Biocontrol Potential of Fungal and Oomycete Phytopathogens by Myxobacterial Strains

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting manuscript that brings new results to science and agricultural practice. The authors examined the in vitro inhibitory activity of 13 strains of myxobacteria against four strains (three species) of common crop pathogens. In pot experiments with cucumber seedlings, they examined the suitability of two myxobacterial strains for controlling Rhizoctonia solani. Furthermore, the survival of one myxobacterial strain in soil with different ratios of rabbit manure was evaluated. Overall, the results are clearly presented. However, it would be better to present the results consecutively, with figure numbers in brackets. Currently, each figure's content is indicated first. Furthermore, the authors provided the formula for calculating the percentage of inhibition. It would be helpful to present all the specific results of these calculations in a table in the Supplement. Currently, terms such as inhibition of above 75%, about 50% inhibition, inhibition between 50-60%, etc., are too often used. In the discussion and conclusion, the authors write that fast-growing fungi are resistant to the myxobacterial inhibitory effects, a finding they also achieved in the current study. The authors completely ignore the following fact: if a biocontrol agent is inoculated earlier (e.g., 7-14 days) and then a rapidly growing pathogen is inoculated, it is often very effectively inhibited. Such conditions can be created in many cases in agricultural practice (biocontrol agent colonizes the substrate earlier). This aspect should be treated more broadly in Discussion. Recently, much research in this area has been conducted on Hymenoscyphus fraxineus (ash decline) and biocontrol fungi. Numerous typographical errors were noted in the manuscript, which must be corrected before publication in Applied Microbiology (see Remarks).

Remarks

Line 25 Rhizoctonia – it should be italic

Line 45 Oomycetes – it should be italic

Line 11 ‘three different phytopathogenic fungi’; line 53 ‘on four common crop pathogens’ – this is an error that requires correction; the data must not be contradictory. The text on lines 78-81 should be included.

Line 62 Cystobacter fuscus….. – bacterial and fungal strains must be written in italic; this applies to the entire manuscript.

Line 64, line 69 – please note that the two strains you mentioned, Corallococcus coralloides and Myxococcus coralloides, are synonyms and cannot be listed in the current manuscript using two different names. I suggest that the first time they appear in the text, they be written as Corallococcus (Myxococcus) coralloides.

Line 64 should be deleted: ‘sp. nov.' – this is only used in publications describing a new species.

Line 116    ‘… 058, covering’

Line 134 in Table 1 – write "Variants/Treatments" rather than "Table."

Line 170 has different pathogenicity and growth rate – pathogenicity was not tested, so on what basis is such a statement made in the Results section?

Line 184-185 – consider revising this sentence.

Line 202, line 413 – it should be Petri dish

Line 206 – it should be (Fig. 2A),

Line 228   ??

Line 250: The text is not clear.

Line 271: Figure 8: ‘Letters A-F indicated’ – the question is, what does the letter G in this figure mean?

Line 283-293: Can the results described here be presented in a table to make the data more specific (were plants bigger in T3, T4?)?

Line 326: ‘In this investigation, the inhibitory effect of 13 myxobacterial strains belonging to four genera’ is incorrect; it should be ‘five genera’ (see lines 62-65).

Line 327: ‘was tested against four different plant pathogens.’ – this text is imprecise; it should be ‘was tested against four strains of three different plant pathogens’. Line 327 'Three of the pathogens were fungi', - requires clarification

Line 332 ‘Myxococcus xanthus’ – it should be M. xanthus

Line 342 'P. ultimatum', there is a mistake, it should be 'P. ultimum'

Line 348 'P. ultimatum', there is a mistake, it should be 'P. ultimum'

Line 368 'E. coli ' – it should be Escherichia coli

Author Response

 Response to comments of Reviewer #1

First, we thank the reviewer for his time and his important comments.

1. The reviewer asks that Fig. numbers be in brackets. We did this as much as possible.
2. The reviewer asks for data collected for the inhibitions to be presented in Suppl. As a Supplementary Table. We agreed and we did provide Table in the supplement as Table 1S. This table contains all the data in the Figures.
3. The reviewer has remarks about fast growing fungi- Yes, we found out that cultures like Rhizoctonia were resistant and in many cases as they were able to cross the barrier myxobacterial line, subsequently, the inhibition drops to zero. However, slow growing fungi like Sclerotinia were inhabited at higher rates. In our vivo trials, however, we added the pathogen first and then the biocontrol and still worked and effectively prevented symptoms. But it might not work if a higher amount of the Rhizoctonia was added to the soil or soil inoculated for several days earlier than inoculation of myxobacteria.
4. Answering the Remarks

Line 25 We made Rhizoctonia italic- Now in Line 26.

Line 45 Regarding making Oomycetes italic. We checked this point very carefully and found that Oomycetes is written without italic in Genbank and in scientific papers. Please check line 518 where cited reference is written without italic.

Line 11 - We changed from three to four in line 11.

Line 64, line 69 We changed and made Corallococcus (Myxococcus) coralloides.

Line 64 We deleted sp. nov.

Line 116 We removed extra space.

Line 134 In the table we changed the Table to Treatments.

Line 170 We removed different pathogenicity as suggested. Please see line 189. However, we added more about the pathogenicity of all four pathogens in the introduction (Lines 30-43).

Line 184-185 As suggested we changed the sentence, and we changed the name the strain number that gave 50% inhibition.  Please see lines 203 and 204.

Line 202 and line 413 We changed petri dish to Petri dish. We also checked the whole document for this error.

Line 206 We changed to Fig.4A, and this is the correct Figure number.

Line 228?? Changed spacing.

Line 250 Text changed.

Line 271 changed to include the letter G. Please see line 289. Thank you.

Lines 283-293- We agreed that a Table was needed, and we included Table 2 that shows inhibition after two weeks. We considered the mycobacterial strains as highly inhibitory only if they were highly effective against all the four pathogens and we considered them low inhibitory only when they had low inhibition against all the four pathogens.

Line 326- I find this sentence near Line 337. We had two names for ATCC 53080 Polyanion brachysporum and Schlegelella brevitalea. The culture was received as Polyanion brachysporum but later it was changed to Schlegelella brevitalea. Because this may cause some confusion, we decided to remove the new name and it and go only with the name that we received. With this change the number of genera is four including Corallococcus. The genera are Cystobacter, Myxococcus Polyanion, and Corallococcus.

Line 327 We changed to four strains and to three species of plant pathogens as suggested.

We also changed fungi to fungal species.  Please see line 368.

Lines 332, 342, 348 and 368 were changed as suggested by the reviewer. Please see lines 373, 384, 390.

 

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors!

Thank you for submitting your article for review. It is devoted to the study of the influence of myxobacteria on the viability of phytopathogenic fungi and bacteria in vitro. Classical microbiological methods were used in the work. The opinion about the work is greatly spoiled by the quality of the article design, and especially the graphical presentation of the results. The article is oversaturated with poorly made graphs. An interesting result in the article is the protective role of myxobacteria when introduced into the cultivation environment of plants infected with phytopathogens.

There are a number of comments and recommendations:

1. Key words should not duplicate words from the title of the article.
2. In the Introduction, I propose to add a short description of the harmfulness of the studied phytopathogens.
3. In the Materials and Methods chapter, there is no paragraph devoted to statistical processing of the results. It is not stated how many biological replicates of the experiments were conducted.
4. In the Results section, I propose to introduce paragraphs devoted to individual sections of the study. In its current form, everything is merged and difficult to navigate.
5. I did not like the statistical processing of the results presented in all the figures. In its current form, it is difficult to assess the reliability of the results. In addition, the graphs are designed carelessly. Often, the lines on the graphs are marked with colors close in shades, which makes it difficult to identify the lines by variants. I recommend that the authors think about presenting their data; perhaps they should be presented as a table with appropriate statistical processing (especially Figure 1). In addition, the graphs are not constructed uniformly. For example, sometimes the graphs start on the X-axis from zero, and sometimes from day 1. I believe that the authors should always have carried out the corresponding measurements at day 0, at the beginning of the experiment.
6. In the figures where Petri dishes are shown, it is better to first present the control dishes, and then the experimental ones. The authors have it the other way around.
7. In Figure 7, it is necessary to align the dishes and make them on the same level. Use appropriate graphic editors for this.
8. When analyzing the results of the studies, it is unclear why the authors present data for exactly 14 days of research. What is the reason for this particular observation period? Is it too long? This point should be explained. Often, on the 14th day, inhibition was reduced to zero, what is the reason for this?
9. The list of references should be formatted according to the rules of the journal.

After a revision, the article can be successfully published in the journal "Appl. Microbiol."

Respectfully Yours, reviewer.

July 18, 2025

Author Response

Response to Reviewer # 2 comments:

First, we thank the reviewer for his time and the important comments.

 

We agree about the graphs needed improvement.  We made some changes in colors that were closely related such as light blue and dark blue. We also made all the graphs uniform, all starting from day 1.  We also added Table 2 which clearly shows the strains that have strong inhibitory actions on all the pathogens. Slimily, the table shows the strains that have weak effect on all the pathogens. We also added a supplementary Table that has all the data used in the graphs and the way we describe them in the text. We added information about each pathogen in the introduction.

Below we answer the specific comments.

1. We changed keywords so that there is no duplication with the words in the title.
2. In the introduction, we added information about the importance of the pathogens and the diseases caused by them in Lines 30-43.
3. In the Materials and Methods there is information about the statistical analyses. We analyzed data using  ANOVA test for the effect of myxobacteria on the seedlings in the presence of Rhizoctonia based on size of the leaves- see lines 160-162. However, for the In Petri dish tests, we did not do statistical tests since the results look very clear from looking at the graphs and the Tables.

As to replicates, the invitro tests on Petri dishes, for each challenge 5 plates were used plus five control plates. Growth in all the plates was measured and then averaged.  – see line 115. Then every challenge was repeated, meaning each challenge between a myxobacterial and a phytopathogen was performed twice and the results of the two trials were averaged- line 126. For the in vivo tests, four plants were used for each treatment see line 154.

4. We divided the results into sections to make it easier to follow the text as the reviewer suggested.
5. The graphs- We changed colors that cannot be easily distinguished. We also made the Figures uniform all starting on day 1. On day zero, when we put the fungal plug, there is no growth of fungus and thus no data and no inhibition. So, the results typically begin on day 1 and that is the reason we made all the Figures to start on day 1. We also made a table in the supplement that has all the numbers used in the graphs. We also made Table 2 that shows which of the myxobacteria strains were effective against all the pathogens. So, texts are supported by graphs and tables. The graphs are important because they show when the pathogen is resistant and crosses the bacterial line and that is shown on the graphs when inhibition goes to zero after 1 or 2 weeks.
6. We agree that the Figures for Petri dishes should have the controls before treatments. Unfortunately, those plates are not available anymore and thus we cannot change.
7. We tried to align but could not align the Figure.
8. The Petri dishes were recorded for a maximum of 14 days because there would not be any changes in the inhibition rate after that. Within 14 the fungus will cover the plate in control, and no more measurements are possible. Also, 14 days is sufficient time for the fungus to grow and cross the bacterial line if they are strong and resistant and the bacterial line. We saw in high growth rate fungus like Rhizoctonia, in many cases Rhizoctonia crossed the line within 14 days and subsequently the inhibition dropped to zero. We added in the methods some explanation why we chose 14 days in lines 119-123.
9. We listed the references according to the rules of the journal. We also added new references and checked the order.

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors!

Thank you for the work you did on the article.

You did not address all of my comments.

The text of the manuscript must be formatted according to the rules of the journal. The font of the text is not uniform.

A paragraph on statistical data processing was not added to the Methodological section of the article.

Previous comments regarding changes to the drawings have not been addressed.

The article and the drawings are poorly formatted, which immediately reduces the impression of the level of work and the authors' respect for the readers.

Respectfully Yours, Reviewer.

03 August 2025

Author Response

We thank the reviewer for his time and the important comments. We provide below our response to the comments:

1. We went through the manuscript and changed all the fonts to one type and size.
2. We included all the statistical analyses we have performed in the materials and methods for each section, and they are in lines 100-103, 126-129, 161-163.
3. We removed 9 drawing graphs (Figures, 2A,2B, 4A.4B, 6A, 6B, 8A, 8B) and replaced them with three Tables 2, 3,4. These tables have all the information that we had in the Figures. We changed text so that it matches the information in the Tables. We changed texts in Materials and Methods, Results and Discussion.