterial abundance. Five key fungal phyla, led by Ascomycota and Basidiomycota, accounted for 88.18%-97.20% of fungal abundance. PGPR genera like Burkholderia and Pseudomonas were identified, with rabbiteye cultivars showing higher symbiotic microbe accumulation. Ericoid mycorrhizal fungi, especially Oidiodendron, were more abundant in Emerald, Premier, O’Neal, and Brightwell cultivars, with Emerald having the highest levels. Rabbiteye cultivars supported more unique and abundant fungal communities. These findings highlight the intricate interplay of genetic factors, root exudates, and environmental conditions in shaping the rhizosphere microbiomes of blueberry plants. Given the current paucity of research on blueberry rhizosphere microbiota, this study provides essential foundational insights.”

All original high-resolution images in the article have been uploaded separately.

Thank you for your attention and valuable comments on this manuscript.