PONE-D-22-01806Fusarium oxysporum infection-induced formation of agarwood (FOIFA): a novel method for inducing the production of high quality agarwoodPLOS ONE

Dear Dr.xiangzhao,

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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: Partly

Reviewer #2: Partly

Reviewer #3: Partly

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

Reviewer #1: N/A

Reviewer #2: I Don't Know

Reviewer #3: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear authors, after a thorough revision of the manuscript titled “Fusarium oxysporum infection-induced formation of agarwood (FOIFA): a novel method for inducing the production of high-quality agarwood”, I would summarize my comments/recommendations as follows -

This study experiments with a particular isolate of Fox and validates its virulency and agarwood formation capacity. It is not clear from the MS why only a single Fox isolate was used in the study and the justifications for choosing the same. The role of other co-occurring microbes in the overall process of virulency and agarwood formation has not been discussed. It is apparent that the roles of those co-occurring microbes were completely ruled out during the experimentation part as well as MS written part. In this way, it is too early to say that only the said strain of Fox is responsible for better agarwood formation. Besides, the environment too plays a considerable effect in this regard, there is a lack of discussion on this too. In many places, the experimental details are not sufficient or not properly written to replicate the same. And the way in which the term “novel” is being used in places needs reconsiderations or justifications in the MS. These issues need to be addressed in the MS in order to make it complete.

1. The title “Fusarium oxysporum infection-induced formation of agarwood (FOIFA): a novel method for inducing the production of high-quality agarwood” mentions this as a novel method, while there are reports of Fusarium spp. induced agarwood formation “Example - Faizal, A., Esyanti, R.R., Aulianisa, E.N. et al. Formation of agarwood from Aquilaria malaccensis in response to inoculation of local strains of Fusarium solani. Trees 31, 189–197 (2017). https://doi.org/10.1007/s00468-016-1471-9”. Therefore, the authors should reconsider incorporating the term “novel” in the title of the MS.

2. Line 65-66 says that “in order to help meet the demand for agarwood production while protecting wild Aquilaria trees, this study had three objectives” but from the MS, it is nowhere discussed how the isolate reported in this study are protecting wild Aquilaria trees. There should be at least a few sentences in the discussion section regarding this.

3. Objective 1 in Line 66-67 says that “i) to identify fungal pathogens of Aquilaria that can induce the production of agarwood”, but the procedures and results describe only one Fusarium strain, and there is no justification why this particular isolate was used out of all the isolates.

4. Line 83: rewrite - “Sequence obtained were used as queries to search in Genbank”

5. Line 84: Mention why only F. oxysporum isolate (AsFo20150101) was used in this study, and give the BLAST results of all other isolates (which you have sequenced) in supplementary data.

6. Line 90-93: Mention the number of replica plants.

7. Line 95: Mention the exact time of harvesting stem tissues in dpi (day post-inoculation).

8. Line 99: “submitted to PCR” – mention which region/gene was amplified.

9. Line 101: The statement “The PCR products were checked using gel electrophoresis” is too vague to speculate what was done afterward. There should be a clear mention of whether the amplicon was sequenced or just visualized with control bands.

10. Line 112: “at 40 mg/ml” Rewrite as “at concentration 40 mg/ml”.

11. Line 117-118: The procedure should be briefly described.

12. Line 133: The conditions under which chromatography was carried out should be mentioned.

13. In Figure 2: The Fox isolate used in this study should be distinctly visualized from the remaining database isolates. Also, mention the strain number of the isolate (which you have assigned), and the accession number of the submitted sequence like those mentioned for database isolates in your phylogenetic diagram.

14. The discussion is a repetition of the results, making the content in this section very small. All the findings should be justified and critically discussed with reference to existing literature.

15. Line 247-249: “In this study, another novel technique for agarwood induction, known as the fungal agarwood inducing technique (Agar-Fit), was invented” – this statement is self-contradictory as earlier in the material method section (Line 93-94) it has been mentioned that the inoculation was done by following a method described by Zhang et al., 2014. Therefore, again reconsider using the term “novel” here.

16. Line 253-254: “the yield of agarwood can be further improved by combining biological inducers and chemical agents” – this sentence is too vague to state as there is no mention of an experiment in which the reported Fox strain was co-inoculated with chemical agents.

Reviewer #2: I have reviewed the paper thoroughly. There is merit in the findings that establishes Fusarium as a strong associate in agarwood -fungus ecology related to agarwood formation. The association of fungus in agarwood formation is a complex phenomenon which is still not clearly understood and has been known to involve several genera of filamentous fungi. The complex ecology also includes insects that probably create injuries for fungi to establish well. Studies that investigate this aspect are welcome as this has potential for artificial induction of agarwood in plantations and spare the natural trees of which only few are left under threat of extinction. But, it must be kept in mind at the same time that the association of Fusarium with agarwood is well known and since agarwood artificial infection is a commercially employed practice with high level of propriety and secrecy, it is quite likely that the same might be in use in formulations used by commercial agarwood producers already. At the same time, the study has a number of scientific and methodological shortcomings which need to be looked into extensively by the authors.

#1. The title needs revision (Ref: Line No. 1). The term FOIFA in line with AgarWit is misplaced since the paper does not establish it as a comprehensive technique for field inoculation. The passing mention is made only in the discussion and no comparative study of the two or different methods of infection has been provided to justify a new improved method.

#2. The information about the organism (Fusarium oxysporum strain) provided is very limited. The isolation, initial number of different isolates, screening from the lot and basis of selection are not presented. As per authors Fusarium solani has the most virulent association ((Ref: Line No. 62)

#3. Material and methods part needs a complete overhaul.

# 3.1. The experimental design for inoculation- how many plants, age of the plants, time period, frequency of success is missing. This should in fact have been clearly presented in a scientific manner and hence and detailed reports on it are required to make any proper assessment. (Ref: Line No. 90)

#3.2. Experimental details like selective media, primer sequence, amplicon size etc are missing (Ref: Line No. 97-101).

#3.3 The inoculation studies and pathogenicity test cannot be differentiated. Are they one and the same? Details are not clear ((Ref: Line No. 102-107).

# 3.4. An important reference resinous agarwood referred to as Wild agarwood (W) has been taken against which the present technique is compared. However its details particularly of source and nature are missing.

#3.5. The HPLC study selected 4 chromones. The basis for such selection is not clarified, source of sample, method of sample preparation are not discussed and source of standards not mentioned making the study weak. ((Ref: Line No. 120-128)

#3.6. The process of alcohol extract and essential oil being critical to the study require explanation and details. The authors have just mentioned a reference (Gao et al 2020). (Ref: Line No. 116-119)

#4. For the results provided I have the following observations:

#4.1. The data on isolation of fungi their screening etc is missing (as in #2)

#4.2. The results on the inoculation and its difference from pathogenicity study are missing (as raised in 3.1 to 3.4)

#4.3. TLC results ((Ref: Line No. 173-181) needs revision. Authors mentions “more constituents and higher contents…” which is not explained in Fig 5 or anywhere else.

#4.4. Chromone study I feel needs a complete revision. The points raised in 3.5 and in Fig 7, chromatogram B, C and D seem to tell a different story than what is mentioned in the manuscript particularly with regard to (if Fig 7D is W).

#4.5. GC results (Ref Table 1)indicate that a major sesquiterpene of agarwood agarospirol is undetected in control (W) which makes the choice of W as a reference for comparison unclear. Hence the claim of better sesquiterpene yield by the FOIFA method is confusing. The gain in B over W seems to be contributed mostly by high alpha selinene for which references are limited for agarwood. Please explain in detail.

#4.6. The figure 9 ABC legends are missing. So without knowing which chromatogram is what it is not possible to assess.

Reviewer #3: The technology used here is a very well known technology. it lacks new knowledge or science. Fu.ngal infection and physical methods and combitaion of both are well known see review CHHIPA and Kaushik ( 2017)

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Reviewer #1: No

Reviewer #2: Yes: Supriyo Sen

Reviewer #3: Yes: Nutan Kaushik

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PONE-D-22-01806R1Fusarium oxysporum infection-induced formation of agarwood (FOIFA): a rapid and novel efficient method for inducing the production of high quality agarwoodPLOS ONE

Dear Dr. xiangzhao,

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 submit your revised manuscript by Sep 25 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Niraj Agarwala, Ph.D.

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. 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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: N/A

Reviewer #2: (No Response)

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear authors, the updated version of the manuscript has been improved in many ways than the previous version. However, there are many aspects in the manuscript where I find the facts presented a bit rimy and superficial. I also feel a lack of general veracity as several of my comments has been deliberately ignored or misunderstood. In general, would like to put my comments as follows:

1. Line 79 – 80: From the description of the subsection “Isolation and identification of fungal isolates” under the section “materials and methods” it is clear that many fungal isolates were isolated from surface-sterilized symptomatic Aquilaria sinensis trees. Among all the isolates the F. oxysporum isolate (AsFo20150101) was selected for the study. But it has not been properly justified why among all, this particular isolate was chosen. The description you are citing (Line 49 – 59) does not justify that. Mention the reason why other isolates were ruled out for the study and why isolate AsFo20150101 was kept; that will suffice.

2. In Figure 2: The accession number of the Fox isolate used in the study is still missing in the phylogenetic tree. It should be mentioned.

3. The discussion section is still not adequate as per the journal requirement. There are many earlier reports where Fox isolates have been reported to induce agarwood formation, where the underlying mechanisms and factors have been deciphered. The discussion section of this manuscript should draw the connections from earlier studies and justify the (possible) mechanisms/roles of the AsFo20150101 for the results shown in the current investigation.

With these modifications, the manuscript will be complete.

Reviewer #2: The Title "novel" should be replaced by "efficient" ; presently both words appear complicating the readability of the title

Reviewer #3: they addressesd all issues. manuscript may be accepted. agarwood research needs more attention as its larg scale application is limited

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Nutan Kaushik

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Fusarium oxysporum infection-induced formation of agarwood (FOIFA): a  rapid and efficient method for inducing the production of high quality agarwood

PONE-D-22-01806R2

Dear Dr. xiangzhao,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Niraj Agarwala, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. 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: Yes

Reviewer #2: Yes

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

Reviewer #1: N/A

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear authors, all the issues have been addressed properly and formatted as per the journal requirements. Therefore, the manuscript is ready now.

Reviewer #2: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Supriyo Sen

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