Cladophialophora guangxiense sp. nov., a New Species of Dark Septate Endophyte, Mitigates Tomato Bacterial Wilt and Growth Promotion Activities
, Yanlu Chen 5, Stanley Nyenje Mataka 1, Xue Jiang 1, Yi Zhou 1, Zhengxiang Sun 1,* and Ling Xie 2,3,4,*
Andres Sanabria-Velazquez
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript “Cladophialophora guangxiense sp. nov, A New Species of Dark Septate Endophyte, Mitigates Tomato Bacterial Wilt and Growth Promotion Activities” is dedicated to the important and current topic of biocontrol of pathogens in agriculturally important plants. The work is well structured and illustrated. In my humble opinion, there are a few points that should be corrected or clarified.
Unfortunately, the lines are not numbered, so it is more difficult to write comments
Minor points
Introduction
“Dark septate endophytes (DSEs) are a class of endophytic fungi characterized by their melanized hyphae, which colonize the root epidermis and cortex both intracellularly and intercellularly.”
Dark septate endophytes (DSE) are not a class of fungi, but a group of endophytic fungi that are characterized by their morphology of melanized, septate hyphae. The term class has a strictly taxonomic meaning. Please correct.
“The study demonstrated the potential of C. guangxiense HX2 as a biocontrol agent.”
This sentence is in the description of the aim of the study, but it does not refer to the aim, but to the conclusion. Please correct.
Material and methods
2.1. Isolation of the endophytic fungal
“The soil samples were taken back to the laboratory as soon as possible and stored at 4 ℃.”
How many samples were examined?
“Tomato seedlings were used as bait plant to capture endophytic fungi from soil samples [30].”
How many seedlings were used as bait plants?
“Purified endophytic fungal strains were preserved at the Institute of Plant Protection, Guangxi Academy of Agricultural Sciences.”
How many strains were isolated and preserved in total?
“The best candidates of fungal isolate were defined and selected for the subsequent assay.”
It is not clear how many isolates were selected for further studies. One? Please clarify.
2.4. Root colonization
“Endophytic fungi was cultured on OMA for 10 days. Sterilized tomato seeds were then transplanted into endophytic fungal colonies and placed within tissue culture bottles, accompanying OMA petri dishes.”
Please change petri to Petri.
Results
3.1. Fungal isolation, endophyte screening and colonization observation
“A total of 142 fungal isolates were obtained from 5 soil samples using baiting method.”
Information on the number of samples is better moved to the Methods section.
“For endophyte screening, eighteen isolates were randomly selected as reprentative from each species or group, and the priority was given to slow-growing isolates that usually start developing after at least 7 to 10 days of incubation.”
Information on the number of isolates is better placed in the Methods section.
“Thus, isolate HX2 were selected for further practical usage.”
If the isolate was one, please replace it with was.
Figure 1
Please indicate the deciphering of all abbreviations in the figure description. CK?
3.2.1. Taxonomy
“Cladophialophora guangxiense Y.Y. Long, Yanlu Chen & L. Xie, sp. nov. Figure 1; MycoBank 855619.”
It is not clear what Figure 1 means in this sentence. The Figure 1 does not show the fungus itself. Please clarify.
Table 2, 5
In my humble opinion, these tables would be better placed in the supplementary materials
Figure 9
Please explain in the description what two, three and four asterixes mean.
Author Response
Question 1: “Dark septate endophytes (DSEs) are a class of endophytic fungi characterized by their melanized hyphae, which colonize the root epidermis and cortex both intracellularly and intercellularly.”
Dark septate endophytes (DSE) are not a class of fungi, but a group of endophytic fungi that are characterized by their morphology of melanized, septate hyphae. The term class has a strictly taxonomic meaning. Please correct.
Answer: Thank you for your suggestion. We have corrected and revised it. Please check the manuscript for modification.
Question 2: “The study demonstrated the potential of C. guangxiense HX2 as a biocontrol agent.” This sentence is in the description of the aim of the study, but it does not refer to the aim, but to the conclusion. Please correct.
Answer: Thank you for your suggestion. We have corrected and revised it. Please check the manuscript for modification.
Question 3: “The soil samples were taken back to the laboratory as soon as possible and stored at 4 ℃.”How many samples were examined?
Answer: Thank you for your suggestion. We have added “ The 5 soil samples were taken back to the laboratory as soon as possible and stored at 4℃”. Please check the manuscript for modification.
Question 4: “Tomato seedlings were used as bait plant to capture endophytic fungi from soil samples.” How many seedlings were used as bait plants?
Answer: Thank you for your suggestion. We have added “ Fifty of tomato seedlings were used as bait plant to capture endophytic fungi from soil samples”. Please check the manuscript for modification.
Question 5 “Purified endophytic fungal strains were preserved at the Institute of Plant Protection, Guangxi Academy of Agricultural Sciences.”How many strains were isolated and preserved in total?
Answer: Thank you for your suggestion. We have added “ Finally, a total of 142 fungal isolates were obtained and preserved at the Institute of Plant Protection, Guangxi Academy of Agricultural Sciences”. Please check the manuscript for modification.
Question 6“The best candidates of fungal isolate were defined and selected for the subsequent assay.”It is not clear how many isolates were selected for further studies. One? Please clarify.
Answer: Thank you for your suggestion. We have corrected and revised it “only one”. Please check the manuscript for modification.
Question 7“Endophytic fungi was cultured on OMA for 10 days. Sterilized tomato seeds were then transplanted into endophytic fungal colonies and placed within tissue culture bottles, accompanying OMA petri dishes.”
Please change petri to Petri.
Answer: Thank you for your suggestion. We have corrected and revised it. Please check the manuscript for modification.
Question 8“A total of 142 fungal isolates were obtained from 5 soil samples using baiting method”. Information on the number of samples is better moved to the Methods section.
Answer: Thank you for your suggestion. We have revised it. Please check the manuscript for modification.
Question 9“For endophyte screening, eighteen isolates were randomly selected as reprentative from each species or group, and the priority was given to slow-growing isolates that usually start developing after at least 7 to 10 days of incubation.” Information on the number of isolates is better placed in the Methods section.
Answer: Thank you for your suggestion. We have revised it. Please check the manuscript for modification.
Question 10“Thus, isolate HX2 were selected for further practical usage.”If the isolate was one, please replace it with was.
Answer: Thank you for your suggestion. We have corrected and revised it. Please check the manuscript for modification.
Question 11:Figure 1 Please indicate the deciphering of all abbreviations in the figure description. CK?
Answer: Thank you for your suggestion. We have indicated the deciphering of all abbreviations in the Figure 1. Please check the manuscript for modification.
Question 12“Cladophialophora guangxiense Y.Y. Long, Yanlu Chen & L. Xie, sp. nov. Figure 1; MycoBank 855619.”It is not clear what Figure 1 means in this sentence. The Figure 1 does not show the fungus itself. Please clarify.
Answer: Thank you for your suggestion. We have corrected and revised it, it is Figure 2, not Figure 1. Please check the manuscript for modification.
Question 13:Table 2, 5 In my humble opinion, these tables would be better placed in the supplementary materials
Answer: Thank you for your suggestion. For overall considerations, We have placed Table 2 in the supplementary materials and Table 5 in the methods section. Please check the manuscript for modification.
Question 14: Figure 9 Please explain in the description what two, three and four asterixes mean.
Answer: Thank you for your suggestion. We have explained “Error bars represent mean standard deviation of triplicate experiments. ** P＜0.05 ****P＜0.001”in the Figure 9. Please check the manuscript for modification.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsIn this research, the authors identified a new species of dark septate endophyte, Cladophialophora. This fungus demonstrated a 76.7% control effect on tomato bacterial wilt caused by Ralstonia solanacearum and promoted tomato growth. The fungus enhances the activity of antioxidant and defensive enzyme systems in tomato plants, helping them resist bacterial wilt. The study used morphological properties, DNA sequence analysis, transcriptomic, and qPCR approaches to understand the fungus's effects and mechanisms. The article provides a report on a new biocontrol agent and should be published. However, before publication, key points that improve the quality and readability of the work should be addressed. Since the authors did not add numbers to the lines, comments were made on the document. Authors should address each comment in a letter before publication. Attached is the document with comments.
Comments for author File: Comments.pdf
Author Response
Question 1:“C. guangxiense HX2 was demonstrated a controlling effect of 76.7% on tomato bacterial wilt and promoted tomato seedling height” and “Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment revealed that tomato can produce …… to the strain HX2 infection” Provide quantitative data or specific metrics.
Answer: Thank you for your suggestion. We have provided quantitative data for this section. Please check the manuscript for modification.
Question 2: “soil-dwelling” to “soil-born”
Answer: Thank you for your suggestion. We have corrected and revised it. Please check the manuscript for modification.
Question 3: “This study was conducted to isolate and characterize DSE HX2 from the rhizosphere soil” What HX2？
Answer: Thank you for your suggestion. We have corrected and revised it. Because this section belongs to the introduction. For the sake of the article's logic, we ultimately chose to delete HX2. Please check the manuscript for modification.
Question 4: “This study was conducted to isolate and characterize DSE from the rhizosphere soil, identified as a new species of Cladophialophora, confirmed through a combination of morphological examinations and ……. Thus, the study aims to evaluate the potential of C. guangxiense HX2 as a biocontrol agent”Will be helpful if you list the specific objectives like i), ii) etc.
Answer: Thank you for your suggestion. We have corrected and revised it. Please check the manuscript for modification.
Question 5:2.3.1 Tomato culture “tomato seeds (Guihong II)” why this cultivar as chosen?
Answer: Thank you for your suggestion. Because the Guihong II susceptible tomato bacterial wilt. Please check the manuscript for modification.
Question 6: “For the experiment, tomato plants with similar growth vigor were selected and treated with endophytic fungal fermentation liquid at three concentrations (1×108 spores/mL, 1×106 spores /mL, 1×104 spores /mL),”provide more information about how this fermentation was produced?
Answer: Thank you for your suggestion. We have added the relevant references and introduction. Please check the manuscript for modification.
“The strain HX2 was cultured on PDA medium at 28°C for 14 d. Then, the colonies were washed with sterile water to collect the spores, and the spore concentration was adjusted to 1×108 spores/mL, 1×106 spores /mL and 1×104 spores /mL.”
Meng J X., Wang Q L., Ma G B., et al.. Screening, identification and evaluation of an acidophilic strain of Bacillus velezensis B4-7 for the biocontrol of tobacco bacterial wilt. Frontiers in Plant Science, 2024, 151360173-1360173.
Question 7: “2.3.3 Effect of endophytic fungal against R. solanacearum infection on tomato” “R. solanacearum” should be “R. solanacearum”
Answer: Thank you for your suggestion. We have corrected and revised it. Please check the manuscript for modification.
Question 8:Table 1 provide enough information so the reader can interpret the table without the main text; provide the company and concentration of “Thiediazole copper”
Answer: Thank you for your suggestion. We have provided enough information. Please check the manuscript for modification.
Question 9: “Sterilized tomato seeds were then transplanted into endophytic fungal …..OMA Petri dishes” should be “Desinfested tomato seeds ……”
Answer: Thank you for your suggestion. We have corrected and revised it. Please check the manuscript for modification.
Question 10: “Enzyme extractions and activity measurements were performed using a kit”Provide more information about the kit.
Answer: Thank you for your suggestion. We have provided more information about the kit. “Enzyme extractions and activity measurements were performed using a kit from Solarbio: Phenylalanine Ammonialyase (PAL) Assay Kit (Cat#BC0210); Catalase (CAT) Activity Assay Kit (Cat#BC0200); Peroxidase (POD) Activity Assay Kit (Cat#BC0090); Superoxide Dismutase (SOD) Activity Assay Kit (Cat#BC0170); Polyphenol Oxidase (PPO) Activity Assay Kit (Cat#BC0190)”. Please check the manuscript for modification.
Question 11: “2.8 Statistical analyses Duncan’s test” provide a rationale for using Duncan test.
Answer: Thank you for your suggestion. We have provided “and Duncan’s test was used to determine significant differences”. Please check the manuscript for modification.
Question 12:Figure 1 :provide enough information so the reader can interpret the figure without the main text; improve the quality of the picture.
Answer: Thank you for your suggestion. We have improved the quality of the picture and added relevant details in the description. Please check the manuscript for modification.
Question 13: “The strain HX2 is similar to Cladophialophora based on morphological analysis. However, the strain HX2 exhibits a unique species form characterized by non-septate, melanized, curled conidia arranged in coherent and unbranched chains” were there positive controls? A pure culture of Cladophialophora already described?
Answer: Thank you for your suggestion. C. inabaensis and C. tortuosa were added as positive controls in the revised manuscript. All of the described Cladophialophora species including the new species described in this research was based on a pure culture. Please check the manuscript for modification.
Question 14: Figure 2 :it will be good to show picture of know Cladophialophora and point out the difference with the new described isolates; Describe the image better, what means each pannel? What media is that? How old is the culture? What are the key differences?
Answer: Thank you for your suggestion. It has been modified according to the reviewer's opinion in the revised manuscript. Please check the manuscript for modification.
Question 15:Figure 4 is too small; Describe the figures with more details.
Answer: Thank you for your suggestion. We have enlarged the image and added relevant details in the description. Please check the manuscript for modification.
Question 16: Figure 5 provide more details about the figure
Answer: Thank you for your suggestion. We have added relevant details in the description. Please check the manuscript for modification.
Question 17: Table 3 provide enough information so the reader can interpret the figure without the main text.
Answer: Thank you for your suggestion. We have added enough information in the description. Please check the manuscript for modification.
Question 18: Figure 6 provide enough information of figure.what kind of cells are these? How was the image obtained?
Answer: Thank you for your suggestion. We have added enough information in the description. “a-b: the colonization of endophytic fungal within the tomato root tissue was observed using an Olympus BX53 microscope, following staining with lactic acid cotton blue. Intracellular (black arrows) and intercellular (red arrows) colonization of hyphae”. Please check the manuscript for modification.
Question 19: Figure 7 too small, Explain the points and the bars; avoid acronyms in figures.
Answer: Thank you for your suggestion. We have enlarged the image and added relevant details in the description. Please check the manuscript for modification.
Question 20: Figure 8 too small.
Answer: Thank you for your suggestion. We have enlarged the image. Please check the manuscript for modification.
Question 21: “To assess the expression levels of disease-resistant and pathogenic genes across various treatment groups, and subsequently subjected to quantitative real-time PCR analysis.”and table 5 should be described in the methods section.
Answer: Thank you for your suggestion. We have described the sentence and Table 5 in the methods section. Please check the manuscript for modification.
Question 22: Figure 9 provide enough information of figure.
Answer: Thank you for your suggestion. We have added relevant details in the description. Please check the manuscript for modification.
Question 23: “Molecular phylogeny showed that C. guangxiense strain HX2 formed a distinct clade with C. inabaensis (EUCL1) based on a combined dataset, however, the ITS sequences similarity was only 92.07%” C. guangxiense HX2 is morphologically and genetically distinct, but thediscussion lacks an in-depthcomparison with other closely related Cladophialophora species. Providing a more detailedanalysis of morphologicaland genetic differenceswould strengthen the claimof novelty for HX2. For example, what nucleotidesare different?
Answer: Thank you for your suggestion. It has been modified according to the reviewer's opinion in the revised manuscript. Please check the manuscript for modification.
Question 24: “Explain which specific geneswere upregulated and howthey relate to HX2's mechanism of action ininducing resistance
Answer: Thank you for your suggestion. We have added “the expression of ERF1 and Hqt were substantially enhanced. Zou [69] et al. re root-knot nematode ([70, 71] ”. Please check the manuscript for modification.
Jinping Z., Xinlin C., Chenxu L., et al. Autophagy promotes jasmonate-mediated defense against nematodes. Nature communications, 2023,14(1):4769-4769.
Pu G., Zhou B., Xiang F.. Isolation and functional characterization of a Lonicera japonica hydroxycinnamoyl transferase involved in chlorogenic acid synthesis. Biologia, 2017, 72(6): 608-618.
Xiaoxiao P., Weidong L., Wenquan W., et al.. Cloning and characterization of a cDNA coding a hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase involved in chlorogenic acid biosynthesis in Lonicera japonica. Planta medica, 2010, 76(16): 1921-6.
Question 25:Did not mentioned the limitations of the results observed. In addition mention practical applications of this finding, such as field-based biocontrol strategies, commercialization potential, or integration into existing tomato disease management practices.
Answer: Thank you for your suggestion. We have added relevant details “However, the underlying mechanism remains elusive. This study demonstrates that strain HX2 has the commercialization potential to be used as a biocontrol agent”. Please check the manuscript for modification.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors adressed all the comments and made the suggested changes. I recommed this article for publication
