Effect of Epiphytic Bacteria from Citrus against Green Mold Post-Harvest Diseases of Citrus
Round 1
Reviewer 1 Report
This study describes a method for the biological control of post-harvest diseases in citrus, providing a new approach to disease control in citrus fruit that reduces the use of pesticides, decreases production costs and protects the environment, which is novel and suitable for this journal. However, there are still major problems, please modify.
1. In the background section, it is recommended to add information on the incidence of citrus diseases caused by P. digitatum, economic losses and other specific figures.
2. For both in vitro and in vivo inhibition experiments, pictures of microorganisms growing on media and citrus should be provided in addition to statistical charts of inhibition rates.
3. Please improve the clarity of the picture.
4. The meaning of the lowercase letters a, b, c, d, e.... in Table 2 or Figure 1 should be made clear in the notes to the figure or table.
5. Using PCR, three strains were identified, B15, P17 and S13, but in Figure 2 there are only images of B15 and why not the other two.
6. Only the molecular identification and metabolite detection of B15 were performed in the text, why P17 and S13 were not included, and the reasons should be explained in the text.
7. “Based on a partial 16S rDNA sequence analysis, the B15 isolate was assigned to the genus Bacillus, and more in detail, according to BLAST comparison the sequences of the isolate S15 showed a high similarity (98%) with B. cereus (JF895480.1) Table3. ” S15 should be replaced by B15 in this sentence.
8. Although B15 produces Iturin A, which has clearly antibacterial activity, in this experiment, if the inhibition activity against P. digitatum is determined inhibitory activity, not only the products of B15 alone should be tested, but the content of metabolites after co-culture of B15 with P. digitatum should be increased and, at the same time, compared with the type and content of metabolites of B15 alone, and only when a substance, after the presence of P. digitatum, its content is significantly increased, can it be determined to be associated with P. digitatum inhibitory activity was related.
Comments for author File: 
Comments.docx
This study describes a method for the biological control of post-harvest diseases in citrus, providing a new approach to disease control in citrus fruit that reduces the use of pesticides, decreases production costs and protects the environment, which is novel and suitable for this journal. However, there are still major problems, please modify.
1. In the background section, it is recommended to add information on the incidence of citrus diseases caused by P. digitatum, economic losses and other specific figures.
2. For both in vitro and in vivo inhibition experiments, pictures of microorganisms growing on media and citrus should be provided in addition to statistical charts of inhibition rates.
3. Please improve the clarity of the picture.
4. The meaning of the lowercase letters a, b, c, d, e.... in Table 2 or Figure 1 should be made clear in the notes to the figure or table.
5. Using PCR, three strains were identified, B15, P17 and S13, but in Figure 2 there are only images of B15 and why not the other two.
6. Only the molecular identification and metabolite detection of B15 were performed in the text, why P17 and S13 were not included, and the reasons should be explained in the text.
7. “Based on a partial 16S rDNA sequence analysis, the B15 isolate was assigned to the genus Bacillus, and more in detail, according to BLAST comparison the sequences of the isolate S15 showed a high similarity (98%) with B. cereus (JF895480.1) Table3. ” S15 should be replaced by B15 in this sentence.
8. Although B15 produces Iturin A, which has clearly antibacterial activity, in this experiment, if the inhibition activity against P. digitatum is determined inhibitory activity, not only the products of B15 alone should be tested, but the content of metabolites after co-culture of B15 with P. digitatum should be increased and, at the same time, compared with the type and content of metabolites of B15 alone, and only when a substance, after the presence of P. digitatum, its content is significantly increased, can it be determined to be associated with P. digitatum inhibitory activity was related.
Author Response
Respectfully,
We are thankful to the reviewers and editor for useful comments on our submission, which have improved the manuscript. All the comments/suggestions have been addressed/incorporated. Revisions/changes have been indicated in Red font color.
1.In the background section, it is recommended to add information on the incidence of citrus diseases caused by P. digitatum, economic losses and other specific figures.
A paragraph regarding this topic was added.
2. For both in vitro and in vivo inhibition experiments, pictures of microorganisms growing on media and citrus should be provided in addition to statistical charts of inhibition rates.
The figures were added.
3. Please improve the clarity of the picture.
It has been done.
4. The meaning of the lowercase letters a, b, c, d, e.... in Table 2 or Figure 1 should be made clear in the notes to the figure or table.
A description has already been written regarding their significant differences; we add (a–j) to clarify the range of differences.
5. Using PCR, three strains were identified, B15, P17 and S13, but in Figure 2 there are only images of B15 and why not the other two.
As we analyzed the antifungal metabolism of the most effective strain B15, we preferred to just discuss B15 in the paper, but according to reviewer comments, we added the PCR results of other strains.Top of Form
6. Only the molecular identification and metabolite detection of B15 were performed in the text, why P17 and S13 were not included, and the reasons should be explained in the text.
As we only analyzed the antifungal metabolic of the most effective bacteria, we preferred to put the information about B. cereus but according to reviewer comments we added the other bacteria information
7. “Based on a partial 16S rDNA sequence analysis, the B15 isolate was assigned to the genus Bacillus, and more in detail, according to BLAST comparison the sequences of the isolate S15 showed a high similarity (98%) with B. cereus (JF895480.1) Table3.” S15 should be replaced by B15 in this sentence.
Thank you; unfortunately, there was confusion in this paragraph as we deleted some data, but it has been edited.
8. Although B15 produces Iturin A, which has clearly antibacterial activity, in this experiment, if the inhibition activity against P. digitatum is determined inhibitory activity, not only the products of B15 alone should be tested, but the content of metabolites after co-culture of B15 with P. digitatum should be increased and, at the same time, compared with the type and content of metabolites of B15 alone, and only when a substance, after the presence of P. digitatum, its content is significantly increased, can it be determined to be associated with P. digitatum inhibitory activity was related.
In this study, our aim was to identify epiphytic bacteria with antagonistic activity and find the most effective antifungal metabolic strain, but it's an excellent idea that could be considered for future studies.
Author Response File: Author Response.docx
Reviewer 2 Report
This manuscript described screening of epiphytic bacteria from citrus fruits. Three isolates were identified and tested against green mold disease in vitro and in vivo. Results are interested but some detail should be clarified.
1. In abstract, metabolites were extracted from isolate H7 but this isolate was not mention anywhere in the manuscript. Where is this isolate come from?
2. Table 1 and the last paragraph on page 4 gave different number of isolate. There are 14 (6+5+3) in the text but only 13 in table.
3. Figure legend for figure 1 should be "Figure 1" and the subtitle 3.3 Molecular Identification..... should be separated from the figure legend.
4. Figure 2, agarose gel showed PCR result from only one isolate (B15) while the figure legend said 3 isolate. Where are the gel for the other two isolates?
5. Table 3 is not necessary. The data is clearly shown in the text.
6. Figure 3, chromatogram of control and B15 should be shown in the same scale especially the X-axis in order to compare the retention time.
7. Subtitle 4.2 Identification most effective isolate.....It would be good if metabolites from the other 2 isolates (S13 & P17) were also analyzed. They might show good antifungal inhibition property and synergism with Iturin A.
8. Conclusion, Bacillus cereus is a human pathogen. Application of this bacterium may not safe for human consumption. Biosafety measure should be strictly followed for handling this bacterium.
9. Reference format is not consistent. e.g. ref. no.3 & 17 are different from others.
10. Pictures of in vitro test (mycelium growth inhibition) and in vivo test (on citrus fruits) should be shown in the manuscript to demonstrate the finding result.
Comments for author File: 
Comments.pdf
none
Author Response
Respectfully,
We are thankful to the reviewers and editor for useful comments on our submission, which have improved the manuscript. All the comments/suggestions have been addressed/incorporated. Revisions/changes have been indicated in Red font color.
1.In abstract, metabolites were extracted from isolate H7 but this isolate was not mention anywhere in the manuscript. Where is this isolate come from?
Thank you. It was edited.
2.Table 1 and the last paragraph on page 4 gave different number of isolate. There are 14 (6+5+3) in the text but only 13 in table.
It has been corrected.
3.Figure legend for figure 1 should be "Figure 1" and the subtitle 3.3 Molecular Identification..... should be separated from the figure legend.
It has been corrected.
4.Figure 2, agarose gel showed PCR result from only one isolate (B15) while the figure legend said 3 isolate. Where are the gel for the other two isolates?
As we only analyzed the antifungal metabolism of the most effective bacteria, we preferred to put the information about B. cereus, but according to reviewer comments, we added the other bacteria's information.
5.Table 3 is not necessary.
The table was deleted.
6. Figure 3, chromatogram of control and B15 should be shown in the same scale especially the X-axis in order to compare the retention time.
The figure has been edited.
7. Subtitle 4.2 Identification most effective isolate.....It would be good if metabolites from the other 2 isolates (S13 & P17) were also analyzed. They might show good antifungal inhibition property and synergism with Iturin A.
This paper is part of a project aimed at comparing the effects of the most effective bacteria with the effects of the most effective plant extracts. So, we decided to just analyze the antifungal metabolite of the most effective strain.
8. Conclusion, Bacillus cereus is a human pathogen. Application of this bacterium may not safe for human consumption. Biosafety measure should be strictly followed for handling this bacterium.
Bacillus cereus is known to produce a wide range of secondary metabolites, including antibiotics, which have antimicrobial activity against various plant pathogens. In this study, our aim was to identify epiphytic bacteria with antagonistic activity and find the most effective antifungal metabolic strain, but for sure, before using this strain as a fungicide, some toxicity tests need to be done in future studies.
9. Reference format is not consistent. e.g. ref. no.3 & 17 are different from others.
It has been edited.
10.Pictures of in vitro test (mycelium growth inhibition) and in vivo test (on citrus fruits) should be shown in the manuscript to demonstrate the finding result.
The picture has been added.
Author Response File: Author Response.docx
Reviewer 3 Report
The authors identified microflora that they obtained from oranges and report microorganisms isolated from the materials. They tested these isolates for the efficacy on the development of green mould on intact oranges and suggest the potential of the isolates for biocontrol agents on this disease. The results can be expected to contribute to the use of these isolates for the suppression of the fungi. Considering the potential value of their results, this manuscript may be considered for the publication in the journal. Comments written below hopefully will be available for revising the manuscript.
Replicate
The most serious problem in this manuscript is the lack of the description on replicate. The authors must mention precisely how they made replicate in each experiment and how they did. Without the explanation, the manuscript would not be published.
Abstract and Introduction
These sections are well written, focusing on what should be studied and what was done in the study.
Materials and Methods
The authors should refer to the time they did anything for the experiment in this study. For example, they should mention the date when they collected the fruit sampling(s), when they prepared samples for the experiments and when they carried out the experiments.
Measuring the growth
The authors should explain how they measured the growth and evaluated the wounds.
The denominator of the formula for disease reduction should be “a”, not “b”. If “b” is taken for the denominator, the reduction, DR, can be infinitive as “b” become smaller.
Results and Discussion
It should be Results.
Describe briefly in Materials and Methods how the isolates were identified.
pH=2 should be written as “pH2”.
The authors used t-test to compare the effects between treatments. As long as this test is used, the authors should correct the probability for the significance in each test according to the number of the pairs in any given experiment. More sophisticatedly, they should consider to use multi-comparison tests.
For the data in Table 2, the authors used Duncan’s test. The authors should use other methods, since this comparison is more likely to lead Type I errors.
Figure 1
Add the errors.
Table 3
This table can be deleted, since the same information is given in the text.
Extraction of samples and HPLC must be explained in Materials and Methods.
Figure 3
The scale of the abscissa of the two panels must be correspondent.
Only minor correction of English may be needed.
Author Response
Respectfully,
We are thankful to the reviewers and editor for useful comments on our submission, which have improved the manuscript. All the comments/suggestions have been addressed/incorporated. Revisions/changes have been indicated in Red font color.
Replicate
The most serious problem in this manuscript is the lack of the description on replicate. The authors must mention precisely how they made replicate in each experiment and how they did. Without the explanation, the manuscript would not be published.
In both parts of In Vitro Screening of Isolates and Effect of Isolated Bacteria on Orange Fruits that we mentioned, it has already been mentioned three repetitions, and it has been highlighted.
Abstract and Introduction
These sections are well written, focusing on what should be studied and what was done in the study.
Thank you.
Materials and Methods
The authors should refer to the time they did anything for the experiment in this study. For example, they should mention the date when they collected the fruit sampling(s), when they prepared samples for the experiments and when they carried out the experiments.
The time of collection was added, but regarding the time of analysis, we already described it (the fruits were then transported to the laboratory without delay for further analysis).
Measuring the growth
The authors should explain how they measured the growth and evaluated the wounds.
Sentences edited.
The denominator of the formula for disease reduction should be “a”, not “b”. If “b” is taken for the denominator, the reduction, DR, can be infinitive as “b” becomes smaller.
Thank you, it has been edited.
Results and Discussion
It should be Results.
It has been edited.
Describe briefly in Materials and Methods how the isolates were identified.
It has been added.
pH=2 should be written as “pH2”.
By writing pH = 2, we wanted to mention pH level, so we did some paraphrasing in the sentence to make it clearer.
The authors used t-test to compare the effects between treatments. As long as this test is used, the authors should correct the probability for the significance in each test according to the number of the pairs in any given experiment. More sophisticatedly, they should consider to use multi-comparison tests.
For the data in Table 2, the authors used Duncan’s test. The authors should use other methods, since this comparison is more likely to lead Type I errors.
Thank you. The statistical description part has been edited.
Figure 1
Add the errors.
It has been done.
Table 3
This table can be deleted, since the same information is given in the text.
It has been done.
Extraction of samples and HPLC must be explained in Materials and Methods.
We made some editions on this part and made them separate to be more clarified.
Figure 3
The scale of the abscissa of the two panels must be correspondent.
It has been edited.
Comments on the Quality of English Language
Only minor correction of English may be needed.
Some editions have been done.
Author Response File: Author Response.docx
Reviewer 4 Report
This study highlights the potential of epiphytic bacteria as a biological control method against green mold post-harvest disease in oranges. The isolated Bacillus, Streptomyces, and Pseudomonas spp. demonstrated strong antifungal properties against P. digitatum, both in vitro and in vivo. The detection of Iturin A in Bacillus cereus indicates the presence of inhibitory compounds that can be further explored for commercial applications. The work is important and interesting, I think it can be accepted after minor revision.
At present, chemical pesticides are the main measures for pest control in the market. Biological control has not yet been accepted by the public. It is recommended to provide more examples in the introduction to explain the commercial varieties of biological control and their advantages.
Nowadays, the middle character "–" is preferred between the numbers.
All ml should change to mL.
Spaces need to be added between data and units.
What are the toxicity and hazards of Epiphytic bacteria?
Pay attention to the unified reference format, as some literature lacks page numbers.
Please find attached the file for more comments!
Comments for author File: Comments.pdf
can be improved
Author Response
Respectfully,
We thank the reviewers and editor for their valuable comments on our submission, which have improved the manuscript.
At present, chemical pesticides are the main measures for pest control in the market. Biological control has not yet been accepted by the public. It is recommended to provide more examples in the introduction to explain the commercial varieties of biological control and their advantages.
We added paragraphs and references.
Nowadays, the middle character "–" is preferred between the numbers.
All ml should change to mL.
Spaces need to be added between data and units.
They have been done.
What are the toxicity and hazards of Epiphytic bacteria?
Epiphytic bacteria are generally considered to be non-pathogenic and have low toxicity. They are commonly found on the surfaces of plants and do not pose significant health hazards to humans or animals. Many epiphytic bacteria play beneficial roles, such as promoting plant growth, protecting plants from pathogens, and contributing to nutrient cycling in ecosystems. However, it is important to note that some bacteria, including certain epiphytic strains, may have the potential to cause infections or produce toxins under certain circumstances. the toxicity and hazards associated with epiphytic bacteria are generally low but for sure for using and presenting this bacteria in the market as a replacement for chemical pesticides further studies such as toxicity should be done but the current study aimed to identify the epiphytic bacteria of citrus with antifungal and antagonistic activity against post-harvest diseases.
Pay attention to the unified reference format, as some literature lacks page numbers.
It has been done.
Reviewer 5 Report
This study investigated epiphytic bacteria isolated from Thompson Navel sweet oranges in northern Iran, focusing on their potential for controlling post harvest green mold disease caused by Penicillium. 23 strains of epiphytic bacteria were screened and their ability to inhibit Penicillium digitatum was evaluated. It was found that the isolates B15 (Bacillus sp.), P17 (Pseudomonas sp.) and S13 (Streptomyces sp.) showed good inhibitory effects on P. digitatum in vitro and in vivo. Among them, B15 has the highest inhibitory effect. Through 16S rRNA sequence analysis, B15 was identified as B. cereus. Metabolic analysis shows that the inhibitory antifungal substance of B15 is Itulin A. This study provides a theoretical basis for the prevention of post harvest green mold disease in sweet oranges. However, there are still many obvious writing errors in the paper, and relevant experiments need to be supplemented to confirm the conclusions in the article.
Major comments:
1. Bacillus can produce antimicrobial peptides through non ribosome synthesis. In addition to Iturin A, there are also different types of antimicrobial peptides, mainly due to differences in the amino acid sequences of peptides. The direct inference in this article is Ituin A, and the conclusion lacks necessary evidence to support it.
2. Lack of activity experiments on B15 isolated products to verify the author's findings.
3. Although the author detected peak signals from B. cereus metabolites by HPLC and the retention time was similar to Itulin A, it cannot be proven that the isolated substance is Itulin A and further mass spectrometry identification is needed.
4. Are there any other metabolites of B.cereus that have antibacterial activity besides Iturin A?
Minor comments:
1. There is an error in the annotation of Fig1.
2. There is an error in the serial numbers of titles 2.7 and 2.8.
3. Figure 5 needs to improve clarity.
Author Response
Respectfully,
We thank the reviewers and editor for useful comments on our submission, which have improved the manuscript.
Bacillus can produce antimicrobial peptides through non ribosome synthesis. In addition to Iturin A, there are also different types of antimicrobial peptides, mainly due to differences in the amino acid sequences of peptides. The direct inference in this article is Ituin A, and the conclusion lacks necessary evidence to support it.
Bacillus species may have different mechanisms when used as biocontrol agents to control disease and promote plant growth and also produce different types of antimicrobial peptides but identification of these antimicrobial peptides was not the aim of this study. The aim of this study was to isolate and molecular identify the epiphytic bacteria with antagonistic activity against post-harvest diseases and just for confirming the antagonistic potential of the isolated bacillus we study the presence of Iturin A compounds in it.
2.Lack of activity experiments on B15 isolated products to verify the author's findings
The aim of this study was to find out the antagonistic activity of isolated strains on Penicillium and we tested and analyzed the antagonistic activity of strains in vitro and in vivo conditions.
Although the author detected peak signals from B. cereus metabolites by HPLC and the retention time was similar to Itulin A, it cannot be proven that the isolated substance is Itulin A and further mass spectrometry identification is needed.
Comparing the retention time and peak characteristics of the Iturin standards with the peaks observed in the Bacillus sample is one of the different methods that confirm if the peaks match in terms of retention time and shape, indicating the presence of Iturin in the sample. It's understandable that different researchers use different methods for their analysis.
Are there any other metabolites of B. cereus that have antibacterial activity besides Iturin A?
Yes, besides Iturin A, there are other metabolites produced by B. cereus that have been found to possess antibacterial activity such as Cerein. B. cereus is known to be a versatile bacterium capable of producing a range of secondary metabolites with different biological activities but Exploring and characterizing these metabolites are not the aim of this study.
There is an error in the annotation of Fig1.
It has been done
There is an error in the serial numbers of titles 2.7 and 2.8.
it has been edited
Figure 5 needs to improve clarity.
We already changed the picture
Round 2
Reviewer 1 Report
The author has modified the article according to the suggestions for modification, which is recommended to be accepted
Minor editing of English language required
Author Response
Respectfully,
We thank the reviewers and editor for valuable comments on our submission, which have improved the manuscript.
We recheck the English grammar of the paper and made editions of some sentences.
We made changes to the conclusion.
Reviewer 2 Report
Authors have made substantial changes to the revised manuscript. Some figures were added to show experimental results. The manuscript is acceptable for publication. However, minor changes are required such as reference no.3 (author name).
none
Author Response
Respectfully,
We appreciate the reviewers and editor for their valuable comments on our submission, which have improved the manuscript. we recheck the paper's English grammar and made some corrections.
we made editions on reference no.3.
