Review Reports

Comment #1

It would be interesting to know the wheat production in the region or territory for which the research is being conducted. What impact on production is attributed to the aforementioned diseases/pathogens? Or what the problem is (data) regarding the levels of toxins these pathogens produce in wheat? With these data, the relevance of the research would be better demonstrated.

Response #1

Some information and relevant references added in the revised version.

Comment #2

Row 90. указать производителя и место производства. It must be translated into English.

Response #2

Corrected.

Comment #3

Row 110. Further details are needed. In what volume was the consortium suspension diluted for seed application? In what volume was the consortium suspension diluted for foliar application in plants?

Response #3

The relevant information added in the appropriate place: “To make a working solution, thus produced bacterial concentrate was diluted with water in a ratio of 2:8, and this suspension was applied at the rate of 2 liters per a ton of seeds or per hectare for foliar applications”.

Comment #4

Row 395 – 396. Rh. arrhizus. Change to R. arrhizus.

Response #4

Corrected.

Comment #5

The discussion is not adequately structured; it was basically presented as "duplicate results," with no scientific explanation for these results.

Response #5

We are afraid we cannot fathom how to discuss results without mentioning the latter in some way: otherwise how a reader can understand what is discussed. In this section we used sometimes the wording like “The finding that… ” , and discuss the mentioned aspect which we deem important. As a minimum we believe we manage to place our results within the relevant framework.

Comment #6

… is the exposure of seeds during treatment with the bacterial consortium sufficient to change the soil microbiota (fungi/bacteria) in a single application? Why would this be the case? If it is stated that bacteria with fungicidal activity were used, were we expected to find more changes in the mycobiome than in the bacteriome?

Response #6

May be sufficient, may be not: nobody can say for sure, as it depends on many factors shaping the context of any study. We did not have any certain opinion on the issue, but wanted to check if such could be the case.

Comment #7

… it is reported that the Phylum Ascomycota, where Fusarium belongs, was not affected using fungicides. Background information indicates that species of this genus are responsible for the main problems in wheat, one of the reasons why the study was conducted. How could these results be interpreted/explained?

Response #7

The Ascomycota phylum, accounting for half of the fungal sequence reads, is the ultimate keystone phylum in this study (as well as in many other soil mycobiome studies) and it could by no means be expected to change its relative abundance in the rhizosphere soil due to fungicide application to seeds and leaves. If anywhere, the phylum relative abundance in the internal plant environment, i.e. inside grain, leaves, roots etc. might have been affected. For instance,  (where Ascomycota can also be dominating, like in tomato roots, but this an avenue for further more detailed research.

Comment #8

Furthermore, considering the relative abundance data of F. oxysporum and Gibberella sp. and the favorable results on grain productivity and quality reported. In this sense, what is the reason for the average 24% increase in yield with the application of bacterial fungicides? What properties of the microorganisms in the biofungicide could have influenced this?

Response #8

The increased grain yield might have resulted from the plant-growth promoting properties of P. allii, but most likely from the compounds, produced by all bacterial components, disrupting some essential biochemical processes like expression of keystone genes, for instance. As we stated in the reviewed manuscript, “An antifungal mechanism of the agents, comprising the novel fungicide tested in this study, should be investigated in detail”.

Comment #9

In the introduction it is stated as an objective …“We decided to test the efficacy and ecological safety of a novel biological fungicide preparation, composed of the endophytic microorganisms, isolated from natural environments in the Kuzbass”… However, no conclusive data were found on its efficacy and ecological safety. What does this mean?

Response #9

We believe the fact that grain yield was increased under the novel fungicide treatment is a prove about the efficacy of the fungicide. As for the ecological safety, as a multifaceted notion we did not address it and did not make any conclusions in this respect, though implicitly one can judge about it from the fact that the bacteria constituting the novel preparation, were isolated from the local soils and that during and after the fungicide application no environmental contamination and hazards for human and ecosystem health were revealed. However, as we did not address the issue in the manuscript, we prefer to remove mentioning it at all in the revised version.

Comment #10

In this regard, did the authors consider that P. allii is an onion pathogen when proposing it as part of the bacterial fungicide formulation? What advantages would there be in using P. allii over the commercial fungicide based on Bacillus subtillis?

Response #10

Being an onion pathogen does not necessarily suggest being a wheat pathogen as well, bacterial functionality often being shaped by environmental context. Many microorganisms are opportunistic pathogens, being at the same time considered to be plant growth promoters. One of the advantages of the novel preparation, if produced locally, might be its lower cost and relative independence from the all-country market fluctuations.

Comment #11

It is considered that a diagnosis of the phytopathogenic fungi of interest or their toxins could have provided more conclusive information.

Response 11

You are right, and we believe that our omission of doing so is a drawback in the study, but right now it could not be helped. However, in the revised manuscript we added some text to the following effect:  “Prior to starting the experiment, the wheat seed infection rate with fungi was estimated; however, specific fungal species or toxins were not determined, and after the experiment as well. This may be regarded as a shortcoming of the study, preventing a more substantiated conclusion about the mechanism of the fungicidal action in the field, albeit the study was not aimed at elucidating the mechanism per se”.

Comment #12

As a general rule, when the scientific name of an organism is written for the first time in a document, it is not necessary to rewrite it in full in subsequent texts. Example: Bacillus subtilis - B. subtilis.

Response #12

Corrected in the Results and Discussion section, leaving the full names in the Abstract, M&M, tables and Conclusion for clarity.

Comment #13

Use only one type of nomenclature to write the probability value. It is more common to write it as p ≤ 0.05.

Response #13

The probability value may be indicted as we did, that is the exact value received by performing a statistical test, or simply a comparison with a certain level of probability value is provided. We always try to show the exact probability values as in such a case a reader themselves can judge about the probability that a statistical summary of the data (the sample mean difference between two compared groups) would be equal

to or more extreme than its observed value (Wasserstein & Lazar, 2016). Comparing with a certain p-level does not provide such opportunity, and therefore is less informative. Besides, one should not forget that «There is nothing mathematically magic about 0.05, it was chosen rather arbitrarily during the early days of statistics; people could have agreed upon 0.04, or 0.025, or 0.071 as the conventional significance level» (McDonald, 2014).

References

Wasserstein R. L. & Lazar N. A. (2016) The ASA Statement on p-Values: Context, Process, and Purpose, The American Statistician, 70:2, 129-133, DOI: 10.1080/00031305.2016.1154108

McDonald J.H. Handbook of Biological Statistics (3rd ed.). Sparky House Publishing, Baltimore, Maryland, 2014, p. 16-23. (https://www.biostathandbook.com/index.html)

Comment #1

There is a mention in the manuscript of the use of “saline-alkaline stress conditions,” but it does not specify the salt composition or concentration used in treatments. This is essential for reproducibility. Kindly provide the missing relevant details.

Response #1

Unfortunately, we did not manage to find any mention of the phrase “saline-alkaline stress conditions” in the manuscript file you reviewed, and otherwise there is no mention of salt and alkaline stress conditions in the manuscript. The study was performed on the rather common agricultural Chernozem, as stated in M&M section.   Therefore, we could not undertake any measures.

Comment #2

Consider mentioning in the manuscript (preferably in the Methods section) how control and treatment groups were established—particularly whether controls included only growth medium or a non-inoculated soil/plant setup. This will add clarity and weightage to the study.

Response #2

The fungicides, used in the study, were applied at the rate of 2 liters of suspension per a ton of seeds or per hectare for foliar applications, as is indicated in the M&M section. However, as you rightfully noted, we did not explicitly specified how we arranged the control treatment: we added that appropriate info “the control treatment received the equal amount of water instead of suspensions”, highlighted in green in the revised version of the manuscript.

Comment #3

The type of statistical test to assess the data obtained in the study (ANOVA, t-test, etc.) is not explicitly mentioned in the Methods. Include details about the software and post-hoc tests used.

Response #3

The Lines 180-184 in the manuscript you revised contain the following text: ”Statistical analyses (descriptive statistics, ANOVA, PCA, ANOSIM and PER-MANOVA) were performed by using Statistica v.13.3 (TIBCO Software Inc., Palo Alto, CA, USA) and PAST [21] software packages. Post-hoc comparisons of differences between treatment means (by Fisher’s LSD test) were considered statistically significant at the p ≤ 0.05 level.

Comment #4

The Abstract consists of statements regarding the novel fungicide increased yield comparably to commercial products; however, later on, the Discussion (Section 4.1) states that this effect did not translate into improved grain quality. This should be reflected consistently across both sections to avoid confusion and misinterpretation.

Response #4

Corrected by adding the following phrase in Abstract: “without affecting its quality”.

Comment #5

The Abstract includes statements regarding no effect of application mode, but Results (Section 3.3, Figure 5) mention PERMANOVA p = 0.042 and 0.015, indicating significant differences among certain treatments. Please clarify this contradiction.

Response #5

Indeed, the Abstract contains such statement. There is no contradiction, as we meant the relative abundance of individual taxa, rather than multivariately. In order to respect the Journal rule of no more than 200 words in Abstract, we decided to remove the words “and rhizosphere microbiome”.

Comment #6

The fungicide treatments (F0–F3, F3(1), F3(2)) are used in Results and Tables; however, they are not clearly defined earlier in the Methods section. Each treatment combination (seed-only, seed + 1 foliar, etc.) should be explicitly described with dosages for ease of understanding and flow.

Response #6

The Methods section contains the following text concerning the treatments: “There were six different treatments in the experiment: without any fungicide (F0), with the chemical fungicide (F1), with the biological fungicide (F2) and with the novel preparation (F3), the latter with three modes of application: seeds treatment only (F3(0)), seeds treatment followed with one foliar application (F3(1)) and seeds only followed by two foliar applications at the panicle-producing stage with 12-days interval, F3(2).” After this text, we added a text, describing the dosages in subsection 2.2, as well; also highlighted in green.

Comment #7

Mention important details, such as environmental conditions (soil type, weather during field trial), which are missing from the manuscript text. These influence microbiome composition and yield and should be added to add value to the study.

Response #7

We absolutely agree with you, and added the relevant information, highlighted in green in the revised version.

Comment #8

The manuscript claims sequencing data are deposited (BioProject PRJNA1320511); however, the accession appears incomplete or inaccessible — confirm submission and consider adding full SRA link.

Response #8

We checked this link to this bioproject, provided in the manuscript, and it works OK. But before there were problems (not related to us)

Comment #9

Rarefaction curves are described as “not shown.” For transparency, include them as supplementary figures.

Response #9

We are afraid we do not agree with you here, as addition the graphs will add nothing essential to the statement made in the manuscript.

Comment #10

The Methods mention OTU analysis via UPARSE but omit thresholds for sequence quality filtering or chimera removal — this needs to be specified for better understanding.

Response #10

The recommended information added and is highlighted in green in the revised version.

Comment #11

The Results section uses letter superscripts (e.g., a, b, c) in Table 2 for significance. However, these letters need to be specified as to which factor (ANOVA + LSD test) they refer to. Add an explanatory note under each table in the footnote.

Response #11

The footnote for Table 2 specifies that “Different letters in rows indicate statistically significant differences (P≤0.05) by Fisher’s LSD test”.  This is the pairwise comparison between all the values in a row.

Comment #12

Section 4.1 acknowledges no difference in grain quality between treatments, yet provides a statement regarding overall positive performance. This is over-generalized and should be toned down to match the data.

Response #12

We are afraid we are not with you on the issue. The phrases we used in this section (like “was similar to”, “did not differ from…”, “the novel fungicide did not seem to provide any gains…”, “possibly had somewhat lower quality”) already suggested rather toned down discussion of the results, did not they?

Comment #13

The Discussion speculates that microbial shifts are “random” or “difficult to interpret.” A more evidence-based explanation or literature support (e.g., known roles of Mortierella, Gibberella) should be included.

Response #13

We indicated in the manuscript that the Gibberella genus contains both pathogenic and saprophytic members, and thus its changes abundance cannot be unambiguously interpreted; we substituted “difficult to interpret” with “cannot be unambiguously interpreted”. We also added some speculation about Mortierella (lines 443-452 in the revised manuscript, also highlighted in green), which, frankly speaking, does not help much with difficulty in interpretation.

Comment #14

The Authors mention “good prospects as a fungicide,” but since strains were not detected in the rhizosphere, field persistence and mechanism of action remain unproven. Recommend a more cautious conclusion or support the good prospects as a fungicide.

Response #14

The aim of the study was to test the novel preparation in the filed for grain yield and rhizosphere microbiome composition and diversity. At this stage we were far from being as ambitious as to elucidate the exact mechanism of the fungicidal effect; the more so since the conclusion about the effect is done indirectly, based on the increased grain yield, not on the fact that a certain fungus was eliminated after a certain activity of the agents from the novel preparation.

Comment #15

Define all abbreviations (F0–F3, F3(1), F3(2)) in each caption in the footnote to the caption along with other abbreviations (if present).

Response #15

Done as recommended and highlighted in green.

Comment #16

Units are missing in several tables (e.g., Table 2 yields t ha⁻¹ is fine, but grain density g L⁻¹ lacks decimals or error). Kindly check once. PCA and PERMANOVA plots lack axis variance percentages — add them to avoid confusion and for clarity.

Response #16

The number of decimals in Table 2 depends on the accuracy of the analyses, therefore grain density is shown as integers, whereas ash or fiber content are shown with two decimals.

Comment #17

Several references are outdated or lack some information, such as missing DOI in a few references and inconsistent journal formatting—align with Applied Microbiology style. Additionally, some background statements in the Introduction (e.g., pesticide persistence, microbial diversity effects) need additional or recent supporting citations to support these claims.

Response #17

We carefully checked the reference list in the manuscript, and are afraid we did not understand what you mean by “outdated”? We believe the first report of the fact should be mentioned, should it not?

As for the DOI and formatting, we corrected where needed, but not all cited publications have one, that is why DOI is missing (two articles).

Comment #1

When I mentioned that the 'p' in the probability value was not written correctly, I was specifically referring to the fact that the letter 'p' was found in the document written inconsistently with both uppercase and lowercase letters. I apologize for the lack of clarity.

Response #2

We are sorry for inadequate response to that comment. We checked the manuscript for consistency in spelling the “p”-value, and corrected where needed. The changes are highlighted in turquoise.

Thank you very much for your review!