Characterisation of Metarhizium majus (Hypocreales: Clavicipitaceae) isolated from the Western Cape Province, South Africa

Letodi L. Mathulwe; Karin Jacobs; Antoinette P. Malan; Klaus Birkhofer; Matthew F. Addison; Pia Addison

doi:10.1371/journal.pone.0240955

Peer Review History

Original SubmissionOctober 5, 2020
28 Oct 2020 Decision Letter - Ebrahim Shokoohi, Editor PONE-D-20-31254 Characterization of Metarhizium majus (Hypocreales: Clavicipitaceae) isolated from the Western Cape province, South Africa PLOS ONE Dear Dr. Malan, 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 28 November 2020. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Ebrahim Shokoohi Academic Editor PLOS ONE Additional Editor Comments: Dear Dr Malan I have received the referee comments. The referee comments and the AE comments are provided below. According to the referee comments, your manuscript needs major revisions. However, after point by point addressing all comments of the AE and the referee. Overall, the paper's morphology, molecular, and phylogeny were provided in a very brief text that needs to be expanded with the most available pieces of information. 1-What model has been used for the phylogeny? Why has NJ been used instead of ML? Do they produce the same result? What is the purpose of Bayesian to be used in this study? 2-In Table 1, if another species sequenced from South Africa needs to be added. 3-Why in Table 1, some locations noted as Brazil and some Brazil, GO? it needs to be clarified. Besides, the outgroup must be added to the table 1. 4-In the result section, the morphological needs to be improved. The authors must compare with any other morphology or morphometrics of the M. majus reported by others. Similar species need to be discussed, as well. Also, the authorship of the species is missed by the authors. Information on the morphology and morphometric must be given in a better format and full. 5-In the phylogenetic, the result needs to be rewritten to indicate the clade. The blast result must be provided in which the similarity should be given. 6-I would suggest adding the Genetic Pairwise distance to compare your sequence and the others for the similarity. 7-In Figure 1 (B and C), the scale is not indicated. By the way, the scale should be the same format for all the figures part. 8-The figure's legends need to be improved with such information as a model, frecA, etc. 9-The quality of Figures 2 and 3 needs to be improved. 10-In Phylogenetic trees, the clades must appear on the figures. 11-Please check all the citations, figures, and the overall format to be in the PLOS ONE style. Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why. 3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ****** 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: Yes ****** 4. 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 ****** 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: The authors of the manuscript titled Characterisation of Metarhizium majus (Hypocreales: Clavicipitaceae) isolated from the Western Cape province, South Africa have described the occurrence of an entomopathogenic Metarhizium majus previously unknown to occur in South Africa. The authors carried out pathogenicity assays on two insect pests in addition to morphological and molecular characterization of the fungus. - Authors should start off by writing in full biological control. This may be shortened later to biocontrol. - Authors should rewrite the abstract. When rewriting this section, authors should take into consideration the relevant parts of an academic abstract. - If If Metarhizium spp. are well-studied entomopathogens, why are the authors not providing examples of other commercialized species of the fungus? The authors should also specifically state whether this is a newly identified Metahizium spp., or newly identified as an entomopathogen? Further more, what are the potential benefits of characterizing this new spp. to South Africa and global agriculture? - Its interesting that throughout the manuscript authors did not consider stating from which orchards soil samples were collected except in line 127. The authors are encouraged to categorically provide the details of the crop and location of the orchards from where soil samples were collected in Western Cape for isolating Metarhizium spp. Were the soil samples bulked, or analysed separately? - Line 61: Authors should indicate the common name for Tenebrio molitor - Line 67: Authors should indicate the duration of time for the 75% ethanol and distilled water rinses were carried out. - How economically important are these insects to South African agriculture? I expect the authors to have focused on characterizing this entomopathogenic fungus using various economically important insect pests. For example, do these two insect pests cut across all the agricultural production regions of South Africa? Are they important constraints to the production of food staples? Do they vector important diseases? These pests are in short not representative enough. - Line 81: Authors should write in full. - Line 92: Percent concentration of agarose gel? Voltage used and duration of time for which it was run. What imaging system was used to document the results of the electrophoresis? - Line 95: BLASTn analysis was carried out on... - Lines 96-97: Fungal cultures were deposited in the fungal collection of the Biosystems Division of the Agricultural Research Council (ARC), Pretoria. - What about the partial sequences of the tubulin (BtuB) - Line 115: What taxonomic key was used for morphological identification? - Lines 152-153: Authors should provide a citation _ Line 166: Authors should italicize M. majus ****** 6. 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 [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. https://doi.org/10.1371/journal.pone.0240955.r001
Revision 1
3 Dec 2020 Author Response Response to Reviewers Additional editors comments: 1-What model has been used for the phylogeny? Why has NJ been used instead of ML? Do they produce the same result? What is the purpose of Bayesian to be used in this study? - The data were analysed using NJ method using uncorrected “p”. Many fungal studies used NJ for phylogenetic comparisons, as it is more robust and can be combined with Bayesian analysis. The purpose of Bayesian analysis is commonly done to test the probability of nodes forming. The ML tree, Bayesian tree and NJ tree had similar topologies in all cases. 2-In Table 1, if another species sequenced from South Africa needs to be added. - Page 7: Line 128. An additional M. majus isolated, from another locality, have been added to Table 1. 3-Why in Table 1, some locations noted as Brazil and some Brazil, GO? it needs to be clarified. Besides, the outgroup must be added to the table 1. - Page 7: Line 128. GO next to Brazil deleted. Outgroup added to the table. 4-In the result section, the morphological needs to be improved. The authors must compare with any other morphology or morphometrics of the M. majus reported by others. Information on the morphology and morphometric must be given in a better format and full. Similar species need to be discussed, as well. - Page 8: Line 132-141. - Also, the authorship of the species is missed by the authors. - Page 3: Line 57. Authorship is indicated in the introduction section at the first mention of the species name. 5-In the phylogenetic, the result needs to be rewritten to indicate the clade. The blast result must be provided in which the similarity should be given. - Page 9: Line 157-167. The different clades were described in the text and the Blastn results provide for identify and coverage. -I would suggest adding the Genetic Pairwise distance to compare your sequence and the others for the similarity. - Page 10: Line188-188. Table included on genetic pairwise analysis. 7-In Figure 1 (B and C), the scale is not indicated. By the way, the scale should be the same format for all the figures part. - Corrected – missing scales indicated and made uniform. 8-The figure's legends need to be improved with such information as a model, frecA, etc. - Legends improved 9-The quality of Figures 2 and 3 needs to be improved. - Improved quality. 10-In Phylogenetic trees, the clades must appear on the figures. - Clades indicated in figures 11-Please check all the citations, figures, and the overall format to be in the PLOS ONE style. - Formatting checked Reviewers comments Reviewer #1: The authors of the manuscript titled Characterisation of Metarhizium majus (Hypocreales: Clavicipitaceae) isolated from the Western Cape province, South Africa have described the occurrence of an entomopathogenic Metarhizium majus previously unknown to occur in South Africa. The authors carried out pathogenicity assays on two insect pests in addition to morphological and molecular characterization of the fungus. - Authors should start off by writing in full biological control. This may be shortened later to biocontrol. Corrected - Authors should rewrite the abstract. When rewriting this section, authors should take into consideration the relevant parts of an academic abstract. Page 2: Line 19-30. Abstract adapted - If If Metarhizium spp. are well-studied entomopathogens, why are the authors not providing examples of other commercialized species of the fungus? Page 3: Line 44-50. Information added The authors should also specifically state whether this is a newly identified Metahizium spp., or newly identified as an entomopathogen? Further more, what are the potential benefits of characterizing this new spp. to South Africa and global agriculture? Page 11: Line 214-215. Information added. Page11: Line 207-209. Stated that it is a new report for SA, not a new species. - Its interesting that throughout the manuscript authors did not consider stating from which orchards soil samples were collected except in line 127. The authors are encouraged to categorically provide the details of the crop and location of the orchards from where soil samples were collected in Western Cape for isolating Metarhizium spp. Were the soil samples bulked, or analysed separately Page 4: Line 69-75. Information added - Line 61: Authors should indicate the common name for Tenebrio molitor Page 4: Line 78. Information added - Line 67: Authors should indicate the duration of time for the 75% ethanol and distilled water rinses were carried out Page 5: Line 83. Information added - How economically important are these insects to South African agriculture? I expect the authors to have focused on characterizing this entomopathogenic fungus using various economically important insect pests. For example, do these two insect pests cut across all the agricultural production regions of South Africa? Are they important constraints to the production of food staples? Do they vector important diseases? These pests are in short not representative enough. Focus are on characterisation and occurrence of M. majus not the insects. - Line 81: Authors should write in full. Page 5: Line 97. Corrected - Line 92: Percent concentration of agarose gel? Voltage used and duration of time for which it was run. What imaging system was used to document the results of the electrophoresis? Page 6: Line 109-110. Corrected - Line 95: BLASTn analysis was carried out on... Page 6: Line 112. Corrected - Lines 96-97: Fungal cultures were deposited in the fungal collection of the Biosystems Division of the Agricultural Research Council (ARC), Pretoria. Page 6: Line 113-115. Corrected - What about the partial sequences of the tubulin (BtuB) The sequences of Btub were included in the dataset of fig 2. Few sequences available. - Line 115: What taxonomic key was used for morphological identification? Page 8: Line 132-142.Information added, however, we relied mainly on molecular identification of the EPF isolate, as the M. anisopliae species complex species mostly look similar morphologically. - Lines 152-153: Authors should provide a citation Page 10: Line 193. Corrected - Line 166: Authors should italicize M. majus Page 11: Line 206. Corrected Journal Requirements: In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why. Page : Line 71-73. Information added Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0240955.r002
4 Jan 2021 Decision Letter - Ebrahim Shokoohi, Editor PONE-D-20-31254R1 Characterization of Metarhizium majus (Hypocreales: Clavicipitaceae) isolated from the Western Cape province, South Africa PLOS ONE Dear Dr. Malan 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 01/02/2021. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Ebrahim Shokoohi Academic Editor PLOS ONE Additional Editor Comments (if provided): Dear Authors I have received the comments from the referee stating that all the issues were not addressed in the revised version of your manuscript. Therefore, I am asking you to check and address all the concerns deeply. The main issue raised by the referee is that the discussion is not written correctly. It needs to be significantly improved. Additionally, the morphology needs to be cited with the proper citation. The language and the manuscript style also needs to be checked carefully. The referee comments attached below. Kind regards, [Note: HTML markup is below. Please do not edit.] 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: (No Response) ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 Response) ****** 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: Lines 26-27: M. majus and T. molitor should be written in full the first time they are being introduced. Lines 44-46: Similar to orthoptera, hemiptera and thysanoptera, cite examples for lepidoptera, coleoptera, diptera and hymenoptera. Line 69: ...from the surveyed plum, apricot, and quince orchards in Tierhoek farm. Soils were obtained at a depth of 15 cm from under the tree canopy. Line 73: It may not be relevant mentioning the name of the orchard owner at this juncture. Lines 76-77: wax moth repeated twice Lines 93-100: what is the taxonomic reference (identification key) that was used for the morphological identification? Although the authors have indicated their reliance on molecular identification, morphological identification is an important aspect of taxonomic classification. If it is an experiment carried out in this study, it carries similar importance as other experiments. Line 104: The polymerase chain reaction (PCR) was conducted... Line 110: From what template were the sequences generated? It appears a critical step in the methodology has been ommitted. Line 118: ...combining 'concatenate' may be a better word to use. In addition, the same synonyms used in the methods section should be used for ITS, BtuB, and EF-1α genes. Lines 127-128: the same headers listed in line 128 should be used as headers for table 1. In addition include footnotes to table 1 describing in full ITS, BtuB, and EF-1α Line 166: 'The South African M. majus did not differ in base similarity from the type strain (KJ398801) of the EF-1α gene, and differs by ...% (indicate percentage) from the most closely related M. guizhouense. Lines 185-189: include footnotes in table 2. For example what does 1 to 14 represent? The manuscript should be thoroughly checked for grammatical errors. The style of writing and formatting used in other relevant manuscripts an be followed. Articles published in this reputable journal will be suitable. ****** 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 [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. https://doi.org/10.1371/journal.pone.0240955.r003
Revision 2
7 Jan 2021 Author Response Reviewer #1: Lines 26-27: M. majus and T. molitor should be written in full the first time they are being introduced. - Page 2: Line28-30. Corrected Lines 44-46: Similar to orthoptera, hemiptera and thysanoptera, cite examples for lepidoptera, coleoptera, diptera and hymenoptera. - Page 3: Line 47-49. Corrected Line 69: ...from the surveyed plum, apricot, and quince orchards in Tierhoek farm. Soils were obtained at a depth of 15 cm from under the tree canopy. - Page 4: Line 72-73. Changed as suggested Line 73: It may not be relevant mentioning the name of the orchard owner at this juncture. - Name of the orchard owner removed. Lines 76-77: wax moth repeated twice - Corrected Lines 93-100: what is the taxonomic reference (identification key) that was used for the morphological identification? Although the authors have indicated their reliance on molecular identification, morphological identification is an important aspect of taxonomic classification. If it is an experiment carried out in this study, it carries similar importance as other experiments. - Page 5; Line 105-106. Morphological of identification of the entomopathogenic fungi was done by according to the key Humber 2012. Add to reference list: 14. Humber RA. Identification of entomopathogenic fungi. In: Lacey LA, editor. Manual of techniques in invertebrate pathology. 2nd ed. Burlington: Elsevier; 2012. pp. 151-187. https://doi.org/10.1016/B978-0-12-386899-2.00006-3 Line 104: The polymerase chain reaction (PCR) was conducted... - Page 5: Line 110. Corrected Line 110: From what template were the sequences generated? It appears a critical step in the methodology has been ommitted. - Page 5-6: Line 110-112. A polymerase chain reaction (PCR) was conducted, using the KAPA2G Robust HotStart ReadyMix [KAPA Taq EXtra HotStart DNA Polymerase, KAPA Taq EXtra Buffer, dNTPs (0.3 mM of each dNTP), MgCl2 (2 mM at 1X) and stabilisers] PCR kit. Line 118: ...combining 'concatenate' may be a better word to use. In addition, the same synonyms used in the methods section should be used for ITS, BtuB, and EF-1α genes. - Page 6: Line 127. Corrected as suggested Lines 127-128: the same headers listed in line 128 should be used as headers for table 1. In addition include footnotes to table 1 describing in full ITS, BtuB, and EF-1α - Page 7: Line 138-139. Corrected throughout text Line 166: 'The South African M. majus did not differ in base similarity from the type strain (KJ398801) of the EF-1α gene, and differs by ...% (indicate percentage) from the most closely related M. guizhouense. - Page 9: Line178-181. Corrected as suggested Lines 185-189: include footnotes in table 2. For example what does 1 to 14 represent? - The cell-column with the numbers have been merged with those of the species name to indicate that the number correspond with the horizontal heading of the matrix, which show the standard error in the above diagonal. The manuscript should be thoroughly checked for grammatical errors. The style of writing and formatting used in other relevant manuscripts an be followed. Articles published in this reputable journal will be suitable. - Language edited by a professional language editor, style thoroughly checked and the discussion improved Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0240955.r004
12 Jan 2021 Decision Letter - Ebrahim Shokoohi, Editor PONE-D-20-31254R2 Characterization of Metarhizium majus (Hypocreales: Clavicipitaceae) isolated from the Western Cape province, South Africa PLOS ONE Dear Dr. Malan, 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 30/1/2021. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Ebrahim Shokoohi Academic Editor PLOS ONE Additional Editor Comments (if provided): Dear Dr Malan I have checked the manuscript, and despite the improvement of many parts of the manuscript, the Discussion still needs to be improved. The referee commented on the Discussion to be improved based on your finding. Revisions 2 and 3 submitted previously have not significant differences. Therefore, I will suggest you improve the Discussion for finalizing your manuscript. Kind regards, Ebrahim Shokoohi [Note: HTML markup is below. Please do not edit.] [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. https://doi.org/10.1371/journal.pone.0240955.r005
Revision 3
19 Jan 2021 Author Response The statement "Soil samples were collected from the orchards surveyed plum, apricot and quince, at a depth of 15 cm, from under the tree canopy on Tierhoek farm (-33˚13'19.687''S 19˚38'44.281'') (- 33,711596; 19,790730), in Tierhoek Valley near Robertson in the Western Cape province. A permit for collection of soil samples at the farm was issued by the farm owner and manager B.K.C. Gilson." has been added at the beginning of the Methods section of the manuscript as suggested by Anna Mentsl. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0240955.r006
1 Feb 2021 Decision Letter - Ebrahim Shokoohi, Editor PONE-D-20-31254R3 Characterization of Metarhizium majus (Hypocreales: Clavicipitaceae) isolated from the Western Cape province, South Africa PLOS ONE Dear Dr. Malan, 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 Mar 18 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Ebrahim Shokoohi Academic Editor PLOS ONE Journal Requirements: Additional Editor Comments (if provided): Dear Prof Malan I have checked up the manuscript submitted by you and your colleagues. I am pleased to inform you that your manuscript was accepted for publication in PLOS ONE. Regards, [Note: HTML markup is below. Please do not edit.] Reviewers' comments: [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. Attachments Attachment Submitted filename: Manuscript corrected.docx https://doi.org/10.1371/journal.pone.0240955.r007
Revision 4
3 Feb 2021 Author Response Ebrahim Shokoohi Dear Prof Malan I have checked up the manuscript submitted by you and your colleagues. I am pleased to inform you that your manuscript was accepted for publication in PLOS ONE. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0240955.r008
5 Feb 2021 Decision Letter - Ebrahim Shokoohi, Editor Characterization of Metarhizium majus (Hypocreales: Clavicipitaceae) isolated from the Western Cape province, South Africa PONE-D-20-31254R4 Dear Dr. Malan, 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, Ebrahim Shokoohi Academic Editor PLOS ONE Additional Editor Comments (optional): Dear Prof Malan I have checked the manuscript. Based on the referee and Editor comments that have been implemented in the submitted manuscript, I am pleased to inform you that your manuscript was accepted for publication in PLOS ONE. Regards, E Reviewers' comments: no comment https://doi.org/10.1371/journal.pone.0240955.r009
Formally Accepted
9 Feb 2021 Acceptance Letter - Ebrahim Shokoohi, Editor PONE-D-20-31254R4 Characterisation of Metarhizium majus (Hypocreales: Clavicipitaceae) isolated from the Western Cape Province, South Africa Dear Dr. Malan: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Ebrahim Shokoohi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0240955.r010

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