Broad Spectrum Antimicrobial Activity of Bacteria Isolated from Vitis vinifera Leaves

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study investigates bacteria isolated from Vitis vinifera leaves as potential biological control agents against downy mildew and other grapevine pathogens. The researchers identified several bacterial strains with antimicrobial activity, particularly B. subtilis 'BLG_B1.1.1', which showed promising results against P. viticola. The work contributes to developing sustainable alternatives to chemical pesticides in viticulture.
The manuscript presents valuable research with sound methodology and significant findings. However, it requires minor revisions to improve clarity and presentation. Specific attention should be given to:

• Introduction: Consider adding hypothesis statement. 
• L380: Report exact p-values.
• L384-386: Add statistical test details
• Line 446: Clarify "no significant differences".
• Lines 455-463: Expression analysis results could benefit from additional quantitative detail.
• L584-605: Consider addressing potential limitations of the in vitro testing approach.
• L616-641: Safety assessment discussion could be expanded.
• L644-661: Conclusions are well-supported by the data but could better address future research directions.
• Figures: Improve image resolution! 
• Discussion: Expand comparison with similar studies.
• References: Add page numbers for all journal articles; check for consistant formatting; add DOIs where missing. 

Author Response

Comment 1. Introduction: Consider adding hypothesis statement.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we inserted in the introduction the following sentence: “. Since previous works demonstrated a correlation between the phyllophere microbiota of grapes and the resistance toward P. viticola [https://doi.org/10.3389/fmicb.2023.1149307],”

Comment 2. L380: Report exact p-values.

Response 2: Done

Comment 3. L384-386: Add statistical test details

Response 3: Thank you for the suggestion, data were added. 

Comment 4. Line 446: Clarify "no significant differences".

Response 4: Thank you for pointing this out. In agreement with this comment, the following sentence (Lines 447-485), “Significant differences in terms of effectiveness against downy mildew were found between the tested bacterial strains (F value = 5.5758, p-value = 0.006965). Leaf treatment with B. velenzensis strain ‘BLG_B2’ didn’t affect the grapevine downy mildew in terms of DS, compared to the control, confirming the ineffectiveness of this bacterial strain. In contrast, leaves treated with P. pleuroti strain ‘BLG_B5’, P. psychotolerans strain ‘BLG_B1.3’, and B. velenzensis strain ‘BLG_B4’ exhibited a reduction in DS of approximately 41.2-47.1% compared to the control, though these differences were not statistically significant. B. subtilis strain ‘BLG_B1.1.1’ demonstrated the highest efficacy, reducing DS by 100% compared to untreated leaves.”

Comment 5. Lines 455-463: Expression analysis results could benefit from additional quantitative detail.

Response 5: We thank you for this comment. In agreement we added quantitative details in the text paragraph 3.4 (Lines 489-496) and we added results from qPCR in Table S3.

Comment 6. L584-605: Consider addressing potential limitations of the in vitro testing approach.

Response 6: Thank you for pointing this out. In agreement with this comment, the following sentence was added (Lines 708-710): “However, it is important to point out that the conclusions reported so far represent preliminary indications, which require further confirmation which can only be obtained through experimental tests carried out in vivo. “

Comment 7. L616-641: Safety assessment discussion could be expanded.

Response 7: Thank you for pointing this out. In agreement with this comment, the following sentence was added (Lines 661-665): “Further investigations are needed to verify the inability of these bacterial strains to cause human infections and allergies. Infection case reports have been published for Bacillus thuringensis, Bacillus sphaericus and Bacillus cepacia. Septic arthritis caused by Pantotea agglomerans after such injuries were revised by Kratz and co-authors”. Additional references were added: 10.1002/ajim.20086; 10.1053/jhin.2001.0995; 10.1128/JCM.41.8.3548-3558.2003; 10.1136/adc.88.6.542

Comment 8. L644-661: Conclusions are well-supported by the data but could better address future research directions.

Response 8: We thank you for this comment. In agreement we modified the Conclusion at lines 680-689.

Comment 9. Figures: Improve image resolution!

Response 9: Done

Comment 10. Discussion: Expand comparison with similar studies.

Response 10: We thank you for your comment, we revised the Discussion session.

Comment 11. References: Add page numbers for all journal articles; check for consistant formatting; add DOIs where missing.

Response 11: Done

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,  

Thank you for submitting your manuscript for review. Your study presents an interesting investigation into bacterial isolates from Vitis vinifera leaves as potential biological control agents (BCAs) against Plasmopara viticola. While the research contributes to sustainable viticulture, several areas require clarification, stronger justification, and improved organization to enhance the manuscript's scientific rigor. Below are my detailed comments and suggestions.  

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Introduction 

Lines 36-41: "The grapevine hosts numerous microorganisms on its compartments that influence the plant's health status, plant growth and wine's organoleptic characteristics."  

Comment: While this provides useful context on the microbiome, it does not sufficiently explain why grapevine leaves (as opposed to other plant compartments) are a particularly promising source of antimicrobial bacteria. The connection to the study’s focus should be more explicitly stated.  

 

Line 43: "Grapevine leaves microbiome follows a cyclic course: it comes together in spring, changes during the vegetative season and disassembles during leaf senescence."  

Comment: This seasonal variation in the microbiome is interesting but does not appear directly relevant to the identification of bacterial strains with antimicrobial activity. If seasonal effects are important, this should be explicitly linked to how they influence bacterial potential as BCAs. Does the cyclic nature of the microbiome affect the antimicrobial properties of the bacterial strains? If so, how? 

 

Line 48: "During the last decades, the necessity to explore novel niches that host microorganisms able to produce bioactive compounds and to investigate plant microbiomes as a rich source of microbes able to secrete molecules with relevant biotechnological interest, including antimicrobial properties, has arisen."  

Comment: This is a broad statement without a clear indication of what specific knowledge gap this study addresses. The introduction should clarify why grapevine leaf microbiomes, in particular, have been underexplored and what is currently unknown about their antimicrobial potential. What specific gaps in knowledge about grapevine leaf microbiomes does this study aim to address? Have previous studies identified antimicrobial bacteria in grapevine leaves, or is this a novel investigation?  

 

Lines 51-59: "Plant-associated microbes synthesize an array of metabolites... [list of compounds]... These characteristics give plant-associated microbes the potential to be employed in numerous ways, including more sustainable and ecologically friendly farming methods."  

Comment: While this statement highlights the role of microbial metabolites, it does not explicitly explain how these metabolites contribute to biological control in grapevine disease management. The relevance to viticulture should be emphasized more clearly.  

 

Lines 60-72: "Indeed, nowadays, to meet both qualitative and quantitative production standards, plant diseases, including those affecting grapevine, are controlled by an intensive schedule of strategies among which the use of chemical compounds is the most common and effective."  

Comment: While pesticide risks are well-documented, this section does not specifically address the limitations of current downy mildew control strategies. A stronger connection to why biological control is necessary for managing P. viticola would improve the argument.  

 

Lines 82-86: "Different bacteria genera have been identified as producers of antimicrobial metabolites with broad-spectrum activity such as Agrobacterium, Bacillus, Pantoea, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces." 

Comment: While this section introduces relevant bacterial genera, it does not explain why these specific bacterial strains were chosen from grapevine leaves. Were they selected based on previous literature, known effectiveness, or their abundance in V. vinifera?  

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Results and Discussion 

Lines 343: "Accordingly to our previous data [32] we selected specific niches for the isolation of microorganisms directly linked to genotypes more tolerant to P. viticola."  

Comment: The authors claim that microbial isolation was based on grape varieties with different tolerance levels, but the rationale for selecting only three cultivars (Dawn Seedless, Blush Seedless, Argentina) from the 19 analyzed is not well explained. The assumption that more tolerant varieties harbor better BCAs is not explicitly supported. Please answer these questions: (1) How were the three cultivars chosen from the 19 analyzed? (2) Were the microbial populations of the selected cultivars significantly different from the others? If so, what evidence supports this?  

 

Lines 350: "Since the D value is close to 1 (D=0.6277021), we can reject the null hypothesis... and conclude that our data set contains meaningful cluster."  

Comment: The Hopkins statistic (D = 0.627) is used to assess clustering tendency, but the threshold for defining “meaningful clusters” is unclear. Additionally, the use of K-means clustering is justified solely based on silhouette width and Dunn index without explaining why other clustering methods (e.g., hierarchical clustering, DBSCAN) were not considered. Please address these questions: (1) Why was K-means clustering selected over other clustering methods? (2) How robust were the clusters? Did you perform a validation test (e.g., bootstrapping) to confirm their reliability?  

 

Lines 381: "Six bacterial strains reduced the leaf disc area covered by P. viticola sporulation but not in a statistically significant way compared to the control."  

Comment: Reporting non-significant trends does not add scientific value unless accompanied by potential explanations. The authors later discuss these strains in characterization studies without clarifying why they were included despite their lack of statistical significance. Please answer: (1) Why were strains with non-significant effects included in further characterization? (2) Would excluding these strains have improved the study’s focus on truly effective BCAs? 

 

Lines 387: "The most effective bacterial strains ‘BLG_B1.3’, ‘BLG_B1.1.1’, and ‘BLG_B4’, together with the moderately effective bacterial strain ‘BLG_B5’ and the ineffective one ‘BLG_B2’, were selected for further characterization studies."  

Comment: The criteria for categorizing bacterial strains as moderately effective versus ineffective are unclear. The authors provide percentage inhibition values, but there is no statistical test (e.g., ANOVA, post-hoc comparisons) to justify why some strains are considered effective while others are not. Please address these questions: (1) What statistical threshold was used to differentiate between effective, moderately effective, and ineffective bacterial strains? (2) Was a post-hoc test (e.g., Tukey’s test) performed to compare bacterial strains? If not, would such an analysis strengthen the conclusions?  

 

Lines 507: "At the time, it is known that microorganisms colonizing plant surface and inner tissue play an essential role in shaping our planet: from our natural vegetation to intense agricultural production systems up to human health."  

Comment: This statement is too broad and does not relate specifically to the study's findings. While microbial communities are important, the discussion should focus on the role of bacterial strains isolated from V. vinifera in plant disease suppression rather than making a generalized ecological statement.  

 

Lines 532: "Results of the leaf disc assay show that of the five most effective bacterial isolates, four (indicated with the acronym ‘BLG’) were obtained starting from the phyllosphere of the most tolerant cultivar ‘Blush Seedless’." 

Comment: While this suggests a correlation between host genotype and bacterial antagonism, the study does not demonstrate causality. Please address these questions: (1) Did you perform microbial community profiling to confirm that Blush Seedless harbors a distinct microbiome enriched in antagonistic bacteria? (2) Could environmental factors (e.g., vineyard management, soil conditions) have influenced the microbial composition more than the plant genotype itself? 

 

Lines 619: "In this work we evaluated the effectiveness of the selected bacterial strains against several grapevine fungal pathogens and their ability to degrade blood erythrocytes."  

Comment: The study does not discuss its limitations, such as the small number of bacterial strains tested, the lack of field validation to confirm effectiveness under vineyard conditions, and the need for formulation studies to determine practical application methods. What are the key limitations of this study? How could future research address these limitations, especially regarding field trials and formulation development? 

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Additionally, several typographical and grammatical errors were noted throughout the manuscript. A thorough proofreading is recommended to enhance clarity and readability.

Comments on the Quality of English Language

A thorough proofreading by a native English speaker or a professional language editor is highly recommended to ensure clarity, coherence, and accuracy in scientific communication.

Author Response

Comment 1. Lines 36-41: "The grapevine hosts numerous microorganisms on its compartments that influence the plant's health status, plant growth and wine's organoleptic characteristics." Comment: While this provides useful context on the microbiome, it does not sufficiently explain why grapevine leaves (as opposed to other plant compartments) are a particularly promising source of antimicrobial bacteria. The connection to the study’s focus should be more explicitly stated.

Response 1: Thank you for pointing this out. In agreement with this comment, the introduction has been revised to better explain the importance of grapevine leaves as promising source of antimicrobial bacteria. The following sentence was inserted at the lines 44-51: “The phyllosphere represents the biomarkers for the phytosanitary status of plant and the exploration of its microbial community has proven to be essential to understand the balance between phytopathogens and beneficial microorganisms. Specific metagenomics studies, conducted on grapevine leaves, revealed a high abundance of bacteria belonging to the Enterobacteriaceae family. These bacteria play an important role in vineyards as they act as beneficial microorganisms thought specific mode of action, such as induction of host resistance and production of glucanase, chitinase and protease [10.1007/s00253-006-0798-3]”.

Additional reference (doi: 10.1007/s00253-006-0798-3) has been included.

Comment 2. Line 43: "Grapevine leaves microbiome follows a cyclic course: it comes together in spring, changes during the vegetative season and disassembles during leaf senescence.” Comment: This seasonal variation in the microbiome is interesting but does not appear directly relevant to the identification of bacterial strains with antimicrobial activity. If seasonal effects are important, this should be explicitly linked to how they influence bacterial potential as BCAs. Does the cyclic nature of the microbiome affect the antimicrobial properties of the bacterial strains? If so, how?

Response 2: Thank you for pointing this out. We agree with this comment. The introduction has been revised to better explain the effects of the seasonal variation in the leaf microbiome. The following sentence was inserted at the lines 55-63: “The seasonal variation in the grapevine leaves microbiome appears to influence the bacterial potential as Biological Control Agents (BCAs). In a recent study, the structure of leaf microbiome community at harvesting period was associated with phenotype resistance toward Plasmopara viticola. Authors highlighted how the leaves in this specific phenological stage were very rich in bacterial species belonging to the genus Paroccoccus, which has previously been negatively correlated with downy mildew disease severity caused by P. viticola. Furthermore, other abundant genera were Devosia and Rhizobium, that shown to have plant growth promotion traits.” 

Additional references (doi: 10.1128/AEM.00415-14; 10.1093/femsec/fiaa053) have been included.

Comment 3: Line 48: "During the last decades, the necessity to explore novel niches that host microorganisms able to produce bioactive compounds and to investigate plant microbiomes as a rich source of microbes able to secrete molecules with relevant biotechnological interest, including antimicrobial properties, has arisen." Comment: This is a broad statement without a clear indication of what specific knowledge gap this study addresses. The introduction should clarify why grapevine leaf microbiomes, in particular, have been underexplored and what is currently unknown about their antimicrobial potential. What specific gaps in knowledge about grapevine leaf microbiomes does this study aim to address? Have previous studies identified antimicrobial bacteria in grapevine leaves, or is this a novel investigation? 

Response 3: We agree with this statement and in accordance with the reviewer's suggestions. We added in the introduction the following sentence at the lines 64-71: “During the last decades, the necessity to explore novel niches hosting microorganisms capable of producing bioactive compounds has increased, particularly in the search for BCAs to combat plant pathogens. In particular, the microbiome present on the grapevine phyllosphere has been explored in search of beneficial microorganisms against airborne pathogens such as P. viticola. Several bacterial species isolated from grapevine leaves have a negative effect on P. viticola under experimental conditions. The genus Bacillus, including B. subtilis and B. pumilus, predominates among P. viticola antagonistic bacteria ”

Additional references (doi: 10.3389/fmicb.2019.02726; 10.1016/j.cropro.2011.02.031) have been included.

Comment 4. Lines 51-59: "Plant-associated microbes synthesize an array of metabolites... [list of compounds] ... These characteristics give plant-associated microbes the potential to be employed in numerous ways, including more sustainable and ecologically friendly farming methods." Comment: While this statement highlights the role of microbial metabolites, it does not explicitly explain how these metabolites contribute to biological control in grapevine disease management. The relevance to viticulture should be emphasized more clearly.

Response 4: Thank you for pointing this out. Accordingly, the introduction has been revised

to better explain the role of microbial metabolites in biological control, particularly in the context of grapevine disease management. We have explicitly highlighted how plant-associated microbes produce bioactive compounds with antimicrobial properties and how these compounds contribute to pathogen suppression and plant defense induction (see lines 72-79). The revised sentence now states: "Microbes on plants produce many useful compounds, including terpenoids, alkaloids, and tannins, which help them compete and support plant defense. These metabolites can exhibit antimicrobial activity, either by directly inhibiting pathogens or by inducing plant resistance mechanisms, making them promising candidates for biological control strategies in viticulture. In particular, some microbial-derived compounds, such as lipopeptides from Bacillus spp. and phenazines from Pseudomonas spp., have been reported to suppress fungal and oomycete pathogens, including those affecting grapevine. "

Additional reference (doi: 10.1146/annurev-phyto-081211-172908) has been included to support the relevance of these microbial metabolites in biological control strategies for viticulture. 

Comment 5. Lines 60-72: "Indeed, nowadays, to meet both qualitative and quantitative production standards, plant diseases, including those affecting grapevine, are controlled by an intensive schedule of strategies among which the use of chemical compounds is the most common and effective." Comment: While pesticide risks are well-documented, this section does not specifically address the limitations of current downy mildew control strategies. A stronger connection to why biological control is necessary for managing P. viticola would improve the argument.

Response 5: We appreciated the suggestion and we therefore improve the introduction to better explain the limitation of current downy mildew control strategies and why biological control methods are necessary for managing P. viticola. The following sentences were added: “Currently, downy mildew is considered one of the most serious diseases in viticulture worldwide. Despite the efforts of geneticists to obtain resistant cultivars and the adoption of cultural practices aimed at reducing conditions favoring P. viticola development, chemical control based on the use of organic and inorganic is the most common and effective. (Lines 80-84)”; “Many of the active ingredients used for downy mildew control belong to fungicide group with a single-site mode of action, the use of which is associated with a high risk of selecting resistant strains within the pathogen population. Furthermore, significant risks, including environmental contamination and human health hazards are associated with extensive use of chemicals pesticides (Lines 86-90)”.

Additional reference (10.20870/oeno-one.2016.0.0.1780;  10.3390/microorganisms9010119; 10.1002/ps.6700;  10.1371/journal.pone.0268385) has been included to support why biological control is necessary for managing P. viticola.

Comment 6. Lines 82-86: "Different bacteria genera have been identified as producers of antimicrobial metabolites with broad-spectrum activity such as Agrobacterium, Bacillus, Pantoea, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces." Comment: While this section introduces relevant bacterial genera, it does not explain why these specific bacterial strains were chosen from grapevine leaves. Were they selected based on previous literature, known effectiveness, or their abundance in V. vinifera?

Response 6: Thank you for pointing this out. We agree with this comment; therefore we better explained the source of isolation of the cited bacterial species.  The following sentences were added: “In particular, different species belonging to the Bacillus genera, isolated from grapevine phyllospheres of different cultivars, have been selected for their ability to significantly reduce grapevine downy mildew ” (Lines 107-110); “Also, several Pseudomonas and Pantotea species , isolated from the phyllospere of different crops, are renowned for their ability to produce a wide variety of antibiotic secondary metabolites (such as phenazine co-compounds, antimicrobial volatile compounds, antibiotics and biosurfactants), that play a crucial role in plant protection.” (Lines 113-117).

Additional references (https://doi.org/10.1016/j.cropro.2017.02.018; doi:10.1080/09583157.2011.574208; 10.3390/plants10091815) have been included.

Comment 7. Lines 343: "Accordingly to our previous data [32] we selected specific niches for the isolation of microorganisms directly linked to genotypes more tolerant to P. viticola." Comment: The authors claim that microbial isolation was based on grape varieties with different tolerance levels, but the rationale for selecting only three cultivars (Dawn Seedless, Blush Seedless, Argentina) from the 19 analyzed is not well explained. The assumption that more tolerant varieties harbor better BCAs is not explicitly supported. Please answer these questions: (1) How were the three cultivars chosen from the 19 analyzed? (2) Were the microbial populations of the selected cultivars significantly different from the others? If so, what evidence supports this?

Response 7: Thank you for pointing this out. To answer to the two comments raised by the reviewer please consider the following modified sentences in the Material and Methods and Results:

1. How were the three cultivars chosen from the 19 analyzed? The following sentences were added at lines 395-399: “We randomly selected one cultivar by each cluster to proceed with the isolation activity of potential candidate bacteria as good BCAs. In detail, we selected the cultivar ’Blush seedless’, ’Argentina’ and ’Dawn seedless’, respectively characterized by a high, medium and low level of tolerance to downy mildew infections at the phyllosphere level.”
2. Were the microbial populations of the selected cultivars significantly different from the others? If so, what evidence supports this? The following sentences were added at lines 405-406: “No significant differences were found in terms of bacterial microbiome abundance between the phyllosphere of the three cultivars analyzed”

Comment 8. Lines 350: "Since the D value is close to 1 (D=0.6277021), we can reject the null hypothesis... and conclude that our data set contains meaningful cluster." Comment: The Hopkins statistic (D = 0.627) is used to assess clustering tendency, but the threshold for defining “meaningful clusters” is unclear. Additionally, the use of K-means clustering is justified solely based on silhouette width and Dunn index without explaining why other clustering methods (e.g., hierarchical clustering, DBSCAN) were not considered. Please address these questions: (1) Why was K-means clustering selected over other clustering methods? (2) How robust were the clusters? Did you perform a validation test (e.g., bootstrapping) to confirm their reliability? 

Response 8: Thank you for pointing this out. To answer to the three comments raised by the reviewer please consider the following sections in the Material and Methods: 

1. The Hopkins statistic (D = 0.627) is used to assess clustering tendency, but the threshold for defining “meaningful clusters” is unclear. As explained in Material and Method, section 2.8 “Statistical analysis” (Lines 358-360), the Hopkins statistic measures the probability that a given data set is generated by a uniform data distribution. This statistic is conducted, using 0.5 as the threshold to reject the alternative hypothesis (i.e. the data set is uniformly distributed). As reported in [10.1021/ci00065a010] if H < 0.5, then it is unlikely that D has statistically significant clusters. Put in other words, If the value of Hopkins statistic is close to 1, then we can reject the null hypothesis and conclude that the dataset D is significantly a clusterable data.
2. Why was K-means clustering selected over other clustering methods? To choose the appropriate clustering algorithms for our dataset, we performed an internal validation, calculating three indeces: ‘Connettivity’, ‘Silhoutte coefficient (Si)’ and ‘Dunn index’. Connettivity represents a measure of the distance between items placed in the same cluster; it can assume values ​​from 0 to infinity and must be as low as possible. Silhoutte coefficient (Si) is an index that estimates the average distance between clusters. Si values ​​close to 1 indicate well-separated clusters; Si values ​​close to 0 indicate that an observation is located between two clusters; finally, negative Si values ​​indicate that an observation is in the wrong cluster. Finally, Dunn index represents the ratio between the minimum inter-cluster separation and the maximum intra-cluster distance. The minimum inter-cluster separation is calculated by measuring the distance between an object in one cluster and a corresponding object in another cluster. The minimum distance between these pairwise comparisons is selected for the calculation of the Dunn index. The maximum intra-cluster distance, on the other hand, is calculated by measuring the distance between objects in the same cluster. The highest value of this pairwise comparison is selected for the calculation of the Dunn index, providing indications on the maximum diameter of the cluster. The Dunn index should be as high as possible, as it means that the maximum diameter of the cluster is lower than the distance between the clusters, which is an indication of well-separated clusters. To best summarize these concepts, the sentences present at lines 379-383 has been modified as follows: “To choose the appropriate clustering algorithms for our dataset, ‘Connettivity’, ‘Silhoutte coefficient (Si) and ‘Dunn index’ were calculated and Kmeans with three clusters resulted the best clustering algorithm, as it determined both the lowest (Connettivity) and highest values (Silhoutte coefficient and Dunn index), compared to the other clustering algorithms (Table S1).” An additional Table, in which we report the value of all indices for each clustering algorithm, has been inserted in Supplementary Material.
3. How robust were the clusters? Did you perform a validation test (e.g., bootstrapping) to confirm their reliability? We decided not to perform a validation test, as we believe that the preliminary statistical analyses that led to the choice of using the kmeans clustering algorithm with three clusters are sufficient to justify the reliability of the identified clusters.

Comment 9. Lines 381: "Six bacterial strains reduced the leaf disc area covered by P. viticola sporulation but not in a statistically significant way compared to the control." Comment: Reporting non-significant trends does not add scientific value unless accompanied by potential explanations. The authors later discuss these strains in characterization studies without clarifying why they were included despite their lack of statistical significance. Please answer: (1) Why were strains with non-significant effects included in further characterization? (2) Would excluding these strains have improved the study’s focus on truly effective BCAs?

Response 9: Thank you for pointing this out. To answer to the two comments raised by the reviewer please consider the following sentence at the paragraph 3.4 “Exspression analysis of effector genes of P. viticola“, section ‘Results’ (Lines 463-467): “The selected bacterial strains (B. velenzensis strain ‘BLG_B2’ and ‘BLG_B4’; B. subtilis strain ‘BLG_B1.1.1’, P. psychotolerans strain ‘BLG_B1.3’ and P.pleuroti strain ‘BLG_B5’) were used to perform a bioassay, using whole leaves of the susceptible cultivar ‘Flame seedlees’, to assess their antagonistic effect against downy mildew and their potential mechanism of action.”

Comment 10. Lines 387: "The most effective bacterial strains ‘BLG_B1.3’, ‘BLG_B1.1.1’, and ‘BLG_B4’, together with the moderately effective bacterial strain ‘BLG_B5’ and the ineffective one ‘BLG_B2’, were selected for further characterization studies." Comment: The criteria for categorizing bacterial strains as moderately effective versus ineffective are unclear. The authors provide percentage inhibition values, but there is no statistical test (e.g., ANOVA, post-hoc comparisons) to justify why some strains are considered effective while others are not. Please address these questions: (1) What statistical threshold was used to differentiate between effective, moderately effective, and ineffective bacterial strains? (2) Was a post-hoc test (e.g., Tukey’s test) performed to compare bacterial strains? If not, would such an analysis strengthen the conclusions? 

Response 10: The reviewer's comment is not clear. Both in the section ‘Materials and methods’, paragraph 2.8 ‘Statistical analyses’, and in the captions of figures 3 and 5 we reported that “The two-way ANOVA test followed by the post-hoc Tukey test (p < 0.05) were performed to identify differences in the antagonistic activity of isolated bacterial strains against the different considered pathogens of grapevine”

Comment 11. Lines 507: "At the time, it is known that microorganisms colonizing plant surface and inner tissue play an essential role in shaping our planet: from our natural vegetation to intense agricultural production systems up to human health." Comment: This statement is too broad and does not relate specifically to the study's findings. While microbial communities are important, the discussion should focus on the role of bacterial strains isolated from V. vinifera in plant disease suppression rather than making a generalized ecological statement. 

Response 11: Thank you for pointing this out. We agree with this comment therefore we removed from the discussion : “At the time, it is known that microorganisms colonizing plant surface and inner tissue play an essential role in shaping our planet: from our natural vegetation to intense agricultural production systems up to human health. In summary, plant-associated microorganisms must be considered as key drivers for plant health, productivity, community composition and ecosystem functioning. Much attention has been paid to the exploration of the plant-associated microbiome in search of new microorganisms with antagonistic activity against various phytopathogens.” 

Comment 12. Lines 532: "Results of the leaf disc assay show that of the five most effective bacterial isolates, four (indicated with the acronym ‘BLG’) were obtained starting from the phyllosphere of the most tolerant cultivar ‘Blush Seedless’." Comment: While this suggests a correlation between host genotype and bacterial antagonism, the study does not demonstrate causality. Please address these questions: (1) Did you perform microbial community profiling to confirm that Blush Seedless harbors a distinct microbiome enriched in antagonistic bacteria? (2) Could environmental factors (e.g., vineyard management, soil conditions) have influenced the microbial composition more than the plant genotype itself?

Response 12: Thank you for pointing this out. We agree with this comment and we modified the following sentence (lines 572-576): “T These data results, although represent a further confirmation of our previous co-evolution hypothesis of microorganisms within the growing area, to the extent of involving the genotype of host plant. Further metagenomic studies are necessary to confirm whether ‘Blush seedless’ harbors an enhanced microbiome composition beneficial for disease control”

Comment 13. Lines 619: "In this work we evaluated the effectiveness of the selected bacterial strains against several grapevine fungal pathogens and their ability to degrade blood erythrocytes." Comment: The study does not discuss its limitations, such as the small number of bacterial strains tested, the lack of field validation to confirm effectiveness under vineyard conditions, and the need for formulation studies to determine practical application methods. What are the key limitations of this study? How could future research address these limitations, especially regarding field trials and formulation development?

Response 13: Thank you for pointing this out. We agree with this comment and we improved the Conclusion (Lines 682-691)

Comment 14. Additionally, several typographical and grammatical errors were noted throughout the manuscript. A thorough proofreading is recommended to enhance clarity and readability.

Response 14: We thank you for your comment, we revised all the document and improved clarity and readability. See in particular the introduction and discussion.

Comment 15. A thorough proofreading by a native English speaker or a professional language editor is highly recommended to ensure clarity, coherence, and accuracy in scientific communication.

Response 15: We thank the reviewer; we checked again the new version of the manuscript with professional language editor and proofread the whole document.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

1-  This is an excellent manuscript with some minor revisions:  Line 34 : economic importance is clear, but clarify "social" importance.  Line 65: should be "derived" rather than deriving.  Line 112 (as well as other Lines : clarify "bunches".  

2-  Line 356: Figure S1 ? Please explain. Where is "Figure S1". ? ?

3-  The figures are well constructed, and clear to understand, and well explained, e.g ; Figures 3, 5, 6, and 7. I have no problem with them. 

Author Response

Comment 1. Line 34 : economic importance is clear, but clarify "social" importance.

Response 1: Thank you for pointing this out. We agree with this comment. To clarify the social importance of V. vinifera, we inserted in the introduction the following sentence (Lines 36-37): “In terms of employment, the wine sector provides jobs to around 2.9 million people within the European Union.”

Comment 2. Line 65: should be "derived" rather than deriving.

Response 2: Thank you for pointing this out. This part of the introduction has been modified based on the comments of other reviewers. The term “derived” has been removed and the sentence has been reworded.

Comment 3: Line 112 (as well as other Lines : clarify "bunches". 

Response 3: Thank you. “Bunches” is a synonym of “Clusters”

Comment 4. Line 356: Figure S1 ? Please explain. Where is "Figure S1". ? ?

Response 4: Thank you. All the Figures and Table indicated with ”S” are present in Supplementary Materials

Comment 5. The figures are well constructed, and clear to understand, and well explained, e.g ; Figures 3, 5, 6, and 7. I have no problem with them.   

Response 5: Thank you

Author Response File: Author Response.docx