Biocontrol of Large Patch Disease in Zoysiagrass (Zoysia japonica) by Bacillus subtilis SA-15: Identification of Active Compounds and Synergism with a Fungicide

Round 1

Reviewer 1 Report

The manuscript by Kim et al. entitled ,, Bacillus subtilis SA-15 Controls Large Patch Disease Caused by Rhizoctonia solani AG 2-2 (IV) in Zoysiagrass (Zoysia japonica): Identification of Active Compounds and Synergism with Fungicide” submitted to Horticulturae fits the scope of the journal and shows interesting results on the B. subtilis-driven inhibition of large patch disease. Though, there are some major comments to be addressed before publication of this work. First of all, there was no proof shown in the publication on linkage between the antifungal activity of B. subtilis and its ability to produce cellulases. Such speculations should be avoided. I only observed that this strain is capable for secreting cellulases and no association between this trait and antifungal properties was shown. Secondly, the introduction section should be highly improved. Thirdly, Materials and methods section needs addition of few details in several sections. The more detailed comments are listed below:

Introduction

• The introduction section is too short and does not provide sufficient background for this study. There are no information on large patch disease – how it is important, how often it occurs, what are the environmental/economic outcomes of the disease outbreaks. There are no information on Rhizoctonia solani No previous studies on biological control against this pathogen have been mentioned. Please provide more literature-anchored details on the following assumption ‘…, but also inhibit the mycelial growth of plant pathogens because cellulase can act on cell wall lytic enzymes (CWLs) in fungal mycelia [7]. CWLs include cellulase, chitinase, and β-glucanase, and microorganisms…” -> as far as I realize cell wall lytic enzymes (if we have in mind the plant cell wall degrading enzymes of phytopathogenic fungi) are proteins. These proteins of enzymatic activity cleave main constituents of plant cell wall, thus cellulose, xylan, and pectin. From the above-listed sentences it turns out that the authors suggest that these proteins could be degraded by cellulases(?). Therefore please correct these sentences or rephrase them to make the message clear. Also provide more literature background on the assumption that cellulolytic properties are beneficial for potential biocontrol agents towards combating phytopathogenic fungi – what is the mechanism behind it? Lines 49-52 lack enough details. Please add also some introductory part on the fungicide used – pencycuron.

Materials and Methods.

• Please provide details on the origin of subtilis SA-15. If it was isolated and identified by the authors’ group, please describe the identification methods used. Have this strain been deposited in international collection of bacteria? If so please provide the name of the collection and the attributed collection number.
• There is a lack of description of the experiment of testing antifungal properties of the acquired lipopeptide fraction (line 102). How was it done? What was the strain used etc? What were the control samples used?
• How antifungal activity could have been determined ‘by Sephadex LH-20 column chromatography’? Pathogenicity tests are applied for stating antifungal properties not chromatographic methods. Please describe in details the applied procedures.
• Please provide more information on the artificial inoculation with R. solani in 2014 (line 133), for sure include the inoculum used.
• In majority of the described experiments there is no information on the number of biological and technical repetitions performed.

Results

• Please add some variability measures e.g. standard deviation or standard error to MIR and the measured halo zone in lines 154-155. The same comment to line 187 in the case of mean inhibitory effects.
• Line 159 -> add information whose cellulase activity is shown
• Line 166 -> retention time concerns substances not peaks
• Figure 4 -> please remove ‘compound’ from beneath the bars. Please correct the y axis label
• Tables 2, 3 -> Please add some variability measures e.g. standard deviation or standard error

Discussion

• In the discussion section there is also no linkage between cellulolytic activity of B. subtilis and its antifungal properties (lines 232-233)
• Line 257 -> did you mean ‘culture age’ instead of growth time?
• Line 242 -> please specify. Did you mean form pores in cellular membrane by its destabilization?
• Line 246 -> I suggest to change the beginning to ‘The antifungal activity of fengycin differs depending on the treated pathogenic species…’
• Line 251 -> ‘of spores in fungal cells’ please correct
• Line 254 -> ‘The molecular structure of each lipopeptide is affected by antifungal action at the active site of pathogenic fungi’ -> not true, please correct
• It is uncommon to provide references to figures and tables in the discussion section
• Line 256-258 -> please be more specific – more literature data, name few phytopathogenic species
• Line 266 -> there are no data presented linking CWLs production and B.subtilis-driven control of the large patch disease. It is a speculation.
• Line 272 -> be more specific – name the applied fungicide instead of writing solely ‘control’

linguistic and editorial issues

line 44 -> help to increase; Lines 49-50-> language correction needed; Line 60 -> lack of italics; lines 68, 145 -> of treated sample/of control sample; lines 78, 120 -> SA-15 culture instead of SA-15 broth;  line 116 -> A HPLC; line 126-127 -> please rephrase to ‘SA-15 cells were collected by centrifuging cell culture at 3000 rpm. Then these cells (1%) were mixed with 93% bentonite, 5% surfactant, and 1% white carbon…’; line 129 -> additional comma; line 226 -> to inhibit the growth of R. solani; lines 242,248 -> instead of bio-membranes please use cellular membranes; Line 249 -> ‘it’ concerns mechanism not fengycin – please correct

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript entitled "Bacillus subtilis SA-15 Controls Large Patch Disease Caused by Rhizoctonia solani AG 2-2 (IV) in Zoysiagrass (Zoysia japonica): Identification of Active Compounds and Synergism with Fungicide "submitted for review, presents original research results that I think are very interesting. I think that the research deserves to be published with some minor corrections.

1. The title of the paper is too long and information contained in the title are repeated in the keywords. This should be corrected. I propose : Biocontrol of Large Patch Disease in Zoysiagrass (Zoysia japonica) by Bacillus subtilis SA-15: Identification of Active Compounds and Synergism with Fungicide. Keywords: Rhizoctonia solani AG 2-2 (IV), lipopeptides, pencycuron.....
2. The introduction should give a broader treatment of Large Patch Disease. It is worth describing what the symptoms of the disease are and what the control measures are.
3. L60. In vitro - italics.
4. L60-75- How many times the experiment was repeated, this should be recorded.
5. L120-124- Give details of the experiment. What were the research options? How many times was the experiment repeated? What concentrations were used?
6. L126-145. Please describe in detail the method of inoculation of the plants by R. solani.
7. Please insert photographs documenting the in vivo experiment.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Dear authors,

This manuscript "Bacillus subtilis SA-15 Controls Large Patch Disease Caused by Rhizoctonia solani AG 2-2 (IV) in Zoysiagrass (Zoysia japonica): Identification of Active Compounds and Synergism with Fungicide" need to clearify some parts. The authour must carefully check and improve it.  The comments are annotated in the manuscript.

Kind regards

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Dear authors, I have read with interest the article "Bacillus subtilis SA-15 Controls Large Patch Disease Caused by Rhizoctonia solani AG 2-2 (IV) in Zoysiagrass (Zoysia japonica): Identification of Active Compounds and Synergism with Fungicide". Your work sound promising. There are some suggestions I consider will improve your article.

Reorganize the last paragraph from the Introduction in a manner that will present point-by-point each of your research hypotheses. Make a different sentence or with i), ii) your aim and objectives.

Results - expand your interpretation. You cannot make sub-sections with only 3-4 lines of text. it is not sustainable and the text will look very fragmented. You need to provide enough text for future readers of your article.

Discussion - Move all the text referenced to previous presented tables in the Results section. Do not force the reader to go back in the result section to view a tbale. 

Author Response

Please see the attachment

Author Response File: Author Response.pdf