PONE-D-19-22808

Biodiversity and characterization of culturable endophytic fungi isolated from Securinega suffruticosa in Shell Islands of the Yellow River Delta, and analysis of their antimicrobial activity

PLOS ONE

Dear Wen Du and Chunlong Sun,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please, see below comments:

1. Professional English proofreading of your manuscript is necessary before resubmission; 2. Shorten the title of your manuscript; 3. Rewrite Introduction - paragraphs describing endophytes and Securinega suffruticosa - the same beginning of each sentence, e.g. Endophytic fungi and S. suffruticosa.; 4. Change manuscript according to the reviewers suggestions.

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2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

12 This work was financially supported by Shandong Provincial Natural Science

13 Foundation (ZR2016BL16, ZR2016CL01); Doctor Foundation of Binzhou University

14 (2016Y17, 2016Y02); Science and Technology Development Plan Project of

15 Shandong Province (2014GSF119032); Project of Shandong Province Higher

16 Educational Science and Technology Program (J17KA120); Research Project of the

17 University-level teaching Reform of Binzhou University in 2017 (BYJYYB201736);

18 School-enterprise Co-construction Course Project of Binzhou University in 2017

19 (BYXQGJ201706), China.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Reviewer #1: Methodology:

a) The samples of Securinega suffruticosa were collected during two sampling years (2017 and 2018). It would be interesting to show the results of the culturable endophytic fungal composition between the two sampling periods.

b) Secondly the protocol for endophyte isolation used in this study does not guarantee that all the culturable fungal diversity was obtained. I suggest that in addition to plating the 10 cm tissue sections on PDA agar, you could also crush the tissue samples using motor and pestle and perform serial dilutions. This will allow more surface area for the fungi to grow (slow growing fungi can be obtained efficiently) which may have not been isolated as the fast growing fungi dominates the incision/cut area of the 10 cm tissue.

You can refer to the summary of protocols for endophyte isolation given by “Verma S.K., Kharwar R.N., Gond S.K., Kingsley K.L., White J.F. (2019) Exploring Endophytic Communities of Plants: Methods for Assessing Diversity, Effects on Host Development and Potential Biotechnological Applications. In: Verma S., White, Jr J. (eds) Seed Endophytes. Springer, Cham.”

Results:

a) As stated above, your results could also show the number of fungal isolates obtained between sampling periods.

b) Statistical inferences (e.g. ANOVA) needs to be generated for the data on the dominance (Y) values of endophytic fungi in tissues, antagonism of endophytic fungal isolates, etc.

Reviewer #2: In the manuscript presented for review, the authors present the results of experiments concerning the isolation and partial characterization of endophytic fungi from Securinega suffruticosa. The initial number of isolates was reduced to 57 for which ITS region sequences were obtained.

The description of the research makes it possible to repeat it, but I have a few questions about the methodology.

P7; L4-5 - Whether the strains came from public accessible collections of microorganisms and had identification numbers? Where they a type strains? What was the source of Fusarium oxysporum and Phoma herbarum?

P8; L6-8 – What was the result of surface disinfection? Is it possible to avoid the fungal growth on PDA medium when the surface sterilised parts of plant contain endophytic fungi and their release from tissues is very probable? Why the water from the last rinse was not tested for fungal presence? How long was the control incubated?

P11; L17-18 - What method was used to determine the number of bacteria in the inoculum?

P12; L20 – Is it possible to obtain more specified than total alkaloid content data? Chromatographic data (e.g. securinice) are strongly recommended.

Results:

P15; L7-8 vs. P14; L3-4 – In my opinion the results obtained from phylogenetic studies concern 57 isolates (57 ITS sequences were obtained, not 420). Morphological analysis does not allow for unambiguous classification of isolates.

P18; L19-22 and P19; L1-8 - I suggest rebuilding the entire chapter because it is currently written in a chaotic manner

P19; L1 – Table 5

P19; L5 – the description does not correspond with data presented in Table 6, e.g. “the crude extracts of C. globosum and C. ramotenellum (1.00 mg/mL) were enough to inhibit S. aureus”. For C. ramotenellum the MIC/MBC values are 2.00/2.00.

Strengths of the article:

A large number of isolates.

Diversity analyzes of endophytic fungi.

Interesting discussion of the results .

Weaker manuscript traits:

Only total alkaloid content of some endophytic fungal isolates.

Methodological doubts that require clarification.

The quality of the text should be assessed by native speaker. I am not, but I see fragments that are difficult to understand (especially in the description of the results).

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Reviewer #1: No

Reviewer #2: No

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Diversity and antimicrobial activity of endophytic fungi isolated from Securinega suffruticosa in the Yellow River Delta

PONE-D-19-22808R1

Dear Dr. Sun,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Katarzyna Hrynkiewicz

Academic Editor

PLOS ONE

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