Review Reports - Evaluation of Capsaicin as a Selector for Growth Promotional Bacteria Isolated from <i>Capsicum</i> Peppers

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors The authors raise the ever-important issue of the role of PGPB bacteria in plant development.
Although a number of similar studies have been conducted for many years, each successive study
allows for the potential of these microorganisms to be harnessed in plant development. The subsequent stages of the experiment were logically planned.
The description and analysis of the obtained results raise no objections. However, please justify why the seeds of both tested plants, Trifolium pratense and Poa pratensis,
were sterilized differently before inoculation with bacterial isolates.
Furthermore, please clarify whether there is any certainty that all microbial colonies
isolated from the peppers were analyzed in subsequent tests? Because when all bacterial colonies
were grouped based on colony morphology, some species forming similar-looking colonies
on solid media may have been missed.

Author Response

Thank you very much for providing feedback to our manuscript. Please see the following point-by-point response to your comments:

Comments from Reviewer 1

Point 1:
The description and analysis of the obtained results raise no objections. However, please justify why the seeds of both tested plants, Trifolium pratense and Poa pratensis, were sterilized differently before inoculation with bacterial isolates.

We thank the reviewer for raising this point, which is important to clarify. In Materials and Methods 2.7, we have added a citation to the sterilization protocol we adapted from and added a line about verifying the surface-sterility of seeds (Lines 269-271 in revised manuscript).

Point 2:
Furthermore, please clarify whether there is any certainty that all microbial colonies
isolated from the peppers were analyzed in subsequent tests? Because when all bacterial colonies were grouped based on colony morphology, some species forming similar-looking colonies on solid media may have been missed.

We agree with this point and acknowledge it as a limitation of our research article. It is possible that the initial screening, based on colony morphology, could have inadvertently excluded some isolates. However, we performed our isolations in triplicate and with varying amounts of capsaicin, which should help to strengthen our findings.

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript Title: Use of capsaicin to drive the isolation of plant-beneficial bacteria from different Capsicum peppers has been received for review. The authors investigated whether the capsaicin content of different Capsicum varieties (bell pepper, jalapeño, habanero) impacts the capsaicin resistance and plant growth-promoting bacteria associated with their seeds and fruit tissues. This study assessed growth-promoting traits such as IAA production, phosphate and potassium solubilization and urease activity. Further plant inoculation experiments on Trifolium pratense and Poa pratensis were conducted to assess functional benefits of capsaicin resistance and plant growth-promoting bacteria. Overall study is interesting, and manuscript is written well. However, some issues must be addressed before the manuscript is considered suitable for publication.

Major comments

(1) The initial hypothesis is on the assumption that increased capsaicin concentration in fruit tissues imposes a selective pressure on seed-associated microbial communities. However, the manuscript later acknowledges that capsaicin is mainly concentrated in placental tissues, not within seeds. Therefore, it is not clear how seed microbiota may experience sufficient exposure to capsaicin for selection. This may explain the lack of correlation observed between fruit and capsaicin tolerance among isolated bacteria. The introduction should clearly explain this anatomical constraint earlier and adjust the hypothesis to reflect realistic microbial exposure pathways.

(2) Growth media (peat) contains accessible nutrients; thus phosphate/potassium solubilization effects cannot be conclusively attributed to bacterial activity. Germination improvements should be statistically tested, not qualitatively described.

(3) Some minor language issue should be adjusted.

Minor comments

(4) Line 12-14: "Peppers (Capsicum spp.) are unique among the plant kingdom for producing capsaicin, whose antibiotic property presents an additional selective pressure..."
The claim is overstated that “Peppers are not unique; other plants in the Solanaceae produce capsaicinoid analogs”. Sentence may be improved.

Example: “Capsicum species are notable for producing capsaicin, a compound with antimicrobial activity that may influence microbial communities associated with pepper fruits and seeds.”

(5) Line 18-20: "Minimum inhibitory concentration (MIC) assays showed that jalapeno and habanero peppers do not host bacterial communities that are more capsaicin-resistant, compared to the bacterial communities from bell pepper."

The experimental design does not measure “communities”; it measures cultured isolates? Replace "communities" with “isolates” to avoid misinterpretation.
Example: Minimum inhibitory concentration (MIC) assays indicated that the capsaicin resistance of isolated bacteria did not correlate with the pungency level of the host pepper variety.”

(6) Lines 34-40: “An important perspective in the discourse surrounding sustainable agriculture is the need to minimize the use of synthetic agrochemicals...”

The phrasing “An important perspective in the discourse” adds unnecessary notion. Revise sentence as:

Example: “Sustainable agriculture aims to reduce reliance on synthetic fertilizers and pesticides, which are energy-intensive to produce and contribute to greenhouse gas emissions.”

(7) Lines 69-75: The development of antibiotic resistance is thought to be linked to reduced overall fitness... any bacteria with capsaicin resistance should have a lifestyle more closely associated with the host pepper...”

This assumes direct seed exposure to capsaicin, which is later questioned in the Discussion. Simplify the sentence as:

Example: “If seed-associated bacteria are exposed to capsaicin during fruit development, selection may favor strains with tolerance mechanisms. However, the extent of such exposure varies with fruit anatomy and remains unclear. Therefore, whether pungency influences seed microbial traits remains an open question.”

(8) Lines 81-83:“Ripe commercial bell, jalapeno, and habanero peppers were purchased … stored at 4°C for up to three days.”

Source detail is not clear. Was the produce organic/conventional? Did different storage times occur? These factors can influence microbial community composition.Clarify selection and storage consistency.

Example: “Ripe, conventionally grown bell, jalapeño, and habanero peppers were obtained from a single commercial supplier. All fruits were processed within 72 hours of purchase and stored at 4 °C under identical conditions to minimize variation.”

(9) Lines 249-270 (Plant inoculation design): No indication of soil sterilization confirmation. Seeds soaked only 10 min may not allow adequate colonization. Magenta box environmental conditions lack light intensity / humidity description

Add environmental growth conditions and colonization verification detail.

(10) Lines 304-310: (Number of isolates): “In total, 20 morphologically unique bacteria were isolated…………”

The text indicates community-level diversity, but only culturables were sampled. State clearly that this refers only to culturable isolates.

(11) Lines 432-447: “Our broth microdilution test… does not support the idea that bacteria isolated from peppers of different pungency levels will have different levels of resistance to capsaicin.”

This section correctly states the result but does not adequately acknowledge anatomical constraints (capsaicin localized in placenta, not seed). Clarify limited microbial exposure.

Example: “This outcome is consistent with the anatomical distribution of capsaicin, which is concentrated primarily in placental tissue rather than within the seeds themselves. Therefore, seed-associated microbes may not be exposed to selective pressures related to capsaicin concentration.”

(12) Lines 535-537: “Despite this, we isolated two growth-beneficial Pseudomonas sp.… that solubilized phosphates and produced IAA and showed that these isolates promoted growth…”

This sentence is too long and compresses multiple key findings into a single sentence. It is difficult to read.Split into two concise statements.

Example: “However, several isolates demonstrated strong plant growth–promoting traits, including IAA production and phosphate solubilization. In particular, two Pseudomonas isolates significantly enhanced seedling growth in both Trifolium pratense and Poa pratensis.”

(13) Lines 536-538 “…meaning biostimulants can work on different plants.”

This conclusion is too strong because only two plant species were tested. The claim suggests universal applicability without evidence. Reframe as potential, not proven.

Example: “These findings indicate the potential for these isolates to function across multiple plant hosts; however, further testing is needed to evaluate their effectiveness in additional crop species.”

Author Response

Thank you very much for your feedback on our manuscript. We greatly appreciate the time and consideration you took to help improve our manuscript. Please see the following point-by-point responses to your comments:

Comments from Reviewer 2

Addressing major comments:

Point 1:
(1) The initial hypothesis is on the assumption that increased capsaicin concentration in fruit tissues imposes a selective pressure on seed-associated microbial communities. However, the manuscript later acknowledges that capsaicin is mainly concentrated in placental tissues, not within seeds. Therefore, it is not clear how seed microbiota may experience sufficient exposure to capsaicin for selection. This may explain the lack of correlation observed between fruit and capsaicin tolerance among isolated bacteria. The introduction should clearly explain this anatomical constraint earlier and adjust the hypothesis to reflect realistic microbial exposure pathways.

We appreciate that this is an important point to clarify and thank the reviewer for their recommendation. We have added an explanation in the Introduction (Lines 64-69) that the placenta has vascular tissue through which nutrients and potential bacteria might be vertically transmitted. Consequently, any seed bacteria that have been transmitted from the parent may be exposed to capsaicin in the placenta, at least in transit. However, research on placenta-mediated vertical transmission of bacteria is scarce, so we frame this as an assumption of our research.

Point 2:
(2) Growth media (peat) contains accessible nutrients; thus phosphate/potassium solubilization effects cannot be conclusively attributed to bacterial activity. Germination improvements should be statistically tested, not qualitatively described.

Thank you for the suggestion regarding growth media. We believe our writing is careful not to claim that phosphate/potassium solubilization of Jf1 and Hs7 are solely responsible for growth promotion. However, to more effectively convey this message, we have added some changes to our Discussion on this matter (Lines 538-548). Namely, we replaced the phrase “may be responsible for growth promotion” to “may have contributed to growth promotion” (Line 538). Also, we clarified that bacterial solubilization “may have improved the plant-accessibility of the organic nutrients and contributed to the improved growth of the host, while the non-inoculated plants could only rely on the inorganic, accessible nutrients.” (Lines 547-548)

Unfortunately, we did not obtain replicates data for germination, and so could not statistically test this metric. Due to the lack of statistical rigor, we are opting to relegate Figure 5 (the germination figure) to the supplementary materials Figure A4. Additionally, we state this limitation in the main text (Lines 414 and 525).

Point 3:
(3) Some minor language issue should be adjusted.

Thank you for the suggestion. We have revised our language in multiple areas to be generally more clear and scrupulous with scientific claims, especially in areas that were specifically requested by the reviewer.

Addressing minor comments:

Point 4:
(4) Line 12-14: "Peppers (Capsicum spp.) are unique among the plant kingdom for producing capsaicin, whose antibiotic property presents an additional selective pressure..."
The claim is overstated that “Peppers are not unique; other plants in the Solanaceae produce capsaicinoid analogs”. Sentence may be improved.

Example: “Capsicum species are notable for producing capsaicin, a compound with antimicrobial activity that may influence microbial communities associated with pepper fruits and seeds.”

Thank you for the suggestion. We agree that the example provided by the reviewer is a better statement and more appropriate for the manuscript. We have replaced our line in the Abstract with the provided example (Lines 12-14).

Point 5:
(5) Line 18-20: "Minimum inhibitory concentration (MIC) assays showed that jalapeno and habanero peppers do not host bacterial communities that are more capsaicin-resistant, compared to the bacterial communities from bell pepper."

Thank you, this is a good point for revision. We have made the requested change (Lines 17-19).

Point 6:
(6) Lines 34-40: “An important perspective in the discourse surrounding sustainable agriculture is the need to minimize the use of synthetic agrochemicals...”

The phrasing “An important perspective in the discourse” adds unnecessary notion. Revise sentence as:

Example: “Sustainable agriculture aims to reduce reliance on synthetic fertilizers and pesticides, which are energy-intensive to produce and contribute to greenhouse gas emissions.”

We agree that this is more efficient phrasing. We have condensed our first two sentences in the Introduction and replaced them with the example offered by the reviewer (Lines 33-35).

Point 7:
(7) Lines 69-75: The development of antibiotic resistance is thought to be linked to reduced overall fitness... any bacteria with capsaicin resistance should have a lifestyle more closely associated with the host pepper...”

This assumes direct seed exposure to capsaicin, which is later questioned in the Discussion. Simplify the sentence as:

Thank you for the suggestion. We have revised the last paragraph in our Introduction to include the example offered by the reviewer (Lines 73-76).

Point 8:
(8) Lines 81-83:“Ripe commercial bell, jalapeno, and habanero peppers were purchased … stored at 4°C for up to three days.”

Source detail is not clear. Was the produce organic/conventional? Did different storage times occur? These factors can influence microbial community composition. Clarify selection and storage consistency.

Example: “Ripe, conventionally grown bell, jalapeño, and habanero peppers were obtained from a single commercial supplier. All fruits were processed within 72 hours of purchase and stored at 4 °C under identical conditions to minimize variation.”

Thank you for the suggestion. All fruits were obtained from the same supplier and were stored in identical conditions, which the reviewer’s example accurately represents. We have replaced the first paragraph in the Materials and Methods to elucidate the nature of the produce and the storage times and conditions (Lines 83-86).

Point 9:
(9) Lines 249-270 (Plant inoculation design): No indication of soil sterilization confirmation. Seeds soaked only 10 min may not allow adequate colonization. Magenta box environmental conditions lack light intensity / humidity description

Add environmental growth conditions and colonization verification detail.

We have added a few sentences to describe our protocols to confirm both successful soil sterilization and successful bacterial inoculation (Lines 282-287). We refrained from including a humidity measurement because of difficulty introducing a humidity sensor to the magenta boxes without causing contamination. We have added a description of the light intensity in Line 280.

Point 10:
10) Lines 304-310: (Number of isolates): “In total, 20 morphologically unique bacteria were isolated…………”

The text indicates community-level diversity, but only culturables were sampled. State clearly that this refers only to culturable isolates.

Thank you for this suggestion to improve our text clarity. We changed our Discussion text from “Each community of culturable bacteria…” to “Each group of culturable bacteria…” (Line 441). We are having difficulty locating other lines where we suggest community-level diversity; if any remain, please let us know and we will address them.

Point 11:
(11) Lines 432-447: “Our broth microdilution test… does not support the idea that bacteria isolated from peppers of different pungency levels will have different levels of resistance to capsaicin.”

This section correctly states the result but does not adequately acknowledge anatomical constraints (capsaicin localized in placenta, not seed). Clarify limited microbial exposure.

We agree that this example is a useful addition to our discussion. We have thus implemented it in Lines 473-476. Thank you for the example.

Point 12:
(12) Lines 535-537: “Despite this, we isolated two growth-beneficial Pseudomonas sp.… that solubilized phosphates and produced IAA and showed that these isolates promoted growth…”

This sentence is too long and compresses multiple key findings into a single sentence. It is difficult to read.Split into two concise statements.

Example: “However, several isolates demonstrated strong plant growth–promoting traits, including IAA production and phosphate solubilization. In particular, two Pseudomonas isolates significantly enhanced seedling growth in both Trifolium pratense and Poa pratensis.”

Thank you, we agree with this change and have implemented it in Lines 554-557.

Point 13:

(13) Lines 536-538 “…meaning biostimulants can work on different plants.”

Thank you for the rephrasing, we believe this is a better way to conclude the paper and have implemented it in Lines 557-559.

Reviewer 3 Report

Comments and Suggestions for Authors

In the present study, the authors explore whether capsaicin acts as a selective factor influencing the recovery of plant growth–promoting bacteria (PGPB) from chili fruits and seeds. Once isolated, the bacterial strains were evaluated for their plant growth–promoting traits and subsequently inoculated in Trifolium pratense and Poa pratensis seedlings, yielding encouraging results for their potential application as bioinoculants.

Dear Authors,

Below are a series of comments that may help strengthen your manuscript:

Title and Scope

The title would be more appropriate if the study had clearly demonstrated that capsaicin itself exerts a measurable effect on the microbial composition or the growth-promoting capabilities of the isolated strains. However, this relationship is not clearly supported by the data presented, as also acknowledged in lines 471–475.

Materials and Methods

Section 2.3.2

It is important to specify the origin of the chili fruits used for bacterial isolation. Were they purchased from a local market? Collected directly from the field? Where were they cultivated, and which varieties were used?

Section 2.5. Identification of Bacteria through 16S rRNA Sequencing

The 16S rRNA gene analysis only allows the identification of bacteria at the genus level and, in some cases, the tentative species level. It is strongly recommended to include a phylogenetic tree (dendrogram) comparing the isolates with closely related reference strains obtained from BLAST analyses. Reporting BLAST results alone is not sufficient evidence of taxonomic identity.

Section 2.7 (Line 264)

Please mention the use of peat first and then specify the brand name.

Results

The presentation of results is somewhat confusing and difficult to follow. You report bacterial isolates obtained from media with different capsaicin concentrations; however, Table X combines “capsaicin concentration at maximum inhibition” with bacterial growth-promoting traits. These datasets should be presented in separate sections.

Initially, describe the bacteria recovered and the capsaicin concentrations of the media from which they were isolated. In a subsequent section, present their growth-promotion characteristics. The current layout blends unrelated data and reduces clarity.

Section 3.2

Include a subsection specifically for taxonomic assignment, where the phylogenetic tree and related descriptions are presented.

Ensure that all scientific names throughout the manuscript are italicized.

Section 3.5

Since the bacterial strains were isolated from chili (Capsicum spp.), it would have been logical to test them first on chili plants. The rationale for using other plant species should be more clearly justified.

Table A2 should appear in the main results section rather than the supplementary materials, as tomato and mint bacteria are not mentioned again elsewhere. The manuscript would gain additional value by comparing the plant growth–promoting effects between chili-derived isolates and heterologous (mint/tomato) isolates.

Overall, the results section should be rewritten in a more systematic and structured manner, aligning the order of experiments with the methods described and clearly distinguishing the outcomes of each assay.

Discussion

Lines 434–438

To assert that strain Hs1 can use capsaicin as a nutrient source, at least one culture assay on water agar supplemented exclusively with capsaicin should have been conducted. Without such evidence, it is preferable to state that capsaicin stimulates bacterial growth rather than serving as a nutrient source.

This reasoning further supports the suggestion that Table A2 should not be relegated to the supplementary section.

Additionally, the justification for using T. pratense and P. pratensis as test plants should be strengthened, especially given that the isolates originate from chili.

You might also consider alternative seed germination assays that minimize substrate interference. For instance:

Calvillo et al., 2023. Germination test for the evaluation of plant-growth promoting microorganisms. Journal of Microbiological Methods, 207:106708.

https://doi.org/10.1016/j.mimet.2023.106708

Author Response

Comments from Reviewer 3

Point 1:
Title and Scope

Thank you for the suggestion, we agree that the title should be changed to better reflect our findings. We are proposing a change to our working title to “Evaluation of capsaicin as a selector for growth promotional bacteria isolated from Capsicum peppers”.

Point 2:
Materials and Methods

Section 2.3.2

Thank you, we have added more detail about the origin of the fruits in Lines 83-86, including the fact they were conventionally grown (not organic), and purchased from a single commercial supplier. Unfortunately, our supplier contact could not provide additional details about the exact cultivation methods and exact varieties, beyond that the fruits were bell pepper, jalapeno, and habanero. Due to this limitation, we saw the need to perform HPLC-UV to confirm capsaicin levels for each fruit type, which we detail in our manuscript.

Point 3:
Section 2.5. Identification of Bacteria through 16S rRNA Sequencing

We agree with this change. We have added a paragraph to our Materials and Methods 2.5 (Lines 231-236) detailing our methods for creating a maximum likelihood phylogenetic tree for our four best bacterial isolates. Thank you very much for the suggestion.

Point 4:
Section 2.7 (Line 264)

Please mention the use of peat first and then specify the brand name.

Thank you, we have revised Lines 275-276 as requested.

Point 5:

Results

Thank you for the suggestion, we agree that the results can be presented more clearly. To that end, we added a new Table 2 which details the isolates recovered, the capsaicin concentration of the media they were recovered in, and the capsaicin concentration at maximum inhibition (Line 325).

Point 6:

Section 3.2

Include a subsection specifically for taxonomic assignment, where the phylogenetic tree and related descriptions are presented.

Ensure that all scientific names throughout the manuscript are italicized.

Thank you for the suggestion. We have added a paragraph to our Materials and Methods 2.5 (Lines 231-236) detailing our phylogenetic tree construction. We have also added a paragraph to our Results 3.4, detailing the results of our taxonomic assignment and phylogenetic tree (Lines 368-371). We have also added a new Figure 3, which are the phylogenetic trees for each Jf1, Jf2, Jf3, and Hs7 (Lines 373-375).

Point 7:

Section 3.5

Thank you for the suggestion. We have clarified our rationale for using Trifolium pratense and Poa pratensis in Results 3.5 (Line 390) as well as in our Materials and Methods 2.7 (Lines 260-263). Additionally, we have moved Table A2 into the main text (Line 332) and renamed it as Table 3.

While we appreciate the value of comparing growth-promoting effects between chili-derived isolates and those derived from mint/tomato, we decided not to pursue this as we recovered only a few heterologous isolates, which may not be sufficient for statistical comparisons to be made.

We have reorganized the Results section in a more systematic manner, moving our capsaicin results to the forefront, followed by our growth promotion testing, then our selection of best strains and subsequent additional tests and inoculation experiments. Thank you for the suggestion.

Point 8:

Discussion

Lines 434–438

Thank you, we agree this is a better representation of our findings. We have changed our sentence in Line 451-452 as such.

Point 9:

This reasoning further supports the suggestion that Table A2 should not be relegated to the supplementary section.

Thank you, we agree with this change. We have moved Table A2 into the main text and relabelled it as Table 3 (Line 332).

Point 10:

Additionally, the justification for using T. pratense and P. pratensis as test plants should be strengthened, especially given that the isolates originate from chili.

Thank you, we have reclarified our justification in both the Materials and Methods (Lines 260-263), Results (Line 390), and Discussion sections (Lines 518-519).

Point 11:

You might also consider alternative seed germination assays that minimize substrate interference. For instance:

Calvillo et al., 2023. Germination test for the evaluation of plant-growth promoting microorganisms. Journal of Microbiological Methods, 207:106708.

https://doi.org/10.1016/j.mimet.2023.106708

Thank you very much for the recommendation and the attached reference. Since we simply reported total germination at the end of the experiment, and did not statistically test these results as Calvillo et al. (2023) recommended, we are opting to acknowledge this limitation in our main text (Lines 414 and 525). Additionally, we are opting to move Figure 5 (the germination figure) to supplementary Figure A4, due to the lack of statistical testing.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I have now carefully examined the authors’ responses to the reviewers’ comments. In my assessment, the revisions satisfactorily address the concerns previously raised, and the manuscript can be considered suitable for publication.