High-throughput, image-based phenotyping reveals nutrient-dependent growth facilitation in a grass-legume mixture

Kirsten Rae Ball; Sally Anne Power; Chris Brien; Sarah Woodin; Nathaniel Jewell; Bettina Berger; Elise Pendall

doi:10.1371/journal.pone.0239673

Peer Review History

Original SubmissionJuly 8, 2020
25 Aug 2020 Decision Letter - Roberto Papa, Editor PONE-D-20-20899 High-throughput, image-based phenotyping reveals nutrient-dependent growth facilitation in a grass – legume mixture PLOS ONE Dear Dr. Ball, 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 Oct 09 2020 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. 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Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information [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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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: No Reviewer #2: 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 Reviewer #2: 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: It was not possible to find a URL/accession or a number/DOIs where it was possible to download the data, unlike what was declared. I would like to bring to the attention of the author some passages: row (64-65-66), reference is made to the types of interaction between plants suggesting that there was a sequence between them, which is not the case. In line (102), reference is made to the objectives of the study using a past tense "were", usually a tense is used at present. In row (182), reference is made to the addition of 99 mg N and 11 of P with a ratio of (6: 1), this ratio is incorrect. In line (241, 242), reference is made to the methodology only through the numerical label of the bibliography, in my opinion I do not find it properly correct. Furthermore, I would like to bring to the attention of the author the fact that in some cases the double quotation in the same sentence is separated by a comma (line 87), while in other cases it is separated by a hyphen (line 70). Overall in my opinion it is well done and the concepts are well expressed and connected. Reviewer #2: Overall, the manuscript is well done and highlights important issues, which could be used in further studies on mixtures and are applicable also on other species. Moreover, it underlines hight-throughtput phenotyping potential for studying crop growth patterns and facilitation in different conditions (i.e. nutrient availability) and its needs for careful calibration and validation. It only needs a minor revision in terms of sintax and lexical form. Result discussion is very well done and conclusions are strongly supported by data. I put some advices, comments and corrections in the following lines. 17: delete “To our knowledge” 25-28: focus attention on results achieved in the mixture, rather than indicate species favourite nutritional conditions, that are largely known 53-56: please, better define this point, it’s not so clear 60: biological nitrogen fixation or biological N2 fixation 84: delete “Regardless of the measurement” 88: delete comma after “rapid” 98: delete “Regardless of the technique” 111 and 116: biological nitrogen fixation or biological N2 fixation 123: I suggest “Experimental design and plant material”; this section is not so clear; I put some suggestion in the following lines. 124-126: introduce better the experimental site, indicating GPS coordinates, specify that the experiment was conducted in a greenhouse using Lemnatec Imaging System and move the info about the images in section 2.6 127-130: it could be better to modify this sentence. I propose “The experiment investigated the effects of four fertiliser treatments derived by factorial combination of two levels of nitrogen (LN and HN) and two levels of phosphorus (LP and HP) on one grass (Phalaris aquatica) and one legume (Trifolium vesiculosum) pasture species grown in monoculture and mixture.” 130-131: it could be better to modify this sentence. I propose “The twelve treatments were arranged in a latinized, resolved incomplete-block design with ten replicates, for a total of 120 pots.” 139-143: I suggest to reorganize this part, adding in brackets the nutrient combinations (LNLP, HNHP, HNLP and LNHP) and deleting the reference to the experimental design, which you will move in the main text. I suggest: “Within each replicate (indicated by the solid red box) the four nutrient combinations (LNLP, HNHP, HNLP and LNHP) were assigned to 4 main units arranged in a grid according to the experimental design. The three species combinations [grass only (Gra), legume only (Leg) and mixture (Gra Leg)] were randomized to the three consecutive pots within each main unit. Colored blocks indicate 1 pot containing two halves.” 145: I suggest to rename this part in “Growing conditions” 146: please add a sentence to specify growth condition in general, in order to better introduce the experimental conditions and parameters description. 149-151: please, improve the sentence 174: I suggest “Fertilisation treatments” 178-179: please, adjust the sentence, including punctuation and brackets 182: ratio 9:1 182-183: adjust punctuation and brackets 187: indicate in which table number 361-362: add punctuation and adjust brackets 367: you can add the reference on equation 1 375, 387, 391, 403: correct the figure number 376, 380: I guess you should better introduce the use of ratios in the main text, maybe in the section 2.10 376-382: please, improve this part 393-400: caption is not so clear; you could improve it moving some informations in the main text and reorganizing the text in order to promote immediate understanding 422-424: explain in the caption text what Gra (Leg) and Leg (Gra) mean 443, 454: add a bracket after “shoot N” 456: in all this section, please add tables and figures references. 464: delete “in mixture” 479: add comma after “grasses” 528: “conclusion” should have a separate paragraph ****** 6. 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Revision 1
9 Sep 2020 Author Response Dear Dr Papa, We enclose herewith our revised manuscript entitled ‘High-throughput, image-based phenotyping reveals nutrient-dependent growth facilitation in a grass – legume mixture’ for consideration for publication in the “Plant Phenomics and Precision Agriculture” special edition. We have revised the manuscript accounting for PLOS ONE’s style requirements as requested and have provided repository information for the data used in the manuscript. At present this is a conditional DOI (10.25909/12895121) which will be linked to the article upon publication. Presently, the data can be accessed via this private link: https://figshare.com/s/99e05c190be6cb416164 The updated financial disclosure which has been removed from the acknowledgements section is as follows: “The Australian Plant Phenomics Facility received grant funding from the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS). KB received a Postgraduate Internship Award from the Australian Plant Phenomics Facility towards the completion of this project”. We wish to sincerely thank the reviewers for their valuable assistance in refining the manuscript for publication and provide responses to their comments herein. We hope that you find these responses satisfactory, and we look forward to positive news of our article’s acceptance. Yours sincerely, Kirsten Ball (on behalf of all co-authors) Reviewer #1: It was not possible to find a URL/accession or a number/DOIs where it was possible to download the data, unlike what was declared. RESPONSE: Thank you for this comment, the associated data for the manuscript has now been placed in a repository and the provisional DOI is 10.25909/12895121, which once published will be linked to the article. Until the article is published, the data can be viewed via this private link: https://figshare.com/s/99e05c190be6cb416164 I would like to bring to the attention of the author some passages: row (64-65-66), reference is made to the types of interaction between plants suggesting that there was a sequence between them, which is not the case. RESPONSE: Thank you for this suggestion. This syntax has now been changed to “Interspecific interactions between plants can be negative, in the case of competition for resources [22], neutral, where complementarity ensures that species do not compete for the same resources [11], and positive, where facilitation leads to higher performance of a species when grown in mixture than in monoculture [23]” removing the suggestion of sequence. In line (102), reference is made to the objectives of the study using a past tense "were", usually a tense is used at present. RESPONSE: Thank you. The word “were” was used not to denote tense but because there were multiple aims of the study, hence “the aims were….” We have now changed this to “This study used” to clarify. In row (182), reference is made to the addition of 99 mg N and 11 of P with a ratio of (6: 1), this ratio is incorrect. RESPONSE: Thank for pointing out this typographic error. It has now been rectified. In line (241, 242), reference is made to the methodology only through the numerical label of the bibliography, in my opinion I do not find it properly correct. RESPONSE: The method employed has now been specified in the initial sentence and the following description modified to emphasize the steps involved in the method. These are now lines 248-250. Furthermore, I would like to bring to the attention of the author the fact that in some cases the double quotation in the same sentence is separated by a comma (line 87), while in other cases it is separated by a hyphen (line 70). RESPONSE: Where there are only 2 references in the citation a comma is used, in the case where there are more than 2, a hyphen is used. Overall in my opinion it is well done and the concepts are well expressed and connected. Reviewer #2: Overall, the manuscript is well done and highlights important issues, which could be used in further studies on mixtures and are applicable also on other species. Moreover, it underlines hight-throughtput phenotyping potential for studying crop growth patterns and facilitation in different conditions (i.e. nutrient availability) and its needs for careful calibration and validation. It only needs a minor revision in terms of sintax and lexical form. Result discussion is very well done and conclusions are strongly supported by data. I put some advices, comments and corrections in the following lines. 17: delete “To our knowledge” RESPONSE: Completed. 25-28: focus attention on results achieved in the mixture, rather than indicate species favourite nutritional conditions, that are largely known RESPONSE: Thank you for this comment. We have removed the single-species favored nutrient conditions from these lines and included only the interactions that occurred in mixtures. 53-56: please, better define this point, it’s not so clear RESPONSE: This has been altered to make the point clearer “Plant growth strategies involve trade-offs between increasing both productivity and resource acquisition (interpreted herein as an acquisitive growth strategy), and reducing productivity and conserving resources (interpreted herein as a conservative growth strategy) [14].” 60: biological nitrogen fixation or biological N2 fixation RESPONSE: Completed 84: delete “Regardless of the measurement” RESPONSE: Completed. 88: delete comma after “rapid” RESPONSE: Completed 98: delete “Regardless of the technique” RESPONSE: Completed 111 and 116: biological nitrogen fixation or biological N2 fixation RESPONSE: Completed 123: I suggest “Experimental design and plant material”; this section is not so clear; I put some suggestion in the following lines. RESPONSE: Thank you. We have now changed this to “Experimental design and species” 124-126: introduce better the experimental site, indicating GPS coordinates, specify that the experiment was conducted in a greenhouse using Lemnatec Imaging System and move the info about the images in section 2.6 RESPONSE: We have revised this sentence accordingly: “Our experiment was conducted between the 24th July 2018 and 5th October 2018 at the Australian Plant Phenomics Facility at the University of Adelaide (-34.971298, 138.639627) in a Lemnatec Imaging System” 127-130: it could be better to modify this sentence. I propose “The experiment investigated the effects of four fertiliser treatments derived by factorial combination of two levels of nitrogen (LN and HN) and two levels of phosphorus (LP and HP) on one grass (Phalaris aquatica) and one legume (Trifolium vesiculosum) pasture species grown in monoculture and mixture.” RESPONSE: We have altered the sentence according to the reviewer’s suggestion. 130-131: it could be better to modify this sentence. I propose “The twelve treatments were arranged in a latinized, resolved incomplete-block design with ten replicates, for a total of 120 pots.” RESPONSE: We have altered the sentence according to the reviewers suggestion. 139-143: I suggest to reorganize this part, adding in brackets the nutrient combinations (LNLP, HNHP, HNLP and LNHP) and deleting the reference to the experimental design, which you will move in the main text. I suggest: “Within each replicate (indicated by the solid red box) the four nutrient combinations (LNLP, HNHP, HNLP and LNHP) were assigned to 4 main units arranged in a grid according to the experimental design. The three species combinations [grass only (Gra), legume only (Leg) and mixture (Gra Leg)] were randomized to the three consecutive pots within each main unit. Colored blocks indicate 1 pot containing two halves.” RESPONSE: We have altered the sentence according to the reviewer’s suggestion. 145: I suggest to rename this part in “Growing conditions” RESPONSE: We have altered the header according to the reviewers suggestion. 146: please add a sentence to specify growth condition in general, in order to better introduce the experimental conditions and parameters description. RESPONSE: Thank you or this suggestion however it is the authors belief that the methods section is already quite lengthy, and much attention has already been paid to demonstrating growth conditions using both text and figures. We have therefore not included more explanation as suggested. 149-151: please, improve the sentence RESPONSE: We have now improved the sentence accordingly:” The plants were physically separated aboveground by a white plastic divider, set 5 cm into the soil and oriented north–south on the conveyor system in the greenhouse for consistency with respect to imaging orientation and solar exposure. The divider allowed intermingling of roots belowground (Fig 2). 174: I suggest “Fertilisation treatments” RESPONSE: We have changed the header per the reviewer’s suggestion 178-179: please, adjust the sentence, including punctuation and brackets RESPONSE: Thank you. We have now separated these sentences for the readers ease. 182: ratio 9:1 RESPONSE: Altered. Thank you. 182-183: adjust punctuation and brackets RESPONSE: These alterations have been made, and brackets added around the units of measurement. 187: indicate in which table number RESPONSE: Thank you for this suggestion however this information is not in tabular form but is present and clearly headed in the supplement. 361-362: add punctuation and adjust brackets RESPONSE: These alterations have been made 367: you can add the reference on equation 1 RESPONSE: Thank you. This has been added. 375, 387, 391, 403: correct the figure number RESPONSE: These have been corrected. 376, 380: I guess you should better introduce the use of ratios in the main text, maybe in the section 2.10 RESPONSE: We have now included this part in the section “Calculating overyielding from sPSA DAP 70 values”: We have altered the text accordingly: “Ymix is the observed yield of species i in mixture and Ymon is the observed yield of species i in monoculture expressed as a ratio (Ygra(Mix):Ygra and Yleg(Mix):Yleg). 376-382: please, improve this part RESPONSE: We have broken down the sections and improved the structure accordingly: “At the whole pot level, productivity in mixtures increased in all treatments. On average, the LNHP treatment increased by 27%, followed by LNLP which increased 21%, then HNLP which increased 20% and finally HNHP which was 10% more productive in mixture” 393-400: caption is not so clear; you could improve it moving some informations in the main text and reorganizing the text in order to promote immediate understanding RESPONSE: Thank you for this suggestion. We have now moved the larger proportion of the figure caption up into the section “Calculations of overyielding” and made the caption text clearer. 422-424: explain in the caption text what Gra (Leg) and Leg (Gra) mean RESPONSE: This has now been added to the figure caption. 443, 454: add a bracket after “shoot N” RESPONSE: Added. Thank you. 456: in all this section, please add tables and figures references. RESPONSE: Thank you for this valuable suggestion, we have now added all table and figure references. 464: delete “in mixture” RESPONSE: 479: add comma after “grasses” RESPONSE: Altered. 528: “conclusion” should have a separate paragraph RESPONSE: Thank you. This section is its own separated paragraph with header “Conclusion” Attachments Attachment Submitted filename: PlosOne_Ball_Response to reviewers.docx https://doi.org/10.1371/journal.pone.0239673.r002
11 Sep 2020 Decision Letter - Roberto Papa, Editor High-throughput, image-based phenotyping reveals nutrient-dependent growth facilitation in a grass – legume mixture PONE-D-20-20899R1 Dear Dr. Ball, 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, Roberto Papa, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0239673.r003
Formally Accepted
28 Sep 2020 Acceptance Letter - Roberto Papa, Editor PONE-D-20-20899R1 High-throughput, image-based phenotyping reveals nutrient-dependent growth facilitation in a grass - legume mixture Dear Dr. Ball: 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 Prof. Roberto Papa Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0239673.r004

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