Impact of a Synthetic Microbial Community on Salvia officinalis Under Optimal and Reduced Irrigation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript horticulturae-3530962 describes the results of an experiment on changes in the morphological and biochemical parameters of sage plants under the influence of mild water deficit and the effect of inoculation with a mixture of rhizosphere bacteria on these changes. The authors conducted a fairly large-scale experiment and presented many results. However, I find that there are fragments in the manuscript that the authors should correct and/or clarify.

For example, under what conditions (temperature, light, humidity) were sage plants cultivated in 10-liter pots for 28 days? And to what extent (in the authors' opinion) do these conditions correspond to natural or agrobiotechnological conditions for cultivating sage? This should be clarified, since one of the authors' conclusions is that under some conditions metabolites undesirable for humans (such as histamine) may appear.

Another shortcoming of the manuscript (in my opinion) is that the authors did not demonstrate the interaction of the microbes they used with the sage plants by any method. Since the plants were treated with bacterial cells and exometabolites obtained from a bacterial culture on LB medium with tryptophan, and the plants treated with water served as a control, the “inoculation” effects observed by the authors may be associated with the components of the medium, not the bacteria.

Minor remarks:

Line 173 and Figure 1c: On what basis did the authors conclude that bacterial IAA (indole acetic acid) was produced, if “indole auxins” were determined?

Line 192: Provide more information about the seeds used (source, manufacturer, possibly variety, line or form of plants).

Line 207: Provide a reference to information that the conditions used were optimal for sage.

Line 220: Why was tryptophan added to the LB medium? Strain A12 did not produce indols.

Lines 317-318: Why did the authors use a t-test? The experiment clearly separated two factors: "drought" and "inoculation". Accordingly, a two-way ANOVA with an assessment of the effect of each factor should have been used.

Table 1: Please provide the additional accession numbers for the nucleotide sequences of the five strains obtained in this work. Please provide the percent identity values ​​to the nearest hundredth. Why are the strains not the type strains used as comparison strains, which would allow a correct assessment of the taxonomic position of the bacteria studied?

Are strains A10, A12, A13, A27, and A29 deposited in any microbial collection? This would significantly increase the scientific significance of this publication.

Line 349: Why is there a reference to Figure 3?

Tables 2 and 3: Because the authors used a t-test instead of ANOVA for most of the variant pairs, there is no information on the statistical analysis. As a result, the t-test data are provided for the DNI vs CNI and CI vs CNI pairs, while the Relative Variation (%) data are provided for the DI vs CI pair, for which there is no t-test data.

Line 495 and Figure 4: The authors identified the compound “Penicillin G” among the metabolites of uninoculated plants (both CI and DNI), but the possible origin of this compound in plants and its importance in plant metabolism are not discussed further.

Author Response

Comment 1: Under what conditions (temperature, light, humidity) were sage plants cultivated in 10-liter pots for 28 days? And to what extent (in the authors' opinion) do these conditions correspond to natural or agrobiotechnological conditions for cultivating sage? This should be clarified, since one of the authors' conclusions is that under some conditions metabolites undesirable for humans (such as histamine) may appear.

Response 1: We provided information about the growth conditions as requested by the reviewer (lines 219-226 in the revised manuscript). These conditions are similar to those used in earlier studies for the indoor cultivation of sage, aiming to demonstrate the effect of reduced water supply compared to well-watered controls. As discussed in lines 102-106, a decrease in water supply has been suggested to enhance oil content in medicinal plants, showing a positive effect on the yield observed in certain aromatic species. While we recognize that our controlled conditions do not entirely replicate natural or agrobiotechnological environments, they offered a necessary framework to investigate the specific research question regarding PGPR efficacy under water stress in sage.

Comment 2: Another shortcoming of the manuscript (in my opinion) is that the authors did not demonstrate the interaction of the microbes they used with the sage plants by any method. Since the plants were treated with bacterial cells and exometabolites obtained from a bacterial culture on LB medium with tryptophan, and the plants treated with water served as a control, the “inoculation” effects observed by the authors may be associated with the components of the medium, not the bacteria.

Response 2: In addressing the reviewer's concern regarding potential effects from the components of the LB medium rather than the bacterial cells or their metabolites, it is essential to consider the microbiological context of our experimental design. A bacterial culture grown for 18 hours in a rich medium like LB, under controlled conditions, reaches a stationary phase, effectively depleting the available nutrients of the medium. Consequently, any residual components of the medium in the applied solution would be present at very low concentrations and significantly altered from their original composition. Additionally, the spent medium was diluted to achieve the appropriate cell density used for inoculation (8 × 10⁸ CFU ml^-1), further reducing the concentration of the residual medium components. Furthermore, the spent medium contains exometabolites produced by the bacteria; thus, its composition differs significantly from that of the original LB medium. Previous laboratory trials utilizing LB medium and tryptophan together or separately as controls consistently demonstrated that neither of these components induced shoot rooting in pear (Luziatelli et al., 2020; doi: 10.3389/fmicb.2020.539359) or an increase in tomato yield or lycopene accumulation in tomato fruits (Youssef et al., 2021; doi: 10.26353/j.itahort/2021.1.8899) or any of the plant responses reported in this study. This accumulated experience strengthens our confidence that the effects reported in the manuscript are due to bacterial inoculation and its associated exometabolites rather than trace residues of the medium components.

Comment 3: Line 173 and Figure 1c: On what basis did the authors conclude that bacterial IAA (indole acetic acid) was produced, if “indole auxins” were determined?

Response 3: According to the reviewer's comment, we modified IAA into “indole auxins” in the text at lines 179-180 in the revised manuscript. We also added this information in Figure 1’s legend (line 185 in the revised manuscript).

Comment 4: Line 192: Provide more information about the seeds used (source, manufacturer, possibly variety, line or form of plants).

Response 4: We added information about the seeds used, as requested by the reviewer (lines 203-204 in the revised manuscript).

Comment 5: Line 207: Provide a reference to information that the conditions used were optimal for sage.

Response 5: In response to the Reviewer’s suggestion, we have incorporated relevant references regarding the cultivation conditions into the revised manuscript (lines 222).

Comment 6: Line 220: Why was tryptophan added to the LB medium? Strain A12 did not produce indols.

Response 6: Tryptophan plays a role in the biosynthesis of proteins and serves as a precursor and inducer for various aromatic compounds containing an indole ring, including IAA. We and others demonstrated that tryptophan is a precursor and an inducer of IAA, but that PGPR can produce several tryptophan derivatives. The presence of this amino acid in the culture medium positively impacts microbial growth, regardless of the strain's ability to produce IAA or other tryptophan derivatives. Furthermore, the literature indicates that LB contains a limited amount of this amino acid, and supplementing this medium with limiting factors improves microbial cell growth. The inability of strain A12 to produce IAA does not imply that this strain cannot utilize tryptophan or convert it into other indoles that the plant can then turn into IAA.

Comment 7: Lines 317-318: Why did the authors use a t-test? The experiment clearly separated two factors: "drought" and "inoculation". Accordingly, a two-way ANOVA with an assessment of the effect of each factor should have been used.

Response 7: Thank you for suggesting a two-way ANOVA to analyze the interaction between "inoculation" and "irrigation level." However, we did not utilize ANOVA, as our focus was not on examining the interaction between a problem (plant stress caused by reduced watering) and a potential solution (inoculation with PGPR to alleviate that stress). Our primary goal is to evaluate the effect of SynCom on sage grown under various watering regimes. Therefore, while the two-way ANOVA is informative, it is not the most direct method to address our specific hypotheses. Meanwhile, we agree with the reviewer on assessing whether the relative variations observed in plants inoculated and grown under reduced and optimal water supply conditions were statistically significant. To specifically evaluate the effect of inoculation at each irrigation level, as suggested by the reviewer, we conducted an additional t-test comparing the inoculation treatment under both well-irrigated and reduced-irrigation conditions (see revised Tables 2 and 3). This approach allowed us to directly assess the significance of the inoculation effect within each irrigation regime, aligning with our primary research focus. This revised analysis, along with the clarifications in the manuscript, addresses your concerns and provides a more focused and relevant analysis of our data.

Comment 8: Table 1: Please provide the additional accession numbers for the nucleotide sequences of the five strains obtained in this work. Please provide the percent identity values ​​to the nearest hundredth. Why are the strains not the type strains used as comparison strains, which would allow a correct assessment of the taxonomic position of the bacteria studied?

Response 8: The accession numbers are detailed in paragraph 2.3 (lines 199-201 of the revised manuscript), and the identity values are presented in Table 1 (line 368 in the revised manuscript).

Comment 9: Are strains A10, A12, A13, A27, and A29 deposited in any microbial collection? This would significantly increase the scientific significance of this publication.

Response 9: The deposition of the strains is ongoing and will be completed following publication.

Comment 10: Line 349: Why is there a reference to Figure 3?

Response 10: We removed the reference.

Comment 11: Tables 2 and 3: Because the authors used a t-test instead of ANOVA for most of the variant pairs, there is no information on the statistical analysis. As a result, the t-test data are provided for the DNI vs CNI and CI vs CNI pairs, while the Relative Variation (%) data are provided for the DI vs CI pair, for which there is no t-test data.

Response 11: We added the t-test for DI vs CI and modified the text according to the reviewer’s suggestions (please see Table 2 and Table 3, lines 414-422 and 447-457 of the revised manuscript).

Comment 12: Line 495 and Figure 4: The authors identified the compound “Penicillin G” among the metabolites of uninoculated plants (both CI and DNI), but the possible origin of this compound in plants and its importance in plant metabolism are not discussed further.

Response 12: We added a discussion related to penicillin G (please see lines 713-725 in the revised manuscript).

Reviewer 2 Report

Comments and Suggestions for Authors

Very interesting work related to a promising tool for the agriculture of the future. This is exactly the approach of synthetic microbial communities (SynComs) for a stable microbial community due to the synergistic interaction between its components.
The work should be published. But the question concerning the survival and communication of microorganisms of the synthetic community in the rhizosphere of plants is not disclosed. Are these rhizosphere microorganisms or endophytes?
It is believed that proline is an anti-stress amino acid, an osmolytic, but I did not see it mentioned in the work.

Author Response

Comment 1: Are these rhizosphere microorganisms or endophytes?

Response 1: The SynCom does not contain endophytes.

 

Comment 2: Proline: “I did not see it mentioned in the work”.

Response 2: The abundance of proline does not change in the leaf metabolome of both inoculated and non-inoculated plants and, therefore, was not explicitly discussed. We direct our attention to metabolites whose abundance was influenced by the treatments.

Reviewer 3 Report

Comments and Suggestions for Authors

The article investigates the impact of a synthetic microbial community on Salvia officinalis under both optimal and reduced irrigation conditions. The authors show that microbial inoculation influences plant drought responses, metabolism and biomass. The potential implications for food quality and safety are also discussed. The combined study on drought stress, synthetic microbial communities, and metabolomic profiling in S. officinalis makes this study interesting for readers and adds an aspect of novelty.

The introduction of the paper is well-structured and connects microbial interactions to drought resistance in plants. The aim of the study and research gap it tries to fill are clearly stated.

The Materials and Methods section is detailed and comprehensive. Some additional information is needed.

Line 124: Give geographical coordinates for this place.

Line 145: Give producers (name, country) for all media used in the manuscript.

Line 196: Add more growth conditions such as air humidity or photoperiod.

Line 197: Seedlings size or number of leaves?

Line 198: One plant was one replication?

Line 270: Add temperature and time of drying.

Results are clearly presented and easy to understand. The figure 3 could be better quality.

The discussion interprets the study’s findings, comparing it to the relevant literature and emphasizing the potential of synthetic microbial communities in drought resilience.

The conclusion effectively summarizes the key findings of the study and states their practical applications and future research directions.

The cited literature is adequate.

 

Author Response

Comment 1: Line 124: Give geographical coordinates for this place.

Response 1: We added the geographical coordinates (line 127 in the revised manuscript) in response to the reviewer's comment.

Comment 2: Line 145: Give producers (name, country) for all media used in the manuscript.

Response 2: We included the producers’ names, and the country of the media and chemicals used in this research (lines 173-176 in the revised manuscript) as requested by the reviewer.

Comment 3: Line 196: Add more growth conditions such as air humidity or photoperiod.

Response 3: We included the requested information about the growth conditions (lines 219-226 in the revised manuscript).

Comment 4: Line 197: Seedlings size or number of leaves?

Response 4: The information was included in the revised manuscript (line 209).

Comment 5: Line 198: One plant was one replication?

Response 5: Exactly. The information is detailed in line 210 of the revised manuscript.

Comment 6: Line 270: Add temperature and time of drying.

Response 6: We added the information (line 291-292-285 in the revised manuscript).

Comment 7: Results are clearly presented and easy to understand. The figure 3 could be better quality.

Response 7: We modified Figure 3.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors made several changes to the revised manuscript. And the manuscript is better now. However, I do not agree with some of the authors' answers.

The authors argue that when producing sage, it is necessary to consider the possibility of the formation of metabolites that pose a danger to humans. Yes, some metabolites are unsafe. However, the authors do not provide either the absolute values of potentially dangerous metabolites or the amount of plant material that can cause undesirable effects in humans. In addition, the authors do not discuss the fact that the manuscript describes a model experiment, the conditions of which do not correspond to the conditions of industrial sage cultivation. Accordingly, the qualitative and quantitative composition of sage metabolites will differ from that described in the manuscript.

I also disagree with the authors that after 18 hours of bacterial cultivation, the LB medium loses its nutritional value. The stationary phase of bacterial culture is caused by the accumulation of bacterial waste products. The increase in biomass continues in the stationary phase (up to 3–5 days) without increasing the number of living cells. And since the LB medium is a medium rich in nutrients, especially vitamins, even after culturing bacteria and diluting 20–50 times, it may contain substances that promote plant growth. The references provided in the authors' answer characterize only the specific reactions of certain plants.

The authors have supplemented the manuscript with the necessary results using the t-test. I still think that ANOVA is more suitable for the experiments described in the manuscript, in addition to the fact that ANOVA is a more informative method, and the results are more compact. However, the authors have the right to use the t-test, although it does not allow assessing the effect of SynCom as a whole for the entire experiment, but only in individual pairs of variants.

It is a pity that the authors did not deposit the strains before submitting the manuscript. If published, this will reduce the scientific value of the article.

The authors explain the presence of "Penicillin G" in non-inoculated plants (CNI and DNI) by the production of this metabolite by fungi. However, line 203 indicates that the plants were sterile and placed on a sterile substrate. How did fungi producing "Penicillin G" appear in the experimental system?

Lines 238-239: Describe in more detail how the OD was measured (device, light path length) and the CFU concentration was determined (medium, dilutions, culture conditions). The value 1.2 is not optimal for determining the optical density. Were dilutions used to determine the optical density of the original bacterial cell suspension?

Author Response

Comment 1: The authors do not provide either the absolute values of potentially dangerous metabolites or the amount of plant material that can cause undesirable effects in humans.

Response 1: This study did not aim to quantify biogenic amines. Untargeted metabolomics provides a relative comparison of metabolite levels, while alternative methods can yield absolute data on specific compounds. From this perspective, we observed that under mild drought conditions or following SymCom inoculation, the relative abundance of certain biogenic amines increased.

Our warning about the side effects of insufficient irrigation on sage, whether grown in industrial or sustainable agriculture, relates to the plant's biology. It is beyond our scope to recommend a safety control for the biogenic amine content in sage cultivation. Still, we agree with the Reviewer’s comment that “the qualitative and quantitative composition of sage metabolites can be affected by the cultivation conditions”.

To avoid any confusion on the scope of our work, we revised the manuscript and removed all references to safety implications.

Comment 2: The references provided in the authors' answer characterize only the specific reactions of certain plants.

Response 2: In light of the reviewer’s comment, we reanalyzed the literature on sage grown in soil conditions through a Scopus search. This search did not reveal any publications demonstrating a biostimulatory effect of vitamin solutions onSalvia species cultivated in soil. No evidence suggests that sage behaves differently from the plants we have previously studied.

Comment 3: “Since the LB medium is a medium rich in nutrients, especially vitamins, even after culturing bacteria and diluting 20–50 times, it may contain substances that promote plant growth.”

Response 3: Regarding concerns about the nutritional value of the spent LB medium, although we have not conducted a detailed analysis of the residual nutrients, we believe our experimental findings strongly support our conclusions. Firstly, and most importantly, our data demonstrate that we did not observe any promotion of plant growth. If the exhausted LB medium retained compounds that could stimulate plant growth, a biostimulatory effect should have been observed. Secondly, if residual nutrients (i.e., vitamins) are present in the diluted cell suspension after its application to the plants, these nutrients would likely be preferentially utilized by the bacteria, rendering them unavailable for plant uptake.Recognizing the difficulty in isolating the effects of residual nutrients from exometabolites, we will apply cells resuspended in an isotonic water solution in future experiments.

Comment 4: (ANOVA) However, the authors have the right to use the t-test

Response 4: As mentioned in the Round 1 responses, we believe that the t-test enabled us to directly evaluate the significance of the inoculation effect within each irrigation regime.

Comment 5: It is a pity that the authors did not deposit the strains before submitting the manuscript. If published, this will reduce the scientific value of the article.

Response 5: The deposited strains will become available to the scientific community following publication. Currently, they are not accessible. A deposited strain whose biological significance is supported by a publication is more relevant than an accession number in a culture collection.

Comment 6: How did fungi producing "Penicillin G" appear in the experimental system?

Response 6: The fungal contaminant was not intentionally introduced into the cultivation system; however, fungal spores are natural contaminants found in all environments that include human presence. It can be suggested that the dry heat sterilization failed to completely inactivate the heat-resistant fungal spores present in the soil. Additionally, it can also be hypothesized that contamination occurred accidentally during transplanting. In any case, the contamination level was low, and no macroscopically visible colonies or symptoms of fungal infection in the plants were observed.

Comment 7: How the OD was measured and the CFU concentration was determined.

Response 7: The information was added to the revised manuscript (lines 242-247).