Poly-γ-Glutamic Acid-Induced Assemblage of Root Endophytic Microbiota Enhances Disease Resistance in Chrysanthemum Plants

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

See attached file

Comments for author File: Comments.pdf

Author Response

Materials and Methods

1. Row 107. Please give the details how was done gamma irradiation sterilization of field soil: what source was used with what intensity, time for treatment. Why was used the concentration 50 mg/kg of streptomycin sulfate for antibiotic treatment?

Response: Gamma irradiation sterilization of field soil was performed using a ⁶⁰Co source at an intensity of 50 kGy for 2 h. The concentration of 50 mg/kg of streptomycin sulfate was used for antibiotic treatment as it is a standard, effective dose for eliminating soil bacteria. This concentration is commonly used in soil sterilization studies and has been validated in previous research to provide consistent and reliable results (Line114-116).

2. Row 113. What was the light intensity in 16-hour light period?

Response: It has been modified to “16 h light/8 h dark photoperiod” (Line122).

3. Row 115. Wilt disease was assessed after two weeks of treatments, and the abundance of Foc in roots was determined. Please give the protocol for wilt assessment and determination of Foc in roots.

Response: Thanks for your comments. The detailed description has been added in the revised manuscript: “Wilt disease index was assessed after two weeks of treatments using the formula: Σ (number of leaves at each disease grade × grade value)/(total leaves examined × highest grade). The abundance of Foc in roots was determined by quantitative real-time PCR (qPCR) according to Castillo et al. (2022)”(Line124-129).

4. Row 132. Please give the type of the spectrophotometer, producer and country of origin.

Response: The detail description about the spectrophotometer has been added: “a microplate spectrophotometer (Thermo, USA)”(Line143-144).  

5. Row 138. Please give the type of the liquid chromatography-mass spectrometry, producer and country of origin.

Response: The detail description about LC-MS has been added: “liquid chromatography-mass spectrometry (LC-MS) (Triple Quadrupole 6460, Agilent, USA)” (Line150-151).   

6. Row 148. Bacterial antagonistic activity was evaluated on Potato Dextrose Agar, give the protocol of evaluation. Potato Dextrose Agar (PDA), give producer and country of origin.

Response: Thanks for your comments. The detailed description about the assays of bacterial antagonistic activity and Potato Dextrose Agar (PDA) has been added in the revised manuscript: “Bacterial antagonistic activity was evaluated on Potato Dextrose Agar (PDA) (Solarbio, China). F. oxysporum was cultured on PDA medium and incubated at 28°C for 7 d. A 5 mm mycelial disk of F. oxysporum was then placed at the center of a newly prepared PDA medium. Each bacterial isolate was subsequently inoculated onto the edge areas, 4 cm away from the central pathogen colony. Plates containing only F. oxysporum were used as controls. All treatments were conducted in six replicates. Following a seven-day incubation period at 28°C in darkness, the growth diameter of the pathogen were determined” (Line159-166).

7. Row 157. sterilized soil, give explanation-gamma irradiation sterilization or antibiotic treatment?

Response: “sterilized soil” has been modified to “gamma irradiation sterilized soil” (Line174).

8. Row 161. Wilt disease severity was assessed two weeks after Foc inoculation. How?

Response: The detailed description about wilt disease index has been added as mentioned above (Comment 3: Row 115. Wilt disease was assessed...)

9. Row 167. liquid chromatography on a silica gel column, give type, producer and country of origin.

Response: The detailed description about silica gel has been added: “silica gel plates (Sigma-Aldrich, USA)” (Line189).  

10. Row 171. gas chromatography-mass spectrometry (GC/MS), give type, producer and country of origin.

Response: The detailed description about GC/MS has been added: “gas chromatography-mass spectrometry (GC/MS)(5977B, Agilent, USA)” (Line190).  

 

Results

1. Row 276. Chrysanthemum, correct to italic

Response: It has been italicized in the revised manuscript(Line277).

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The main question addressed in the study concerns the effect of γ-PGA on improving plant disease resistance by modulating the SA pathway and promoting the colonization of beneficial microbiota, especially Burkholderia sp. I especially find significant and relevant the triple interaction between biostimulants, plants and root microbiomes, which significantly affected the health and functionality of plants. The disadvantage of the manuscript is the vagueness of the purpose in the given formulation. The purpose and objectives of the study should be clearly stated and the research hypothesis should be formulated. The authors' research adds to the subject area, in comparison with other published materials, new data on the modulation of the root microbiome and subsequent increase in plant resistance to disease. The methodology does not raise any major questions, but the authors should bring originality to it related to the object of study based on the results obtained. The conclusions are consistent with the results presented, but they could be improved by providing specific, clear data from the study. This will make it easier for other authors to compare data. There are quite a lot of references, but they relate to the topic of the study. One should agree with the authors' opinion about their necessity. The tables and figures are of high quality and the data is presented logically.»  

The research material used was selected appropriately, as well as the statistical methods used to analyze it. Own results and their discussion in the Discussion are properly described and were compared with previous studies by other authors. I have no objections to the substantive and editorial value reviewed article. However, the aim of the study needs editing and a succinct title. It is necessary to distinguish between the aim and objectives of the study.

 In my opinion, the manuscript is well written and has no comments. I reflected this in the report. If you want comments, you should look for another reviewer. I always approach articles critically when reviewing and reject them. But not at this time. The article is good.

 

Comments for author File: Comments.pdf

Author Response

1. “Our study showed that γ-PGA playeda pivotal role in shaping root microbiomes in Chrysanthemum, thereby strengthening the plant's defense against fungal pathogens. The objectives of this research encompassed elucidating the impact of γ-PGA on root microbiomes, unraveling the mechanisms that underlay this modulation, and assessing the resulting enhancement of disease resistance in Chrysanthemum plants.” The aim of the study is written multi-part. The aim and objectives of the study should be clearly defined.

Response: Thanks for your comments. These sentence has been modified to “The main aim of this research was to elucidate the impact of γ-PGA on root microbiomes, unravel the underlying mechanisms of this modulation, and assess the resulting enhancement of disease resistance in Chrysanthemum plants.”. 

2. “The triplicate interplay among the biostimulants, plants, and root microbiomes significantly influenced plant health and functionality. ”. How? It should be specific.

Response: According to your suggestions. This sentence has been modified to “The triplicate interplay among γ-PGA, Chrysanthemum plants and root microbiomes significantly influenced plant disease resistance”.  

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled "Poly-γ-Glutamic Acid-induced Assemblage of Root Endophytic Microbiota Enhances Disease Resistance in Chrysanthemum Plants" is interesting but minor revision is needed.

Please revise the manuscript or answer the following questions.

Title – I propose to add information about pot experiments.

Abstract

Line 17 – “ revealed a marked reduction” – I propose to add information about % value of reduction.

Introduction

Chrysanthemums are associated with ornamental plants. Please briefly mention that they can be used as chrysanthemum tea from the flower heads and the leaves are edible and can be added to salads or cooked like other vegetables.

In the introduction, please explain what poly-γ-glutamic acid and a prebiotic/biostimulant have in common.

Are there any other methods to prevent the development of Fusarium in Chrysanthemums?

Methods

Please provide manufacturer's name and country of origin for all devices/tests used.

Results

Line 200 – 204 – please transfer this information to the methodology.

Line 206 – please provide the disease index values.

Line 206 – please explain in advance what Quantitative PCR is.

Line 221 – Acidobacteria are also abundant at the phylum level. Complete the description of the results.

Line 345 – by how much can it inhibit the germination of fungal spores?

Figure 6 e, f, g, I, j, k - there is no visible information whether the differences are significant or not significant.

Conclusions

The conclusions include the term biostimulant. Please write more precisely what chemical compound the Authors had in mind.

Author Response

Abstract

1. Line 17 – “ revealed a marked reduction” – I propose to add information about % value of reduction.

Response: According to your suggestions, it has been modified to “...a 73.2% reduction in Foc abundance in the roots” (Line19).

Introduction

1. Chrysanthemums are associated with ornamental plants. Please briefly mention that they can be used aschrysanthemum tea from the flower heads and the leaves are edible and can be added to salads or cooked like other vegetables.

Response: According to your suggestions.The briefly description about Chrysanthemum has been added in the section of Introduction: “Chrysanthemum plants are widely cultivated in China and its flowers can be used to make tea, and the leaves are edible, making them a great addition to salads or suitable for cooking like other vegetables. However, Chrysanthemum plants are severely affected by Fusarium wilt, which poses a significant threat to their cultivation” (Line98-102).

2. In the introduction, please explain what poly-γ-glutamic acid and a prebiotic/biostimulant have in common.Are there any other methods to prevent the development of Fusarium in Chrysanthemums?

Response: Thanks for your comments. Poly-γ-glutamic acid (γ-PGA) and prebiotics/biostimulants play crucial roles in promoting plant health and resilience against pathogens. Both γ-PGA and other biostimulants enhance plant growth, improve nutrient uptake, and increase stress tolerance. These substances also stimulate systemic resistance mechanisms, priming plants to effectively combat pathogens. In addition to γ-PGA and biostimulants, several other strategies can help prevent Fusarium wilt. Biological control agents, certain bacteria and fungi (Such as such as Bacillus and Trichoderma), can be used to suppress Fusarium populations in the soil. Genetic resistance through breeding or biotechnological approaches aims to develop chrysanthemum cultivars that are inherently resistant to Fusarium. Cultural practices, including crop rotation with non-host plants and optimizing irrigation and soil conditions, also contribute to disease management by reducing the pathogen's ability to persist in the environment.

 

Methods

1. Please provide manufacturer's name and country of origin for all devices/tests used.

Response: The detailed description about reagent and devices has been added in the section of Materials and Methods.

Results

1. Line 200 – 204 – please transfer this information to the methodology.

Response: Thanks for your comments. This information has been transferred to the section of Materials and Methods (Line116-120).  

2. Line 206 – please provide the disease index values.

Response: This sentence has been changed to “Treatments with γ-PGA caused a 56.8% reduction in the wilt disease index compared to the un-treated group under natural conditions”.

3. Line 206 – please explain in advance what Quantitative PCR is.

Response: “qPCR” has been described in the section of Materials and Methods (Line129).

4. Line 221 – Acidobacteria are also abundant at the phylum level. Complete the description of the results.

Response: Thanks for your comments. This sentence has been modified to “...followed by other phyla such as Acidobacteria, Actinobacteria and Patescibacteria” (Line230-231).

5. Line 345 – by how much can it inhibit the germination of fungal spores?

Response: Thanks for your comments. This sentence has been modified to “...this component (0.1 mM) could markedly inhibit fungal spore germination” (Line355).

6. Figure 6 e, f, g, I, j, k - there is no visible information whether the differences are significant or not significant.

Response: Thanks for your comments. The Figure and the related legends has been modified: “Data were presented as mean ± SD, with significant differences indicated by lowercase letters (Tukey’s HSD test, p < 0.05)”.

 

Conclusions:

7. The conclusions include the term biostimulant. Please write more precisely what chemical compound the Authors had in mind.

Response: Thanks for your comments. “biostimulant” has been instead of “γ-PGA” (Line430-431).