Exiguobacterium acetylicum Strain SI17: A Potential Biocontrol Agent against Peronophythora litchii Causing Post-Harvest Litchi Downy Blight
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript contains valuable information on the biocontrol mechanisms and genome analysis, experimental design is detailed and unique.
As the improvements, I would reccomend to improve the Discussion. In current form, it is very narrow, and repeats the Results part. Give it a more thorough analysis of other scientific data, expand it with more details. It will improve the quality of your obtained important data and overall quality of the manuscript.
Author Response
Comments 1: The manuscript contains valuable information on the biocontrol mechanisms and genome analysis, experimental design is detailed and unique. As the improvements, I would reccomend to improve the Discussion. In current form, it is very narrow, and repeats the Results part. Give it a more thorough analysis of other scientific data, expand it with more details. It will improve the quality of your obtained important data and overall quality of the manuscript.
Response 1: We added some content to the discussion section.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe document presents serious deficiencies in reference to the design of the experiment, these deficiencies complicate the reproduction of the experiment, but above all they put the reliability of the results at risk
1.- The phenological stage of the fruits that allows identifying the moment of application of preharvest treatment in the experiment is not mentioned. for example, days after anthesis?. The incidence of diseases due to phytopathogenic microorganisms can be highly related to pre-harvest factors such as stages of phenological development of the fruit in which it may be more susceptible to the entry of the pathogen, development of the exocarp as a means of protection, among others. For this reason, it is necessary to indicate at least the days after flowering that have elapsed at the time of application of the pre-harvest treatment.
2.- Because we are working with specific microorganisms, it is necessary to fully describe the methodology for the growth and conservation of the microorganisms of interest; it is not advisable to reference another document as is done in lines 115 to 116, where the authors send the reader to check quote 37, this is not correct in a publication of this type. The authors must describe the cultivation of P. litchii SC18 and the biocontrol agent E. acetylicum SI17.
3.-The authors do not define the number of fruits used for each treatment.
4.-The authors do not define what LB means
5.- The authors do not define an experimental design. This compromises the reliability of the results, there is no statistical support to support the veracity of the difference between data.
6.-In the materials and methods section it is not described how they carried out the statistical comparison of data to be able to make a discussion.
7.-The methodology does not specify the humidity conditions during storage, so the experiment would be poorly designed, especially considering that the development of a pathogenic fungus is being evaluated. The humidity requirement for fungi and bacteria is different, therefore, it is a determining factor in the evaluation of the biocontrol effect on the surface of the fruits.
8.- There is no statistical analysis of results data.
9.-It is not defined what CK means placed in figure 3
10.-In lines 231 to 232 the authors mention “findings indicated that SI17-treated fruits had a significantly lower disease index at 60, 72, 231 and 84 hours, but this difference lessened over time”. The authors cannot assert anything without the application of statistical analysis.
11.- In lines 235 to 236 the authors mention “By 24 hpi, pathogen levels in the SI17 treatment group were three times lower than those in the control group, and by 36 hpi, they were six times lower (Figure 3C)”. The authors cannot assert anything without the application of statistical analysis.
12.- On lines 236 to 237 the authors mention “As time progressed, the pathogen growth rate in the control group was much higher than that in the SI17 treatment group. The authors cannot assert anything without the application of statistical analysis.
13.-In lines 248 to 249 the authors mention “Prior to 84 hours, CAT activity in the SI17 treatment group surpassed that of the control group.” However, the authors cannot assert anything without the application of statistical analysis.
14.- In the conclusion section, the authors cannot conclude anything without the establishment of a defined experimental design, and without the application of a statistical analysis
Author Response
Comments 1: The phenological stage of the fruits that allows identifying the moment of application of preharvest treatment in the experiment is not mentioned. for example, days after anthesis?. The incidence of diseases due to phytopathogenic microorganisms can be highly related to pre-harvest factors such as stages of phenological development of the fruit in which it may be more susceptible to the entry of the pathogen, development of the exocarp as a means of protection, among others. For this reason, it is necessary to indicate at least the days after flowering that have elapsed at the time of application of the pre-harvest treatment.
Response 1: The phenological stage of the fruits were added in line 129 “about 65 days after flowering”.
Comments 2: Because we are working with specific microorganisms, it is necessary to fully describe the methodology for the growth and conservation of the microorganisms of interest; it is not advisable to reference another document as is done in lines 115 to 116, where the authors send the reader to check quote 37, this is not correct in a publication of this type. The authors must describe the cultivation of P. litchii SC18 and the biocontrol agent E. acetylicum SI17.
Response 2: The methods of cultivation of E. acetylicum SI17 and P. litchii SC18 were described in line 110-111, line 127-128 respectively.
Comments 3: The authors do not define the number of fruits used for each treatment.
Response 3: The number of fruits used for each treatment in line 134-135, “Each treatment contained 3 replicates, with 30 fruits per replicate”.
Comments 4: The authors do not define what LB means
Response 4: The LB medium was defined in line 110-111, “Luria-Bertani (LB) medium (containing Tryptone 10 g/L, Yeast extract 5 g/L and NaCl 10 g/L)”.
Comments 5: The authors do not define an experimental design. This compromises the reliability of the results, there is no statistical support to support the veracity of the difference between data.
Response 5: The difference analysis has been
The method of data analysis is added in line 163-167, and the difference analysis of data is carried out.
Comments 6: In the materials and methods section it is not described how they carried out the statistical comparison of data to be able to make a discussion.
Response 6: The method of data analysis is added in line 163-167, and the difference analysis of data is carried out.
Comments 7: The methodology does not specify the humidity conditions during storage, so the experiment would be poorly designed, especially considering that the development of a pathogenic fungus is being evaluated. The humidity requirement for fungi and bacteria is different, therefore, it is a determining factor in the evaluation of the biocontrol effect on the surface of the fruits.
Response 7: We simulated the experimental conditions and measured the humidity at 95%, and added in line 132.
Comments 8: There is no statistical analysis of results data.
Response 8: The method of data analysis was added in line 163-167, and the difference analysis of data was carried out.
Comments 9: It is not defined what CK means placed in figure 3
Response 9: The CK means the control, as described in lin 274-275.
Comments 10: In lines 231 to 232 the authors mention “findings indicated that SI17-treated fruits had a significantly lower disease index at 60, 72, 231 and 84 hours, but this difference lessened over time”. The authors cannot assert anything without the application of statistical analysis.
Response 10: The method of data analysis is added in line 163-167, and the difference analysis of data is carried out.
Comments 11: In lines 235 to 236 the authors mention “By 24 hpi, pathogen levels in the SI17 treatment group were three times lower than those in the control group, and by 36 hpi, they were six times lower (Figure 3C)”. The authors cannot assert anything without the application of statistical analysis.
Response 11: The method of data analysis is added in line 163-167, and the difference analysis of data is carried out.
Comments 12: On lines 236 to 237 the authors mention “As time progressed, the pathogen growth rate in the control group was much higher than that in the SI17 treatment group. The authors cannot assert anything without the application of statistical analysis.
Response 12: The method of data analysis is added in line 163-167, and the difference analysis of data is carried out.
Comments 13: In lines 248 to 249 the authors mention “Prior to 84 hours, CAT activity in the SI17 treatment group surpassed that of the control group.” However, the authors cannot assert anything without the application of statistical analysis.
Response 13: The method of data analysis is added in line 163-167, and the difference analysis of data is carried out.
Comments 14: In the conclusion section, the authors cannot conclude anything without the establishment of a defined experimental design, and without the application of a statistical analysis
Response 14: The method of data analysis is added in line 163-167, and the difference analysis of data is carried out.
Reviewer 3 Report
Comments and Suggestions for AuthorsIf I correctly read the paper horticulturae-3115026 “Biocontrol potential of Exiguobacterium acetylicum SI17 against post-harvest litchi downy blight caused by Peronophythora litchi” the authors report data on the genome and biological activity of Exiguobacterium acetylicum strain SI17. This bacterium strain, isolated from the litchi fruit carposphere, has proven biocontrol activity against litchi downy blight (LDB) associated with Peronophythora litchi. The SI17 genome, sequenced, assembled, and annotated, shows a 3157929 bp circular chromosome and four plasmids of 119838, 43790, 165 6694, and 68355 bp. Among the functional annotations, the SI17 genome contains 3541 protein-coding and 101 RNA genes. Based on the whole genome sequences, phylogenetic analysis clustered strain SI17 into Group I (strains capable of thriving at temperatures up to 7 ℃). During pre-harvest treatment against Peronophythora litchii strain SC18 inoculation, SI17 reduces disease index at 60, 72, and 84 hours post strain SC18 inoculation. qRT-PCR demonstrates a low relative quantity of pathogen in the SI17 treated litchi fruit. SI17 treated fruit decreased H2O2 content, catalase (EC 1.11.1.6) and peroxidase (EC 1.11.1.7) activities, while increasing superoxide dismutase (EC 1.15.1.1) activity. No inhibitory effect on the growth of P. litchii SC18 was associated with plate assays performed with SI17 cell suspension, fermentation broth or supernatant. These findings lead to the hypothesis that SI17 enhances litchi fruits resistance to P. litchii and candidate E. acetylicum as a potential biological control agent.
Notwithstanding the scientific sound of this work, the presentation in the form of a manuscript lacks scientific strictness. It is difficult to read, confusing, unclear, and requires adjustments.
Follow the Horticulturae instructions for Authors and template in revision (https://www.mdpi.com/journal/horticulturae/instructions).
The title is not completely pertinent to the topics of the manuscript. Improve.
A possible suggestion:
“Exiguobacterium acetylicum strain SI17: a potential biocontrol agent against Peronophythora litchii causing post-harvest litchi downy blight”
The “abstract” and the “conclusions” are two important parts of the paper, after reading the manuscript I suggest rewriting.
Define all the acronyms used in the first citation.
Abstract:
Lines 17-18: What does “post-harvest” mean? The manuscript reports information on “pre-harvest treatments”.
Line 18: What does “interventions” mean?
Line 20: use “and ” instead of “along with”.
Line 21: delete “that were”
Line 21: Verify “26 Exiguobacterium strains”. Figure 2 shows 29 different strains belonging to 14 diverse Exiguobacterium spp.
Line 24: delete “(SOD)”. The acronym is not necessary because it is not utilized in the abstract.
Line 26: use “It” instead of “In summary, it”.
Line 26: delete “has the ability to”
Line 26: delete “various”
Use pertinent keywords.
The “Introduction” section is not clear. Improve. In particular, the aim/s of the work is confused and not fully pertinent.
It is necessary to improve the litchi downy blight symptoms description associated with Peronophythora litchi infections. A brief morphological description (sporangiophores, sporangia, zoospores, oospores) of P. litchi could help the reader.
Line 44: explain “chemical fungicides”.
Lines 44-45: period not clear. Improve.
Line 55: delete “bacterial”. Paecilomyces sp. is a fungal species.
Lines 64-82: periods not clear. Improve.
Line 83: What does “multiple” mean?
Lines 83-84: period not clear. Improve.
Lines 96-99: periods not clear. Improve.
The “Materials and Methods” section is not clear. Rewrite.
This section should describe the step-by-step applied protocols to allow others to replicate the published results.
Insert information on:
Were E. acetylicum liquid cultures performed under static or shaking conditions?
Platforms used for sequencing.
Catalase, superoxide dismutase, and peroxidase detection kits.
Greenhouse growth conditions.
Perform statistical analysis for disease index, relative expression PlActin, H2O2 content, CAT, SOD and POD activities.
Line 116: use “by Zheng et al.” instead of “earlier”.
Line 117: use “suspension” instead of “culture”.
Line 118: explain that diluted LB broth was used as a control.
Lines 148-160: periods not clear. Improve.
Line 150: What does “Fermentation” mean?
How was fermentation broth obtained?
A proposed “Material and Method” subdivision:
2.1. Exiguobacterium acetylicum bioinformatics analysis
2.2. Antagonistic activity on litchi fruit
2.2.1. Relative quantification of P. litchii SC18 in pericarp
2.2.2. Enzyme activity in the pericarp
2.3. Antagonistic activity on plate assay
The whole “Results” section (lines 162-261) is not clear. Enhance.
This section should provide a concise and precise description of the experimental results. Avoid information on “material and methods” and “discussion”.
Insert information on section “2.6. Plate antagonism and biocontrol effect of fermentation (C), suspension (F), and supernatant (Q) of SI17 against P. litchii”.
Follow the proposed “Material and Method” subdivision to rewrite the “Results” section.
Move Figure S6 from Supplementary Materials to the result section.
Figures and tables must be self-explanatory, clear, and easy to understand without extra explanation. Improve.
Line 176 and Figure S1: Explain “COG” significance. Presume Clusters of Orthologous Genes
Figure 1
Lines 196-204: Improve.
Lines 206 and 209: use “Exiguobacterium acetylicum strain SI17” instead of “SI17”
Figure 2:
Lines 223-234: Improve.
The Figure 2 legend and line 230 report 26 Exiguobacterium species. I read 29 strains/accessions belonging to 14 Exiguobacterium species [E. acetylicum (4 accessions), E. acidotolerans (1 accession), E. alkaliphilum (1 accession), E. antarcticum (2 accessions), E. aurantiacum (3 accessions), E. chiriqhucha (4 accessions), E. enclensi 1( accession), E. flavidum (1 accession), E. indicum (3 accessions), E. marinum (1 accession), E. mexicanum (2 accessions), E. profundum (1 accession), E. sibiricum (2 accessions), and E. undae (3 accessions)]
Figure 3:
Sections B-G: use “Hours post Peronophythora litchi inoculation” instead of “Hours post P. litchi inoculation (hpi)”.
Lines 256-261: Improve.
A possible suggestion:
Figure 3. Interaction between pre-harvest treatment with Exiguobacterium acetylicum strain SI17 and Peronophythora litchi on litchi cv. Feizixiao fruits: A) litchi downy blight symptoms and B) disease index (B) development, C) PlActin expression, D) H2O2 content, E) catalase (CAT), F) superoxide dismutase (SOD), G) peroxidase (POD) activities variation. Litchi fruits were sprayed with E. acetylicum strain SI17 (5×107 CFU/mL) cell suspension (SI17) or a 1/10 dilution of LB broth (CK) until completely covered. After seven days, the fruits were harvested, maintained at 25 °C, with 12-hour day and night cycles. After 24 hours, fruits were sprayed with P. litchii SC18 sporangia suspension (5×104 sporangia/mL, 30 mL).
Improve the discussion section (lines 263-301). In particular lines 290-301.
Line 273: What does “(L.)” mean? If “(L.)” is the Author name abbreviation delete or insert to all the cited organisms.
Rewrite the legend of Figure S6.
Arrange “Reference” following the Horticulturae instructions https://www.mdpi.com/journal/horticulturae/instructions.
Kind regards
Author Response
Comments 1:“Exiguobacterium acetylicum strain SI17: a potential biocontrol agent against Peronophythora litchii causing post-harvest litchi downy blight”
Response 1: We have changed the title according to the comments, in line 2-4.
Comments 2: Lines 17-18: What does “post-harvest” mean? The manuscript reports information on “pre-harvest treatments”.
Response 2: The post-harvest has been deleted in line 17.
Comments 3: Line 18: What does “interventions” mean?
Response 3: We replaced “interventions” with “treatment” in line 18.
Comments 4: Line 20: use “and ” instead of “along with”.
Response 4: We replaced “along with” with “and” in line 20.
Comments 5: Line 21: delete “that were”
Response 5: We deleted “that were” in line 21.
Comments 6: Line 21: Verify “26 Exiguobacterium strains”. Figure 2 shows 29 different strains belonging to 14 diverse Exiguobacterium spp.
Response 6: We verified and revised the “26 Exiguobacterium strains” as “29 Exiguobacterium strains” in line 21.
Comments 7: Line 24: delete “(SOD)”. The acronym is not necessary because it is not utilized in the abstract.
Response 7: We deleted “(SOD)” in line 24.
Comments 8: Line 26: use “It” instead of “In summary, it”.
Response 8: We replaced “In summary, it” with “It” in line 26.
Comments 9: Line 26: delete “has the ability to”
Response 9: We deleted “has the ability to” in line 26.
Comments 10: Line 26: delete “various”
Response 10: we deleted “various” in line 26.
Use pertinent keywords.
Comments 11: It is necessary to improve the litchi downy blight symptoms description associated with Peronophythora litchi infections. A brief morphological description (sporangiophores, sporangia, zoospores, oospores) of P. litchi could help the reader.
Response 11: The description of sporangia, zoospores, oospores of P. litchi and the litchi downy blight symptoms were added in line 41-46.
Comments 12: Line 44: explain “chemical fungicides”.
Response 12: The chemical fungicides mainly farm chemical, as explained in line 50.
Comments 13: Lines 44-45: period not clear. Improve.
Response 13: The period of the applications of chemical fungicides for LDB control is at present, as indicated in line 49.
Comments 14: Line 55: delete “bacterial”. Paecilomyces sp. is a fungal species.
Response 14: We deleted “bacterial”.
Comments 15: Lines 64-82: periods not clear. Improve.
Response 15: Sorry, we don’t what does it mean.
Comments 16: Line 83: What does “multiple” mean?
Response 16: We replaced “multiple” with “sereral”.
Comments 17: Lines 83-84: period not clear. Improve.
Response 17: Sorry, we don’t what does it mean.
Comments 18: Lines 96-99: periods not clear. Improve.
Response 18: Sorry, we don’t what does it mean.
Comments 19: Were E. acetylicum liquid cultures performed under static or shaking conditions?
Response 19: The E. acetylicum liquid cultures performed under shaking conditions, and it was added in line 111.
Comments 20: Platforms used for sequencing.
Response 20: The genome was sequenced on the Nanopore PromethION, added in line 119-120.
Comments 21: Catalase, superoxide dismutase, and peroxidase detection kits.
Response 21: The Catalase, superoxide dismutase, and peroxidase detection kits were obtained from Nanjing Jiancheng Biological Engineering Institute, Nanjing, China (http://www.njjcbio.com/). The website was added in line 162.
Comments 22: Greenhouse growth conditions.
Response 22: The greenhouse maintained at a steady temperature of 25 °C, 95% humidity, with 12-hour day and night cycles in line 132-133.
Comments 23: Perform statistical analysis for disease index, relative expression PlActin, H2O2 content, CAT, SOD and POD activities.
Response 23: The statistical analysis methods were added in line 163-167.
Comments 24: Line 116: use “by Zheng et al.” instead of “earlier”.
Response 24: We replaced “earlier” with “by Zheng et al.” in line 127.
Comments 25: Line 117: use “suspension” instead of “culture”.
Response 25: We replaced “culture” with “suspension” in line 130.
Comments 26: Line 118: explain that diluted LB broth was used as a control.
Response 26: We explained diluted LB broth was used as a control in line 131.
Comments 27: Lines 148-160: periods not clear. Improve.
Response 27: Sorry, we don’t what does it mean.
Comments 28: Line 150: What does “Fermentation” mean?
How was fermentation broth obtained?
Response 27: The fermentation indicated that the bacterial solution after shaking culture in LB broth, which containing suspension and supernatant. And explained in the supplementary material.
Comments 28: A proposed “Material and Method” subdivision:
2.1. Exiguobacterium acetylicum bioinformatics analysis
2.2. Antagonistic activity on litchi fruit
2.2.1. Relative quantification of P. litchii SC18 in pericarp
2.2.2. Enzyme activity in the pericarp
2.3. Antagonistic activity on plate assay
Response 28: We have removed the “Plate antagonism and biocontrol effect of fermentation (C), suspension (F), and supernatant (Q) of SI17 against P. litchii” into the supplementary material as the result only indicated fermentation (C), suspension (F), and supernatant (Q) had no inhibitory effect on the growth of P. litchii SC18. So maybe it is better that the subtitle of “Material and Method” remained unchanged.
Comments 29: Insert information on section “2.6. Plate antagonism and biocontrol effect of fermentation (C), suspension (F), and supernatant (Q) of SI17 against P. litchii”.
Follow the proposed “Material and Method” subdivision to rewrite the “Results” section.
Move Figure S6 from Supplementary Materials to the result section.
Response 29: We have removed the “Plate antagonism and biocontrol effect of fermentation (C), suspension (F), and supernatant (Q) of SI17 against P. litchii” into the supplementary material as the result only indicated fermentation (C), suspension (F), and supernatant (Q) had no inhibitory effect on the growth of P. litchii SC18.
Comments 30: Line 176 and Figure S1: Explain “COG” significance. Presume Clusters of Orthologous Genes
Response 30: The COG analysis classified 23 categories as the results showed in line 183-187.
Comments 31: Figure 1 Lines 196-204: Improve. Lines 206 and 209: use “Exiguobacterium acetylicum strain SI17” instead of “SI17”
Response 31: Thanks for your suggestion, however, we think it is better that the methods and results should be appropriate part while not in figure legends, as review 4 suggested.
We have replaced “SI17” with “Exiguobacterium acetylicum SI17” all through the manuscript.
Comments 32: Figure 2: Lines 223-234: Improve. The Figure 2 legend and line 230 report 26 Exiguobacterium species. I read 29 strains/accessions belonging to 14 Exiguobacterium species [E. acetylicum (4 accessions), E. acidotolerans (1 accession), E. alkaliphilum (1 accession), E. antarcticum (2 accessions), E. aurantiacum (3 accessions), E. chiriqhucha (4 accessions), E. enclensi 1( accession), E. flavidum (1 accession), E. indicum (3 accessions), E. marinum (1 accession), E. mexicanum (2 accessions), E. profundum (1 accession), E. sibiricum (2 accessions), and E. undae (3 accessions)]
Response 32: Thanks for your suggestion, however, we think it is better that the methods and results should be appropriate part while not in figure legends, as review 4 suggested.
We revised the “26 Exiguobacterium spcieces” as “29 Exiguobacterium strains”.
Comments 33: Figure 3: Sections B-G: use “Hours post Peronophythora litchi inoculation” instead of “Hours post P. litchi inoculation (hpi)”.Lines 256-261: Improve.
A possible suggestion:
Figure 3. Interaction between pre-harvest treatment with Exiguobacterium acetylicum strain SI17 and Peronophythora litchi on litchi cv. Feizixiao fruits: A) litchi downy blight symptoms and B) disease index (B) development, C) PlActin expression, D) H2O2 content, E) catalase (CAT), F) superoxide dismutase (SOD), G) peroxidase (POD) activities variation. Litchi fruits were sprayed with E. acetylicum strain SI17 (5×107 CFU/mL) cell suspension (SI17) or a 1/10 dilution of LB broth (CK) until completely covered. After seven days, the fruits were harvested, maintained at 25 °C, with 12-hour day and night cycles. After 24 hours, fruits were sprayed with P. litchii SC18 sporangia suspension (5×104 sporangia/mL, 30 mL).
Response 33: Thanks for your suggestion, however, we think it is better that the methods and results should be appropriate part while not in figure legends, as review 4 suggested.
We have replaced “Hours post P. litchi inoculation (hpi)” with “ours post Peronophythora litchi inoculation (hpi)” in Figure 3B-G.
Comments 34: Improve the discussion section (lines 263-301). In particular lines 290-301.
Response 34: We added some content to the discussion section.
Comments 35: Line 273: What does “(L.)” mean? If “(L.)” is the Author name abbreviation delete or insert to all the cited organisms.
Response 35: We have deleted “(L.)” in line 287.
Reviewer 4 Report
Comments and Suggestions for AuthorsThe manuscript entitled "Biocontrol potential of Exiguobacterium acetylicum SI17 against post-harvest litchi downy blight caused by Peronophythora litchii" isolated a strain of E. acetylicum from the litchi fruit carposphere, sequenced its genome, and analyzed it, finding genes involved in the production of secondary metabolites. Additionally, litchi fruits were treated with E. acetylicum SI17 before harvesting, demonstrating improvements in resistance. The current version of the manuscript is interesting; however, some concerns need to be addressed for it to be considered for publication in the journal Horticulturae.
Major observations:
A concerning aspect of the genomic analyses is the criterion the authors chose to include only 28 genomes belonging to 14 Exiguobacterium species. Currently, there are 327 genomes reported in NCBI (unfortunately, many at the "sp." level). Thus, the results are not yet conclusive to determine the species proposed by the authors. An analysis including the genomes of all possible Exiguobacterium species should be performed. Additionally, an ANI analysis should be conducted to determine the percentage of similarity with the species grouped in the phylogenomic analysis. In fact, the phylogeny is performed with a set of concatenated genes, including the 16S gene. These MLST analyses have been replaced by phylogenomic analyses, so to obtain conclusive results, the authors should perform analyses at the complete genome level, which will give a conclusive result and/or reinforce the result reached in the current version.
Minor details:
· Restructure the abstract according to the author's guidelines. Background information on the research topic is missing, among other details.
· L22: Change "phylogenetic analysis" to "phylogenomic analysis" since you worked with complete genomes.
· L23: It is unclear who belongs to "Group I"; there is no context.
· L83-90: This paragraph contains results and should not be in the introduction. Additionally, it is unclear why bacteria are grouped according to the mentioned temperature; provide clear context.
· L96-99: Restructure the objectives section, highlighting all the experiments performed. The current version only highlights the genomic analysis.
· Section “2.1. Bacterial culture and DNA extraction”: Include the source and method of bacterial isolation.
· L110: The cited article “Zheng et al. [39]”, in “Genome Sequencing, Assembly, and Annotation” section contains analyses not performed in this manuscript. It is important to detail the analyses that were carried out in the current manuscript.
· L124: Specify from which tissue the RNA was extracted.
· L163: The authors mention using Illumina and Nanopore platforms; however, the methodology refers to following the protocol described by “Zheng et al. [39]” which only mentions Nanopore sequencing. Therefore, the authors should detail in the methodology section which platforms, programs, and analyses were used, instead of citing with missing information. In fact, reviewing the sequenced genome metadata in NCBI, only Nanopore sequencing information is present.
· Table 1: Are the table results from data generated by Illumina or Nanopore sequencing? Additionally, it is important to include the genome coverage achieved, as only 716,365 reads were obtained (the platform is unknown).
· L212-213: This information should be in the Materials and Methods section.
· L213: The tree does not contain 26 species; it only has 14 species.
· L267: The tree does not have 25 genomes; review and standardize the information correctly.
· Discussion: In general, the discussion is very brief and does not reflect the detailed information generated in the results. It should be restructured to discuss all evaluated aspects. For example, the authors include an extensive Table 1 describing genomic characteristics, Genomic Islands, Prophage identification, Interspersed Repeat, and Tandem Repeat, yet none of this information is highlighted in the discussion, treating it merely as anecdotal; the same occurs with the supplementary materials. If the authors determined that the direct confrontation between Exiguobacterium acetylicum SI17 and Peronophythora litchii did not produce direct antagonism and attribute its effect to the production of secondary metabolites, they should discuss, based on the genome annotation of Exiguobacterium acetylicum SI17, which metabolites have been reported in the literature for their antimicrobial capacity with fungi of this type and/or in such crops.
Comments on the Quality of English LanguageMinor editing of English language required
Author Response
Comments 1: A concerning aspect of the genomic analyses is the criterion the authors chose to include only 28 genomes belonging to 14 Exiguobacterium species. Currently, there are 327 genomes reported in NCBI (unfortunately, many at the "sp." level). Thus, the results are not yet conclusive to determine the species proposed by the authors. An analysis including the genomes of all possible Exiguobacterium species should be performed. Additionally, an ANI analysis should be conducted to determine the percentage of similarity with the species grouped in the phylogenomic analysis. In fact, the phylogeny is performed with a set of concatenated genes, including the 16S gene. These MLST analyses have been replaced by phylogenomic analyses, so to obtain conclusive results, the authors should perform analyses at the complete genome level, which will give a conclusive result and/or reinforce the result reached in the current version.
Response 1: The phylogenomic analysis showed that most of the same species are clustered together. Exiguobacterium acetyllicum SI17 and Exiguobacterium acetyllicum DSM14481 are clustered together, indicating that there should be no problem with the naming of Exiguobacterium acetyllicum SI17.
In addition, the purpose of phylogenomic analysis is to see which group Exiguobacterium acetylicum SI17 is clustered into, and our results are consistent with Zhang’s findings (Zhang, D.; Zhu, Z.; Li, Y.; Li, X.; Guan, Z.; Zheng, J. Comparative genomics of Exiguobacterium reveals what makes a cosmopolitan bacterium. mSystems 2021, 6, e0038321), who used ANI analysis. Therefore, we believe that the research results of this method are reliable.
Comments 2: Restructure the abstract according to the author's guidelines. Background information on the research topic is missing, among other details.
Response 2: The background information was added, “Litchi downy blight (LDB) caused by Peronophythora litchii destroys 20-30 % of litchi fruit every year and causes significant economic losses.” in line 15-16.
Comments 3: L22: Change "phylogenetic analysis" to "phylogenomic analysis" since you worked with complete genomes.
Response 3: All of the "phylogenetic analysis" in the manuscript have been revised to “phylogenomic analysis”.
Comments 4: L23: It is unclear who belongs to "Group I"; there is no context.
Response 4: Due to the word limit of the abstract, the results of the phylogenetic analysis are simply described, as “In the phylogenomic analysis encompassing the entire genome, SI17 was clustered into Group I.” in line 23-24.
Comments 5: L83-90: This paragraph contains results and should not be in the introduction. Additionally, it is unclear why bacteria are grouped according to the mentioned temperature; provide clear context.
Response 5: Here is the result of the other research, not this research. According to phylogenetic analysis results it was found that strains cluster into Group 1 can live at temperatures below 7 ℃.
Comments 6: L96-99: Restructure the objectives section, highlighting all the experiments performed. The current version only highlights the genomic analysis.
Response 6: The methods were same to that in the cited article Zheng et al. [39] (the artical we published before). Therefore, in order to reduce the repetition rate, most of the methods in the paper refer to this paper.
Comments 7: Section “2.1. Bacterial culture and DNA extraction”: Include the source and method of bacterial isolation.
Response 7: The source and the culture methods were added in line 110-113.
Comments 8: L110: The cited article “Zheng et al. [39]”, in “Genome Sequencing, Assembly, and Annotation” section contains analyses not performed in this manuscript. It is important to detail the analyses that were carried out in the current manuscript.
Response 8: The methods were same to that in the cited article Zheng et al. [39] (the artical we published before). Therefore, in order to reduce the repetition rate, most of the methods in the paper refer to this paper.
Comments 9: L124: Specify from which tissue the RNA was extracted.
Response 9: Total RNA was extracted from the litchi pericarp, in line 140.
Comments 10: L163: The authors mention using Illumina and Nanopore platforms; however, the methodology refers to following the protocol described by “Zheng et al. [39]” which only mentions Nanopore sequencing. Therefore, the authors should detail in the methodology section which platforms, programs, and analyses were used, instead of citing with missing information. In fact, reviewing the sequenced genome metadata in NCBI, only Nanopore sequencing information is present.
Response 10: The platform is Nanopore, we have revised in line 172.
Comments 10: Table 1: Are the table results from data generated by Illumina or Nanopore sequencing? Additionally, it is important to include the genome coverage achieved, as only 716,365 reads were obtained (the platform is unknown).
Response 10: The platform is Nanopore, we have revised in line 172.
Comments 11: L212-213: This information should be in the Materials and Methods section.
Response 11: We have put “A phylogenomic analysis using whole genome sequences of established species was conducted, employing Bacillus subtilis as the out-group.” into the Materials and Methods section.
Comments 12: L213: The tree does not contain 26 species; it only has 14 species. L267: The tree does not have 25 genomes; review and standardize the information correctly.
Response 12: There are 29 Exiguobacterium genomes, we have revised.
Comments 13: Discussion: In general, the discussion is very brief and does not reflect the detailed information generated in the results. It should be restructured to discuss all evaluated aspects. For example, the authors include an extensive Table 1 describing genomic characteristics, Genomic Islands, Prophage identification, Interspersed Repeat, and Tandem Repeat, yet none of this information is highlighted in the discussion, treating it merely as anecdotal; the same occurs with the supplementary materials. If the authors determined that the direct confrontation between Exiguobacterium acetylicum SI17 and Peronophythora litchii did not produce direct antagonism and attribute its effect to the production of secondary metabolites, they should discuss, based on the genome annotation of Exiguobacterium acetylicum SI17, which metabolites have been reported in the literature for their antimicrobial capacity with fungi of this type and/or in such crops.
Response 13: We added some content to the discussion section.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsNo comments, suggestions were taken into account.
Author Response
Thank you for the recognition of our work.
Reviewer 3 Report
Comments and Suggestions for Authors
The revised manuscript horticulturae-3115026-peer-review-v2 remains confused, unclear, imprecise, dispersive, and difficult to read, and requires adjustments.
Use the Horticulturae instructions for Authors and template during revision (https://www.mdpi.com/journal/horticulturae/instructions). See subheadings format properties.
Following the practice adopted by the International Code of Nomenclature for Algae, Fungi and Plants scientific names of all taxonomic ranks should be italicized. For every organism, the full genus name should be included at the first mention. The genus name should be abbreviated to the initial letter if no ambiguity arises, although the full genus name should always be used.
Please use the genus abbreviation along the text.
Pay attention to the table position: Table 1 is between two pages (5 and 6).
Line 16: What does “Exiguobacterium exhibits” mean? I suppose: “Some Exiguobacterium species exhibit” or “Some Exiguobacterium strains exhibit”.
Line 17: use “Exiguobacterium acetylicum strain SI17” instead of “A specific strain, Exiguobacterium acetylicum SI17”.
Line 22: use “strains so far” instead of “strains”.
Lines 28-29: use “litchi plants resistance” instead of “disease resistance in plants”.
As possible new Keywords: Plant growth promotion; Antagonist; Defense activation; H2O2; Catalase; Superoxide Dismutase; Peroxidase.
Lines 41-46: use “P. litchii infects litchi tender leaves, flowers, and mature fruits in the field and postharvest storage. This hemibiotrophic, homothallic and polycyclic oomycete produces oospores spherical in shape, with smooth walls and a light-yellow color. Oospores can survive in the soil or plant debris for several years and germinate directly or indirectly under favorable conditions. Lemon-shaped deciduous sporangia with papillae at the apex are also produced. Sporangium, wind or water dispersal, germinates directly, produces germ tubes and penetration hyphae able to penetrate the receptive organs. Alternatively, sporangia can form secondary sporangia or release zoospores. Zoospores, kidney-shaped, have two lateral flagella, and rapidly germinate in cool wet conditions. P. litchii causes withering and watery brown spots on the infection sites of tender leaf or fruit, and produces downy white sporangiophores, which trigger pre- and postharvest fruit decay [3].” instead of “The sporangia ... [3].”.
Line 50: What does “farm chemical” mean? Are chemicals produced by farmers? A possible suggestion “e.g., mancozeb, metalaxyl, cymoxanil, dimethomorph, flumorph, mandipropamid, azoxystrobin and pyraclostrobin, or other registered products”.
Lines 50-51: sentence not clear. Are the “farm chemical” residues carcinogenic hazards to consumers and contributing to environmental pollution? Usually, active molecules used to control plant pathogens could safe health and the environment.
Lines 70-88: sentences not clear. Improve.
Is the genus Exiguobacterium involved in industry and agriculture …?
What does “facilitation” mean?
A possible rearrangement:
“The genus Exiguobacterium includes species and strains engaged in industry and agriculture, comprising enzyme production, bioremediation, degradation of toxic substances and plant growth promoting properties [17]. Cellulase, pectinase, mannanase, xylanase, and tannase produced by Exiguobacterium sp. VSG-1 makes steam-exploded sugarcane bagasse useful to biofuel fabrication by Saccharomyces cerevisiae fermentation [18]. A protease-producing strain of E. profundum has been employed in the extraction of chitin from shrimp shells [19]. Lipase, amylase, and pullulanase have been isolated from different Exiguobacterium strains, increasing their potential applications [20-23]. Exiguobacterium sp. AO-11, E. alkaliphilum B-3531D, and E. mexicanum M7 metabolize crude oil, suggesting promise for the bioremediation of oil-contaminated marine ecosystems [24-26]. Exiguobacterium strains ZM-2, GS1, PY14, and E. mexicanum from chromite mines reduce Cr (VI) levels in polluted environments [27-30]. Moreover, several Exiguobacterium strains exhibit significant potential for agricultural applications as plant growth promoting bacteria [31-35]. Exiguobacterium has been reported to suppress fungal diseases of cereal crops in Australia (Barnett, S.J., Anstis, S.T., Roget, D.K., Ryder, M.H., 2006. Suppression of Rhizoctonia solani AG-8 induced disease on wheat by the interaction between Pantoea, Exiguobacterium, and Microbacteria. Soil Res. 44, 331–342), and E. acetylicum strain 1P MTCC 8707 inhibited the in vitro growth of Rhizoctonia solani, Sclerotium rolfsii, Pythium sp. and Fusarium oxysporum (Selvakumar, G., Joshi, P., Nazim, S., Mishra, P.K., Kundu, S., Gupta, H.S., 2009. Exiguobacterium acetylicum strain 1P MTCC 8707, a novel bacterial antagonist from the North Western Indian Himalayas. World. J. Microbiol. Biotechnol. 25, 131–137). No applications of Exiguobacterium in pre- and post-harvest plant disease management have not been reported thus far.”
Lines 89-90: delete “, enabling us to conduct a comparative analysis of their genomes”.
Lines 102-105: sentence not clear. Rewrite. Report the main aim of the work and highlight the principal conclusions.
The “Materials and Methods” section remains not clear. Rewrite.
If I correctly read the revised version of the manuscript, the main aims of the work are:
1) Bioinformatics analysis of Exiguobacterium acetylicum SI17
2) Biocontrol (Antagonistic) activity of Exiguobacterium acetylicum SI17 on litchi fruit
Each purpose needs different stages to be achieved.
To carry out the bioinformatics analysis, it is necessary to perform:
1) Bacterial culture and DNA extraction
2) Genome sequencing, assembly, annotation, and phylogenomic analyses
To reach the biocontrol activity on litchi fruit:
1) Inoculation experiments on litchi fruit
2) Relative quantification of P. litchii SC18 in pericarp
3) Enzyme activity in the pericarp
A data analysis should also be performed.
Based on these considerations, I suggest the following “Material and Methods” subdivision:
2.1. E. acetylicum SI17 bioinformatics analysis
2.1.1. Bacterial culture and DNA extraction
2.1.2. Genome sequencing, assembly, annotation, and phylogenomic analyses
2.2. Biocontrol (Antagonistic) activity on litchi fruit
2.2.1. Inoculation experiments on litchi fruit
2.2.2. Relative quantification of P. litchii SC18 in pericarp
2.2.3. Enzyme activity in the pericarp
2.3. Data analysis
The proposed subdivision also remarks your “Results” section arrangement.
Lines 108, 129, 221-222, 222, 272, 274, 280, 281, 283, 283-284, …: use “E.” instead of “Exiguobacterium”.
Line 110: delete “containing”.
Line 127: use “oomycete” or “Chromista” instead of “fungus” or delete “fungus”.
Line 168: use “3.1. Genome sequencing, assembly, and functional annotation” instead of “3.1. … SI17”.
Line 170: add “(Figure 1, Table 1)” between “chromosome” and “with”.
Lines 201 and 199: move “(Figure S5)” between “analysis” and “showed”.
Lines 219 and 229: use “Phylogenomic” instead of “phylogenomic”.
Lines 231-232: rearrange as a subheading, avoiding results description
Move lines 233-237 “To verify the effectiveness of pre-harvest E. acetylicum SI17 treatment against LDB 233 and investigate potential mechanisms, experiments were performed using an LB solution 234 as a control. Litchi fruits were treated with E. acetylicum SI17 SI17 and then inoculated 235 with spores of the LDB pathogen (P. litchii SC18). Disease severity was measured every 12 236 hours from 60 to 96 hpi.” to following “Material and Methods” as integration of lines 126-136.
Lines 233-237: use “Pre-harvest inoculation experiments on litchi fruit” instead of “To verify … findings”.
Comments 32 referred to Figure 2. In my previous suggestions for Figure 2, I reported the numbers of strains and species, recording that the total indicated (26) differed from the actual (29) number of Exiguobacterium strains.
Lines 229-230: sentence not clear. Improve.
A suggestion:
Figure 2. Phylogenomic tree based on whole genome sequences of 29 Exiguobacterium strains.
Bacillus subtilis was used as an out-group. Strain E. acetylicum SI17 is highlighted in bold.
Line 239: use “A, B” instead of “a, b” as reported in Figure 3.
Lines 244-241 and 247-249: rearrange the sentence to avoid “Material and Methods” information.
Figure 3: improve magnification.
Lines 265-270: rearrange the sentence to avoid result information (e.g., “treatment increased the activity of antioxidant enzymes, thereby improving resistance to Phytophthora litchi”); also avoid repetition.
A possible suggestion:
Figure 3. Interaction between Exiguobacterium acetylicum strain SI17 pre-harvest treatment and Peronophythora litchi on litchi cv. Feizixiao fruits: A) litchi downy blight symptoms and B) disease index (B) development, C) PlActin expression, D) H2O2 content, E) catalase (CAT), F) superoxide dismutase (SOD), G) peroxidase (POD) activities. CK = control.
Line 276: What does “25” mean?
Line 278: What does “categorized” mean?
Line 292: What does “real-world” mean?
Line 293: use “A, B” instead of “a, b”.
Line 295: Add information on the effects of H2O2 on plant-pathogens.
Line 302: use “D” instead of “d”.
Line 303: use “E-G” instead of “e-g”.
Lines 304-305: Without references in the “Materials and Methods” and “Discussion” sections, it is not easy to understand the meaning of these results.
Lines 307-309: sentence non clear. Improve.
Line 318: put “Pythium oligandrum” in italics.
Line 319: delete “the fugal pathogen”.
Line 319: What does “[]” mean? Insert the opportune reference.
Figure S1 legend: use “Clusters of Orthologous Groups (COG)” instead of “COG”.
Figure S3 legend: use “Kyoto Encyclopedia of Genes and Genomes (KEGG)” instead of “KEGG”.
Figure S4 legend: use “Carbohydrate-Active enZYmes Database (CAZy)” instead of “CAZy (Carbohydrate-Active enZYmes Database)”.
Figure S5 legend: use “Transporter Classification Database (TCDB)” instead of “TCDB (Transporter Classification Database)”.
Kind regards
Author Response
Dear Editors and reviewers,
Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled "Exiguobacterium acetylicum strain SI17: a potential biocontrol agent against Peronophythora litchii causing post-harvest litchi downy blight” (horticulturae-3115026). We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in blue in the paper, The main corrections in the paper and the responds to the reviewer's comments are as flowing:
Reviewer 3
Comments 1: Following the practice adopted by the International Code of Nomenclature for Algae, Fungi and Plants scientific names of all taxonomic ranks should be italicized. For every organism, the full genus name should be included at the first mention. The genus name should be abbreviated to the initial letter if no ambiguity arises, although the full genus name should always be used. Please use the genus abbreviation along the text.
Response 1: We have revised it all through the manuscript.
Comments 2: Pay attention to the table position: Table 1 is between two pages (5 and 6).
Response 2: We place Table 1 and Table 2 on separate pages.
Comments 3: Line 16: What does “Exiguobacterium exhibits” mean? I suppose: “Some Exiguobacterium species exhibit” or “Some Exiguobacterium strains exhibit”.
Response 3: We have revised as “Some Exiguobacterium strains exhibit considerable promise in both agricultural and industrial sectors.” (in line 16)
Comments 4: Line 17: use “Exiguobacterium acetylicum strain SI17” instead of “A specific strain, Exiguobacterium acetylicum SI17”.
Response 4: It has been revised (in line 17).
Comments 5: Line 22: use “strains so far” instead of “strains”.
Response 5: It has been revised (in line 22).
Comments 6: Lines 28-29: use “litchi plants resistance” instead of “disease resistance in plants”.
Response 6: It has been revised (in line 29).
Comments 7: As possible new Keywords: Plant growth promotion; Antagonist; Defense activation; H2O2; Catalase; Superoxide Dismutase; Peroxidase.
Response 7: The main finding of this study is the biocontrol effect of Exiguobacterium acetylicum SI7 on litchi downy blight (LDB), while not the plant growth promotion. And the H2O2; Catalase; Superoxide Dismutase; Peroxidase are the defense activation of the plant after infection by Peronophythora litchii. So we revised the Key words as: Exiguobacterium acetylicum SI17; litchi downy blight; genome sequencing; defense activation; biocontrol efficacy.
Comments 8: Lines 41-46: use “P. litchii infects litchi tender leaves, flowers, and mature fruits in the field and postharvest storage. This hemibiotrophic, homothallic and polycyclic oomycete produces oospores spherical in shape, with smooth walls and a light-yellow color. Oospores can survive in the soil or plant debris for several years and germinate directly or indirectly under favorable conditions. Lemon-shaped deciduous sporangia with papillae at the apex are also produced. Sporangium, wind or water dispersal, germinates directly, produces germ tubes and penetration hyphae able to penetrate the receptive organs. Alternatively, sporangia can form secondary sporangia or release zoospores. Zoospores, kidney-shaped, have two lateral flagella, and rapidly germinate in cool wet conditions. P. litchii causes withering and watery brown spots on the infection sites of tender leaf or fruit, and produces downy white sporangiophores, which trigger pre- and postharvest fruit decay [3].” instead of “The sporangia ... [3].”.
Response 8: We have revised it (in line 42-53).
Comments 9: Line 50: What does “farm chemical” mean? Are chemicals produced by farmers? A possible suggestion “e.g., mancozeb, metalaxyl, cymoxanil, dimethomorph, flumorph, mandipropamid, azoxystrobin and pyraclostrobin, or other registered products”.
Response 9: We have added the chemical fungicides mainly used in LDB control, “like dimethomorph and propamocarb” in line 57.
Comments 10: Lines 50-51: sentence not clear. Are the “farm chemical” residues carcinogenic hazards to consumers and contributing to environmental pollution? Usually, active molecules used to control plant pathogens could safe health and the environment.
Response 10: Most chemical fungicides are low toxicity, however, in order to achieve better prevention and control effects, farmers tend to use high concentrations of chemical fungicides, which may pose potential risks of chemical fungicides residues.
Comments 11: Lines 70-88: sentences not clear. Improve.
Is the genus Exiguobacterium involved in industry and agriculture …?
What does “facilitation” mean?
A possible rearrangement:
“The genus Exiguobacterium includes species and strains engaged in industry and agriculture, comprising enzyme production, bioremediation, degradation of toxic substances and plant growth promoting properties [17]. Cellulase, pectinase, mannanase, xylanase, and tannase produced by Exiguobacterium sp. VSG-1 makes steam-exploded sugarcane bagasse useful to biofuel fabrication by Saccharomyces cerevisiae fermentation [18]. A protease-producing strain of E. profundum has been employed in the extraction of chitin from shrimp shells [19]. Lipase, amylase, and pullulanase have been isolated from different Exiguobacterium strains, increasing their potential applications [20-23]. Exiguobacterium sp. AO-11, E. alkaliphilum B-3531D, and E. mexicanum M7 metabolize crude oil, suggesting promise for the bioremediation of oil-contaminated marine ecosystems [24-26]. Exiguobacterium strains ZM-2, GS1, PY14, and E. mexicanum from chromite mines reduce Cr (VI) levels in polluted environments [27-30]. Moreover, several Exiguobacterium strains exhibit significant potential for agricultural applications as plant growth promoting bacteria [31-35]. Exiguobacterium has been reported to suppress fungal diseases of cereal crops in Australia (Barnett, S.J., Anstis, S.T., Roget, D.K., Ryder, M.H., 2006. Suppression of Rhizoctonia solani AG-8 induced disease on wheat by the interaction between Pantoea, Exiguobacterium, and Microbacteria. Soil Res. 44, 331–342), and E. acetylicum strain 1P MTCC 8707 inhibited the in vitro growth of Rhizoctonia solani, Sclerotium rolfsii, Pythium sp. and Fusarium oxysporum (Selvakumar, G., Joshi, P., Nazim, S., Mishra, P.K., Kundu, S., Gupta, H.S., 2009. Exiguobacterium acetylicum strain 1P MTCC 8707, a novel bacterial antagonist from the North Western Indian Himalayas. World. J. Microbiol. Biotechnol. 25, 131–137). No applications of Exiguobacterium in pre- and post-harvest plant disease management have not been reported thus far.”
Response 11: We revised according to your suggestion (in line 96-113).
Comments 12: Lines 89-90: delete “, enabling us to conduct a comparative analysis of their genomes”.
Response 12: We have deleted the sentence in line 114.
Comments 13: Lines 102-105: sentence not clear. Rewrite. Report the main aim of the work and highlight the principal conclusions.
Response 13: We simplified this part of the content, and the principal conclusions were listed in the Conclusion part in line 382-384.
Comments 14: The “Materials and Methods” section remains not clear. Rewrite.
If I correctly read the revised version of the manuscript, the main aims of the work are:
1) Bioinformatics analysis of Exiguobacterium acetylicum SI17
2) Biocontrol (Antagonistic) activity of Exiguobacterium acetylicum SI17 on litchi fruit
Each purpose needs different stages to be achieved.
To carry out the bioinformatics analysis, it is necessary to perform:
1) Bacterial culture and DNA extraction
2) Genome sequencing, assembly, annotation, and phylogenomic analyses
To reach the biocontrol activity on litchi fruit:
1) Inoculation experiments on litchi fruit
2) Relative quantification of P. litchii SC18 in pericarp
3) Enzyme activity in the pericarp
A data analysis should also be performed.
Based on these considerations, I suggest the following “Material and Methods” subdivision:
2.1. E. acetylicum SI17 bioinformatics analysis
2.1.1. Bacterial culture and DNA extraction
2.1.2. Genome sequencing, assembly, annotation, and phylogenomic analyses
2.2. Biocontrol (Antagonistic) activity on litchi fruit
2.2.1. Inoculation experiments on litchi fruit
2.2.2. Relative quantification of P. litchii SC18 in pericarp
2.2.3. Enzyme activity in the pericarp
2.3. Data analysis
The proposed subdivision also remarks your “Results” section arrangement.
Response 14: We revised the subdivison of “Materials and Methods” and “Results”.
Comments 15: Lines 108, 129, 221-222, 222, 272, 274, 280, 281, 283, 283-284, …: use “E.” instead of “Exiguobacterium”.
Response 15: We have revised it all through the manuscript.
Comments 16: Line 110: delete “containing”.
Response 16: We have deleted “containing” in line 134.
Comments 17: Line 127: use “oomycete” or “Chromista” instead of “fungus” or delete “fungus”.
Response 17: We have deleted “fungus” in line 152.
Comments 18: Line 168: use “3.1. Genome sequencing, assembly, and functional annotation” instead of “3.1. … SI17”.
Response 18: It has been revised. (in line 195)
Comments 19: Line 170: add “(Figure 1, Table 1)” between “chromosome” and “with”.
Response 19: We have added the contents in line 196-198.
Comments 20: Lines 201 and 199: move “(Figure S5)” between “analysis” and “showed”.
Response 20: We moved “(Figure S5)” between “analysis” and “showed”. (in line 228)
Comments 21: Lines 219 and 229: use “Phylogenomic” instead of “phylogenomic”.
Response 21: They have been revised. (in line 266 and 276)
Comments 22: Lines 231-232: rearrange as a subheading, avoiding results description
Response 22: We have rearranged the subheadings.
Comments 23: Move lines 233-237 “To verify the effectiveness of pre-harvest E. acetylicum SI17 treatment against LDB 233 and investigate potential mechanisms, experiments were performed using an LB solution 234 as a control. Litchi fruits were treated with E. acetylicum SI17 SI17 and then inoculated 235 with spores of the LDB pathogen (P. litchii SC18). Disease severity was measured every 12 236 hours from 60 to 96 hpi.” to following “Material and Methods” as integration of lines 126-136.
Response 23: The sentence “Disease severity was measured every 12 hours from 60 to 96 hpi.” has been moved to “Material and Methods” section in line 159-160.
Comments 24: Lines 233-237: use “Pre-harvest inoculation experiments on litchi fruit” instead of “To verify … findings”.
Response 24: It has been revised. (in line 281-283)
Comments 25: referred to Figure 2. In my previous suggestions for Figure 2, I reported the numbers of strains and species, recording that the total indicated (26) differed from the actual (29) number of Exiguobacterium strains.
Response 25: We checked again, and confirmed their were 29 Exiguobacterium strains.
Comments 26: Lines 229-230: sentence not clear. Improve.
A suggestion:
Figure 2. Phylogenomic tree based on whole genome sequences of 29 Exiguobacterium strains.
Bacillus subtilis was used as an out-group. Strain E. acetylicum SI17 is highlighted in bold.
Response 26: It has been revised. (in line 277-278)
Comments 27: Line 239: use “A, B” instead of “a, b” as reported in Figure 3.
Response 27: It has been revised. (in line 283)
Comments 28: Lines 244-241 and 247-249: rearrange the sentence to avoid “Material and Methods” information.
Response 28: We have deleted the contents with “Material and Methods” information. (in line 286 and 295)
Comments 29: Figure 3: improve magnification.
Response 29: We don’t understand this, is it mean magnify the picture when publish the article?
Comments 30: Lines 265-270: rearrange the sentence to avoid result information (e.g., “treatment increased the activity of antioxidant enzymes, thereby improving resistance to Phytophthora litchi”); also avoid repetition.
A possible suggestion:
Figure 3. Interaction between Exiguobacterium acetylicum strain SI17 pre-harvest treatment and Peronophythora litchi on litchi cv. Feizixiao fruits: A) litchi downy blight symptoms and B) disease index (B) development, C) PlActin expression, D) H2O2 content, E) catalase (CAT), F) superoxide dismutase (SOD), G) peroxidase (POD) activities. CK = control.
Response 30: It has been revised. (in line 311-314)
Comments 31: Line 276: What does “25” mean?
Response 31: It should be 28 and we have revised. (in line 320)
Comments 32: Line 278: What does “categorized” mean?
Response 32: It has been revised as “clustered” in line 322.
Comments 33: Line 292: What does “real-world” mean?
Response 33: It has been revised as “field application” in line 335.
Comments 34: Line 293: use “A, B” instead of “a, b”.
Response 34: It has been revised. (in line 336)
Comments 35: Line 295: Add information on the effects of H2O2 on plant-pathogens.
Response 35: We added the information in line 338-340.
Comments 36: Line 302: use “D” instead of “d”.
Response 36: It has been revised. (in line 348)
Comments 37: Line 303: use “E-G” instead of “e-g”.
Response 37: It has been revised. (in line 349)
Comments 38: Lines 307-309: sentence non clear. Improve.
Response 38: The detailed information of Fermentation (C), suspension (F), and supernatant (Q) were in Supplementary_Materials, is it need to explain here?
Comments 39: Line 318: put “Pythium oligandrum” in italics.
Response 39: It has been revised. (in line 364-365)
Comments 40: Line 319: delete “the fugal pathogen”.
Response 40: We have deleted it. (in line 365)
Comments 41: Line 319: What does “[]” mean? Insert the opportune reference.
Response 41: The reference has been inserted. (in line 366)
Comments 42: Figure S1 legend: use “Clusters of Orthologous Groups (COG)” instead of “COG”.
Figure S3 legend: use “Kyoto Encyclopedia of Genes and Genomes (KEGG)” instead of “KEGG”.
Figure S4 legend: use “Carbohydrate-Active enZYmes Database (CAZy)” instead of “CAZy (Carbohydrate-Active enZYmes Database)”.
Figure S5 legend: use “Transporter Classification Database (TCDB)” instead of “TCDB (Transporter Classification Database)”.
Response 41: They have been revised.
Reviewer 4 Report
Comments and Suggestions for Authors.
Author Response
Thank you for the recognition of our work.
We have revised again according to reviewer 3's suggestion.
