Natural Products Obtained from Argentinean Native Plants Are Fungicidal against Citrus Postharvest Diseases
, José Francisco Pensiero 1,2, Juan Marcelo Zabala 1,2, María Alejandra Favaro 1,2 and Marcos Gabriel Derita 1,3,*Round 1
Reviewer 1 Report (New Reviewer)
This work was performed with skill and competence. The Materials and methods section is complete and well explained. However, the English style needs to be polished and some errors require correction, So, I recommend the paper for publication in Horticulturae previous the revision detailed below:
Introduction
Lines 33-36. Change to “Fruits of Citrus spp. (Rutaceae) are cultivated in more than one hundred countries and their fruits are widely consumed throughout the world. Postharvest handling tries to achieve the highest quality fruits, increasing their postharvest life and reducing production losses, thus obtaining commercially suitable fruits.”
Lines 41, 42: change “citrus fruits, and once infected, they should be discarded from the production lot [1].” to “citrus fruits which, once infected, must be discarded from the production lot [1].”
Lines 62, 64, 74: insert a comma before “which”
Line 77: insert a comma after “health”
Line 81: change “the use of antagonistic microorganisms” to “biological control”
Lines 84, 85: eliminate “for the control of citrus disease”
Line 87: You explained (i) “biological control” in line 82, (ii) “natural products in line 84, 85”. However, the explanation of “thermotherapy” is missing.
Line 91: change “consisted of” to “consisted on”
Materials and Methods
Line 115: eliminate “different”
Lines 116-119, change to: “and successively macerated with 250 mL of dichloromethane (DCM) and 250 mL of methanol (MeOH) under mechanical stirring (3x 24 h each), to obtain the corresponding extracts after filtration and evaporation. These solvents were selected based on their differential extraction capability of less….”
Line 146, change to: “(Biotium Inc., Fremont, CA, USA)”
Lines 166-171: The sentence is too long and difficult to follow: Please change to “Once the mycelium of the control plates completely covered the surface of the medium (approximately 7 days), the mycelium diameter of each plant-treated plate was carried out by scanning the plates with ImageJ® software [19].”
In the temperature values, please do not underline the “ ° ”. For example, in line 182, the authors should write “25 °C”. In addition, please homogenize: In line 144, the authors write “57°C and 72°C” without space between the number and the unit, and in line 182, a space is inserted (“25 °C”). Revise the whole text by adding the missing space in line 144 and others.
Line 188: change “index value” to “sporulation index”
Results
Lines 244-245, change “probably due to the different capacities for phytochemical extraction and the presence of more polar substances such as phenolic compounds, alkaloids, or flavonoid glycosides” to “probably due to the more polar substances extracted by MeOH have greater fungicidal activity than the less polar compounds extracted by DCM”
Line 252: change “maintained P. italicum…” to “maintained their P. italicum…”
Line 254: change “O. virgata, both” to “O. virgata and both”
Line 255: change “A. inundata showing” to “A. inundata, which showed”
Line 282: change “fungicides” to “fungicide”
Line 292: Do not begin a sentence with a number
Line 328: “p” in low-case letter and in italics
Line 349: eliminate “mostly”
Line 382: write either “Albizia spp were used” or “Albizia sp was used”
Line 388: change “anti-Candida” to “anticandidal”
Line 395, 396: eliminate “due to their sedative properties”
References
Line 485: the titles of books must be written in capital letters
The corrections to the English language was detailed in the Comments and Suggestion
Author Response
Responses to Reviewer 1:
Reviewer: This work was performed with skill and competence. The Materials and Methods section is complete and well explained. However, the English style needs to be polished and some errors require correction, So, I recommend the paper for publication in Horticulturae previous the revision detailed below:
Response: Thank you very much for your appreciation.
Reviewer: Lines 33-36. Change to “Fruits of Citrus spp. (Rutaceae) are cultivated in more than one hundred countries and their fruits are widely consumed throughout the world. Postharvest handling tries to achieve the highest quality fruits, increasing their postharvest life and reducing production losses, thus obtaining commercially suitable fruits.”
Response: This sentence was changed.
Reviewer: Lines 41, 42: change “citrus fruits, and once infected, they should be discarded from the production lot [1].” to “citrus fruits which, once infected, must be discarded from the production lot [1].”
Response: It was changed.
Reviewer: Lines 62, 64, 74: insert a comma before “which”
Response: a comma was inserted.
Reviewer: Line 77: insert a comma after “health”
Response: a comma was inserted.
Reviewer: Line 81: change “the use of antagonistic microorganisms” to “biological control”
Response: It was changed.
Reviewer: Lines 84, 85: eliminate “for the control of citrus disease”
Response: It was eliminated.
Reviewer: Line 87: You explained (i) “biological control” in line 82, (ii) “natural products in lines 84, and 85”. However, the explanation of “thermotherapy” is missing.
Response: The explanation of thermotherapy was added.
Reviewer: Line 91: change “consisted of” to “consisted on”
Response: changed.
Reviewer: Line 115: eliminate “different”
Response: It was eliminated.
Reviewer: Lines 116-119, change to: “and successively macerated with 250 mL of dichloromethane (DCM) and 250 mL of methanol (MeOH) under mechanical stirring (3x 24 h each), to obtain the corresponding extracts after filtration and evaporation. These solvents were selected based on their differential extraction capability of less….”
Response: It was changed.
Reviewer: Line 146, change to: “(Biotium Inc., Fremont, CA, USA)”
Response: changed.
Reviewer: Lines 166-171: The sentence is too long and difficult to follow: Please change it to “Once the mycelium of the control plates completely covered the surface of the medium (approximately 7 days), the mycelium diameter of each plant-treated plate was carried out by scanning the plates with ImageJ® software [19].”
Response: The sentence was changed.
Reviewer: In the temperature values, please do not underline the “ ° ”. For example, in line 182, the authors should write “25 °C”. In addition, please homogenize: In line 144, the authors write “57°C and 72°C” without space between the number and the unit, and in line 182, a space is inserted (“25 °C”). Revise the whole text by adding the missing space in line 144 and others.
Response: The whole text was corrected by adding the missing space before °C and the °symbol was not underlined.
Reviewer: Line 188: change “index value” to “sporulation index”
Response: it was changed.
Reviewer: Lines 244-245, change “probably due to the different capacities for phytochemical extraction and the presence of more polar substances such as phenolic compounds, alkaloids, or flavonoid glycosides” to “probably due to the more polar substances extracted by MeOH have greater fungicidal activity than the less polar compounds extracted by DCM”
Response: It was changed.
Reviewer: Line 252: change “maintained P. italicum…” to “maintained their P. italicum…”
Response: It was changed.
Reviewer: Line 254: change “O. virgata, both” to “O. virgata and both”
Response: It was changed.
Reviewer: Line 255: change “A. inundata showing” to “A. inundata, which showed”
Response: It was changed.
Reviewer: Line 282: change “fungicides” to “fungicide”
Response: It was changed.
Reviewer: Line 292: Do not begin a sentence with a number.
Response: A connector was used at the beginning of the sentence.
Reviewer: Line 328: “p” in low-case letters and in italics
Response: changed.
Reviewer: Line 349: eliminate “mostly”
Response: It was eliminated.
Reviewer: Line 382: write either “Albizia spp were used” or “Albizia sp was used”
Response: It was changed to Albizia sp. was used.
Reviewer: Line 388: change “anti-Candida” to “anticandidal”
Response: It was changed.
Reviewer: Line 395, 396: eliminate “due to their sedative properties”
Response: It was eliminated.
Reviewer: Line 485: the titles of books must be written in capital letters.
Response: It was corrected.
Reviewer 2 Report (New Reviewer)
suggestions and comments are included in the manuscript.suggestions and comments are included in the manuscript.
Comments for author File: 
Comments.pdf
Author Response
Responses to Reviewer 2:
Thank you very much for your revisions. As this reviewer made his/her comments on pdf, we now respond to them indicating the line of the request made in the original MS.
Line 34: Current production data and percentages of production damage caused by these diseases were mentioned. We need to say that in the previous submission of the article, there was a detailed analysis of oranges production worldwide, in South America and Argentina, but all the reviewers agreed that we should delete all these sections when resubmitting the manuscript. Anyway, we now respond to this reviewer’s query.
Line 39: Order and taxonomic families of the phytopathogens under study in this article are now mentioned.
Line 42: more citations were added.
Line 55: G. was spelled as Geotrichum.
Line 84 and line 90: This comment is opposite to that made by Reviewer 1, in which he/she asked to compare the advantages of using the different types of natural products (microorganisms and plant extracts) as controllers. So, we decided to maintain a brief explanation of biological control in the introduction.
Line 91: The word study was added.
Line 99: In this paragraph, not only the aim of the study was depicted (only the first sentence) but also a summary of methodologies and results is mentioned. We consider it important to present all the sections of the article in the introduction.
Line 104: For better comprehension, Plant material and Extract preparation were divided into two sections. All the species used for the study are mentioned in Table 1 and the volume of the solvent used is also mentioned in the text (250 ml of DMC and 250 mL of MeOH).
Line 109: Conditions of the suitable environment were mentioned.
Line 114 and line 120: The comments were strengthened with a scientific reference in the result section. References: 1-Derita, M.; Leiva, M.; Zacchino, S. Influence of plant part, season of collection and content of the main active constituent, on the antifungal properties of Polygonum acuminatum Kunth. J. Ethnopharmacol. 2009, 124, 377-383. https://doi: 10.1016/j.jep.2009.05.029. 2-Svetaz, L.; Zuljan, F.; Derita, M.; Petenatti, E.; Tamayo, G.; Cáceres, A.; Cechinel Filho, V.; Giménez, A.; Pinzón, R.; Zacchino, S.A.; Gupta, M. Value of the ethnomedical information for the discovery of plants with antifungal properties. A survey among seven Latin American countries. J. Ethnopharmacol. 2010, 127, 137-158. https://doi.org/10.1016/j.jep.2009.09.034.
Line 124: We cannot change the title of Table 1 as the reviewer did not make a suggestion and we consider it suitable.
Line 125: For better understanding, Fungal strains and Molecular characterization were divided into two sections.
Line 128: The procedures of fungal isolation are described. They are basic microbiological techniques.
Line 132: Those acronyms stand for our Institute affiliation, please see affiliations below the title of the article.
Line 135: The year was deleted.
Line 164 and line 178: We used only one concentration (1000 ppm) because the aim of the work was to realize a high throughput assay using a large number of plant species. That concentration is appropriate to identify if a plant extract will display fungicidal activity or not.
Line 189: Statistical section was added.
Line 194: As the amount of each plant used was fixed (100 g), the yield of each extract is determined by the solvent polarity due to their different extraction capacity. This is a basic concept in organic chemistry.
Line 195: The yield of an extract consists of one number (%) because a mix of dried plants is used to perform it.
Line 200: Please see reference 21 for better comprehension of extract preparations and yields. This topic does not follow the statistical design of other assays. It is a particular field of Pharmacognosy research area.
Line 206: Names were abbreviated.
Line 212: The meaning of ITS was already explained in the material and methods section. The meaning of BLASTn was now added in the same section.
Line 213: Numbers were homogenized.
Line 223: Tables and Figures are as close as possible to the paragraphs. Moreover, this will be set by the editorial process.
Line 226: Percentages of fungal inhibition are now with one decimal, but extract yields are with two decimals due to the precision of both measurements.
Line 246: This paragraph was reordered according to Reviewer 1 comment. Orthosia name was spelled out in full.
Line 263: We referred cite 21 to support this.
Line 271: The reason why we have prepared two tables to show inhibition percentages (at 7 and 14 days) is due to the huge information that they bring. To join both tables into one would make understanding difficult.
Line 272: Statistical analysis section was added in material and methods.
Line 308: Same consideration as the response in line 271.
Line 323: The concentration was the same as the alternative natural product evaluated in this article.
Line 364: References about the chemical contents of these plants are mentioned. We consider it important to depict botanical and geographical data of the six plant species to be consistent with the title of the work.
Line 405: We consider it not suitable to mention the family and taxonomic order of all the microorganisms that are referenced in the literature cited. That is the reason to cite those articles. We cannot explain what other authors have already published, otherwise, the article would be a review.
Line 416 and 435: we comment on references here, so we cannot explain how these metabolites act against fungus because it is not explained in those references.
Line 478: In the case of old references, it is due to the few studies (sometimes old) that present the most active plant species described here. But this could be a good reason to encourage their study.
Author Response File: 
Author Response.docx
Reviewer 3 Report (New Reviewer)
Natural products obtained from Argentinean native plants act 2 as fungicidal against citrus postharvest diseases
Plants are an inexhaustible source of active compounds with remarkable antifungal properties. Finding researchers who are dedicated to harnessing these properties to fight plant pathogenic fungi in plants is a source of satisfaction. It would be interesting to be able to extract all this potential to start substituting many of the chemicals that are applied to foods that will later enter the food chain, either indirectly through animal feed or directly through human consumption. In addition, the application of chemicals severely damages the environment. Everything that can be obtained from nature saves resources and also contributes to minimizing the impact of climate change.
In this sense, my congratulations for the research work presented in this article. In my opinion, it is a very interesting publication because of the findings presented.
General comment
As for the manuscript presented, in general, it is neat and the results are presented in an orderly and clear manner. In terms of material and methods, a section on statistical treatment is missing. The discussion is good but I would recommend adding a paragraph on other natural fungal control techniques to highlight the importance of fungicides from natural plant extracts.
Specific comments
Line 33: Add a cite.
Line 37-41: Sentence too long. Divide in two to a better understanding.
Line 78: “ … market globalization”. The correct expression is “Marketing globalization”
Line 184: cite of scale? Is a own scale? Can you explain on what basis the reference values were proposed?
Questions
Were the extracts used analysed organoleptically? Do they add flavour to the orange? Are they safe for the consumer because of the addition of extraction chemicals?
Personal opinion
Lines 341-343: Very interesting finding. It is much appreciated and interesting.
Line 362-364: I strongly encourage you to continue researching about this plant.
Comments for author File: Comments.pdf
Author Response
Responses to Reviewer 3:
Reviewer: Plants are an inexhaustible source of active compounds with remarkable antifungal properties. Finding researchers who are dedicated to harnessing these properties to fight plant pathogenic fungi in plants is a source of satisfaction. It would be interesting to be able to extract all this potential to start substituting many of the chemicals that are applied to foods that will later enter the food chain, either indirectly through animal feed or directly through human consumption. In addition, the application of chemicals severely damages the environment. Everything that can be obtained from nature saves resources and also contributes to minimizing the impact of climate change.
In this sense, my congratulations for the research work presented in this article. In my opinion, it is a very interesting publication because of the findings presented.
Response: Thank you very much for these comforting words. We greatly appreciate it.
General comment
Reviewer: As for the manuscript presented, in general, it is neat and the results are presented in an orderly and clear manner. In terms of material and methods, a section on statistical treatment is missing. The discussion is good but I would recommend adding a paragraph on other natural fungal control techniques to highlight the importance of fungicides from natural plant extracts.
Response: A section on statistical treatment and the correspondent reference was added. A paragraph on other natural fungal control techniques was added at the beginning of the conclusion.
Specific comments
Reviewer: Line 33: Add a cite.
Response: This sentence was corrected according to Reviewer 1 comments and the corresponding cite is number 1.
Reviewer: Line 37-41: Sentence too long. Divide in two for a better understanding.
Response: This sentence was corrected according to Reviewer 1 comments.
Reviewer: Line 78: “ … market globalization”. The correct expression is “Marketing globalization”
Response: It was corrected.
Reviewer: Line 184: cite of scale? Is an own scale? Can you explain on what basis the reference values were proposed?
Response: The cite was added: Di Liberto, M.G.; Seimandi, G.M.; Fernández, L.N.; Ruiz, V.E.; Svetaz, L.A.; Derita, M.G. Botanical control of citrus green mold and peach brown rot on fruits assays using a Persicaria acuminata phytochemically characterized extract. Plants 2021, 10, 1-13. https://doi.org/10.3390/plants10030425. Here are the bases of the reference values proposed.
Questions
Reviewer: Were the extracts used analyzed organoleptically? Do they add flavor to the orange? Are they safe for the consumer because of the addition of extraction chemicals?
Response: Extracts were not technically analyzed organoleptically, but we sniffed them and we did not appreciate the smell or flavor. Maybe essential oils could add flavor to the oranges, but in this article, we did not use essential oils but fixed extracts. They are completely safe for the consumer because the solvents used for preparation were fully eliminated under evaporation at very low pressure.
Personal opinion
Reviewer: Lines 341-343: Very interesting finding. It is much appreciated and interesting.
Response: Thank you very much.
Reviewer: Line 362-364: I strongly encourage you to continue researching this plant.
Response: We promise it. We will continue with safety topics, chemical formulations, and applications on fruits, among others.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report (New Reviewer)
the authors made the suggested corrections, the manuscript is ready for publication in the form in which it is presented.
Author Response
Academic Editor: Dear Authors,
Your responses have met almost all the reviewer’s requests. However, there are some additional revisions listed below.
Response: Dear Academic Editor. Thank you very much for your revisions, we have responded to each one and you could see the changes in the track changes version of the MS.
Line 112: ‘suitable environment (a dark room with low relative humidity)’. Please indicate the value of relative humidity, temperature and how long (for 24 h, 48 h..?)
Response: These data were added.
Suggestion for Table 1. ‘Plant species evaluated against the orange pathogens Penicillium digitatum, P. italicum, and Geotrichum citri-aurantii. Botanic family, plant scientific name, Voucher specimen, collection data, and place of collection or acquisition are also described.’ Change with ‘Plant species used for extracts preparation to be tested against the orange pathogens Penicillium digitatum, P. italicum, and Geotrichum citri-aurantii. Botanic family, Voucher specimen, collection data, and place of collection or acquisition are also reported.
Response: The title of Table 1 was changed according to this suggestion.
Line 170: ‘containing 1000 ppm of each plant extract’. Have you carried out previous studies for choosing this concentration? If yes and they are published, please, indicate it in the text and insert a reference.
Response: It was indicated and two references were inserted.
Line 183: Why did you test a different concentration (3000 ppm) from the ‘vitro susceptibility test’?
About the concentration used in this article, you also state ‘The concentration was the same as the alternative natural product evaluated in this article.’ In relation to Reviewer2 request at line 331: 3000 ppm aqueous solution of Imazalil®. Where is mentioned the ‘natural product’? It was never mentioned but it is important because you have chosen the concentration in relation to this product.
Response: for the in vivo test, the 3000 ppm concentration of the extract was chosen because we did not have a good response when we treated the fruits with a 1000 ppm concentration. So, we decided to hardly increase the fungicidal concentration resulting from the in vitro assays. This was now mentioned in the revised version.
The expression “natural product” in the response to Reviewer 2 refers to S. pilcomayense methanolic extract. Its concentration was described in treatment 2. The same concentration of the natural product (S. pilcomayense methanolic extract) was used for Imazalil.
Line 190: A point is missing. ‘surface) [20] The sporulation’
Response: The point was added.
Line 194: ‘Remarkably, yields were higher 202 from MeOH extractions than DCM, because of solvent polarity and extraction efficiency ‘
Rev 2 ‘An explanation is needed for this assertion. The polarity of the solvent affects metabolite carryover, while the yield is determined by the amount of plant used, the filtration method, and the solvent recovery time or drying. It is important to justify or discard this assertion.’
Response: This assertion was discarded.
Line 204: ‘O. virgata showed similar crude extract percentages’. E’ giusto quell oche ha scritto il revisore2: To express similarities or differences, it is necessary to perform an ANOVA and a comparison of means. I consider that the focus of the study is not the performance of the extraction, but rather the effectiveness of the various extracts performed.’
Response: That´s right. The yield of an extract is a percentage calculated from the mass of the crude extract obtained (once the solvent was completely eliminated) referred to as the mass of plant dry material initially processed.
Ls 207-209: ‘This fact may support the hypothesis that the place of collection and the environmental conditions could determine the composition and quantity of secondary bioactive metabolites in plants [21].’ You are talking about yield, not about composition. However, this sentence is more suitable in Discussion section.
Response: This sentence was inserted in the Discussion section.
In accordance with the Reviewer 2, you must decide whether to use one or two decimal places in all the numbers throughout the document. Just some examples, in line 235 you have one decimal (80.0, 79.3, and 75.2%), in line 222 two decimals (99.43%). Choose one type and applied it in all text. If you choose two decimals due to the precision of
Measurements, two decimals should applied in all document.
Response: We decided to use one decimal throughout the document. It was carefully corrected.
Lines 252-254: This sentence is more suitable in Discussion section. In Result section only the presentation of the data obtained. Please, if there are any other sentence like this, include them in Discussion section.
Response: This sentence was removed and commented in Discussion section.
Table 3-4: You cannot comment these tables without statistical analysis.
Response: Statistical analysis was done. It was explained in section 2.7 and significant differences are now shown in Tables 3 and 4.
Line 234: You cannot state this: ‘but the most active ones after 7 days of incubation were obtained from T. tricholepis, A. inundata and P. stelligerum’ there is no statiscal analysis in the table to support it.
Response: Now statistical analysis was performed.
Line 236: ‘P. italicum inhibition by DCM extracts resulted in lower’, At the beginning of a sentence all latin name must be specify, no abbreviations. There some mistake in this sentence, probably ‘Pennicilium italicum inhibition by DCM extracts resulted lower’. Anyway, to state this, you need the statistic for supporting it.
Response: Penicillium was full-spelled and the word “in” was deleted.
‘3.3. Statistical analysis of the antifungal screening’. The name of this paragraph is wrong. Statistical analysis section is presented in materials and methods. Paragraph 3.3 must renamed.
Response: Paragraph 3.3 was renamed.
Discussion section:
I agree with Reviewer 2, The discussion section should not review the botanical characteristics of the plant. Instead, it would be better to mention references about the compounds found in the south American plants. If there are no reports, look for something related to the genus to which they belong. If you still do not find anything, mention the botanical families to which they belong. Otherwise you can search for literatures concerning compounds composition and biological activities of other South American plants/plant extracts (more recent articles!). This is important to support your findings. Probably, you will find some articles about antifungal activities of these extracts against plant and human pathogens and their mode of action against pathogens and you can discuss it.
Response: Botanical characteristics of the plants were removed. Botanic family, common name, pharmacological activities, mode of action when it was found, and isolated compounds were the topics mentioned for each species, genera, or family. Moreover, more recent references were added and old ones were eliminated.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
This paper describes important research on naturally occurring fungicides with commercial potential. Why was the orange production data analysed and presented? This data is interesting, but it seems out of place in this manuscript.
It is recommended to confirm the identities of the fungal isolates using molecular techniques.
The descriptions of the plant species would normally be in the methods section. I understand that this information is presented in the discussion because the authors have only described six of the species and the information was presented in context with the results.
Minor edits include:
Line 16 – suggest replacing ‘inadequate’ with ‘improper’ since ‘inadequate’ implies that not enough of the fungicide was applied, but the opposite is typically the case when resistance develops.
Line 21 – spell out what DMSO stands for
Line 22 – replace ‘tested samples’ with ‘products tested’
Line 23 – replace ‘resulted’ with ‘were’
Line 24 – replace ‘plants’ with ‘products’
Line 25 – ‘pathogens were extracted from the following plants: ….’
Line 26 – replace ‘This species was‘ with ‘These products were’
Line 27 – ‘treatments’
Lines 28-29 – ‘The products extracts from native plants have fungicide potential, but further studies are required.’
Line 33 – ‘widest fruits’ is misleading, suggest changing to ‘one of the most widely consumed fruits’
Line 43 – ‘cause citrus diseases by invading wounds in the fruit rind. Wounds occur during harvest and …’
Line 48 – replace ‘turning out’ with ‘becoming’
Line 50 – ‘In the meantime …’
Line 57 – ‘pulp’
Line 63 – ‘employed postharvest by the’
Line 74 – ‘the most widely used treatments for more’
Lines 88 -100 – I suggest revising this paragraph to outline the objectives of the study, rather than summarising the method.
Line 109 – ‘Each plant sample was identified …’
Line 139 – replace ‘by’ with ‘in’
Lines 145-146 – ‘A negative control was prepared…’
Lines 146-147 – ‘… and the solvent DMSO without plant extract served as a positive (growth) control.’
Line 169 – delete ‘in’
Lines 185-187 – ‘Yields were higher from MeOH extractions than DCM, because of solvent polarity and extraction efficiency.’
Line 190 – replace ‘respond to’ with ‘support’
Line 214 – replace ‘by’ with ‘in’
Line 215 – ‘… using the agar…’
Line 239 – ‘phytochemical’
Lines 244-45 – ‘…extracts were not as high as P. digitatum …’
Line 249 – what does ‘being not negligible the activity’ mean?
Line 258 – ‘metabolite’
Lines 252-253 – ‘...where plants were collected from two different locations…’
Line 259 – replace ‘by’ with ‘in’
Lines 266-267 – ‘The results were divided into the following categories for statistical analysis: …’
Line 270 – replace ‘resulted to be’ with ‘were rated as’
Line 276 – ‘replace ‘being’ with ‘with’
Line 279 – replace ‘resulted’ with ‘were’
Line 283 – replace ‘resulted’ with ‘were’
Line 292 – ‘have potential as fungicides given their fungicidal activity was maintained for more than 14 days.’
Line 309 – remove ‘s’ from ‘percentage’
Line 316 – remove ‘s’ from ‘percentage’
I suggest changing the label on the Y axis of Figures 3 and 4 to ‘% extract activity’.
Line 319 – replace ‘of’ with ‘from’
Figure 5 is an excellent way to illustrate the results and rating system.
Line 345 – replace ‘consequently with’ with ‘due to’
Line 346 – replace ‘resulted’ with ‘more’
Line 359 – leaf species’
Line 369 – ‘…liver protectant and for…’
Line 392 – delete ‘provided’
Line 416 – ‘the bioactivity of P. alliacea leaf extracts could …’
Line 438 – replace ‘presents’ with ‘contains’
Line 464 – replace ‘provide’ with ‘contain’
Line 466 – replace ‘diminish’ with ‘reduce’
Line 471 – replace ‘resulted to be’ with ‘was’
Line 472 – replace ‘over’ with on’
Reviewer 2 Report
The manuscript investigated the effect of natural produces extracted from native plants in Argentinean on controlling postharvest diseases in citrus. The topic is interesting and provide a new idea for inhibiting postharvest disease in fruit by using fungicides alternatives. However, the design and organization of this manuscript were disordered, and the lack of some basic background in the introduction made the meaning and innovation of this study unclear. The discussion was not good enough to explain the results obtained. Therefore, I am regretted for having to reject this paper.
General comments
1. Tediously introduction of postharvest diseases in citrus caused by P. digitatum, P. italicum and G. citri-aurantii as well as fungicides used to control the diseases should be abridged. In the introduction, investigation of using naturel extracts to control fungal diseases in fruit needs to be concluded. Please rewrite it.
2. Author used two solutions, dichloromethane and methanol, to extract natural compounds from plants. Except extraction yield, the chemical composition in each extraction obtained by the two solutions should be different, which need to be clarified. In this case, I suggestion that qualitative and quantitativeanalysis of the components in plant extract solutions should be carried out by GC-MS.
3. Except radial growth, mycelial growth, spore germination, minimum inhibitory concentration and EC50 are also important parameters to investigate antifungal activity of each extract. Please add the results. In addition, more experiments should be done in vivo experiment, including incidence, lesion diameter.
4. In the discussion, author need to compare the results with those of others and discuss possible relevant mechanisms. Please rewrite it.
Specific comments
1. Please delete ‘2.1 World, South American, and Argentinean orange production data from 1980 to 2020’. It is unnecessary.
2. How many plants of each species were collected and used in the experiment should be clarified.
3. The extraction process of natural produces from plants must be clarified since it’s the most important part of the experiment.
4. In figure 6, it’s better to show the whole morphology of each species of plants, including roots, leaves, stems and flowers, rather than parts of the plant.
5. The antifungal components of the plant extract solutions obtained by different extraction solutions are different. In vivo and vitro experiments, which one was used ? DCM extracted or MeOH extracted? Why?
6. Which fungus was used in vitro experiment ? The preparation of spore suspension should be clarified.
7. Line 143-144: ‘A conidia concentration between 104 and 105 CFU mL-1 was inoculated…’ The accurate concertation of spore suspension should be used in experiment, not a range. Moreover, the concentration of each plant extractedsolution should be expressed as ul/ml or % instead of ppm on line 145.
8. Please use spore/ml instead of CFU/ml
9. Artificial injury and fungal inoculation in orange fruit should be described in detail.
10. Fruit is usually treated by exogenous solutions and then inoculated with fungal pathogen. In this experiment, orange fruit was inoculated by fungus, and then treated by plant extracted solution. Please explain it. Moreover, why only S. pilcomayense methanolic extract was used in vivo experiment?
11. Pictures of fungal growth on plate with different extracts treatment should be better exhibit the antifungal activity. Please add it.
12. Fig 3 and 4 are very confused. Please reorganize the data and show the results in another way.
13. English of the manuscript should be improved and scientific language must be used.
Reviewer 3 Report
The manuscript studies the effects of the extracts obtained from Argentinean native plants on the development of some postharvest citrus fruit pathogens. The topic is important because of the need to replace synthetic fungicides with safer ones. The authors tested a large number of species, in some cases the same species was collected in different environments to evaluate the environmental effects on the fungicidal capacity. Nevertheless, this manuscript looks more like a preliminary research. It would have been interesting and would have represented a real advance in the knowledge to correlate the profile and concentration of the main secondary metabolites with the fungicidal activity. Unfortunately, the profile and the concentration of the main secondary metabolites are completely lacking, I suggest the authors to add this data to improve the manuscript and resubmit it considering also the following issues.
The following are some other major and minor corrections:
- Please be more precise in the description of the preparation of the extracts. In particular, be more precise about the biomass solvent ratio. It is not completely clear the extraction time: 3 x 24h does mean that the extraction time is 72 h or 3 refer to the replications??At what temperature the extraction has been carried out????
- Line 145. Are you sure that the concentration of the extract is 1000 ppm???It sounds weird because normally the extracts’ concentration in in vivo experiments are larger than in vitro ones, but in your paper is the contrary.
- Line 163 please indicate the UR inside the storage room.
- The paragraph 3.1 does not give any additional information about the fungicidal activity and is out of the topic of the paper. I would ask the authors to delete it.
- Tables 3 and 4. What do you mean with “by quadruplicate”??? if the value is the average of the inhibition percentage of the four sectors in which the petri dish was divided, please write it more clearly
- Please modify figure 2 some photos of the in vitro tests (control and treatments). Figure 2 how it was designed is of poor interest considering that the pathogens tested are extremely well known
- line 253 it is not specified whether plants were collected only in different places or even in different dates. This is quite important since the concentration of phenolic compounds is also affected by temperature and by the plants’ developmental stage.
- Line 258 please add a citation
- why did you use for in vivo experiments an extract concentration of 300 ppm??
- Line 344 “and this fact is consequently with the number and type of molecules that can be 345 extracted by the solvent methanol” this is a plausible but not confirmed hypothesis since no analyzes of this type have been made. Please change or delete this sentence.
The discussion is fully focused on the botanical aspects of each species but it gives very few inputs on the fungicidal activity of the extracts and on the possible causes of this activity. I suggest the authors to write it again.
Reviewer 4 Report
In this manuscript, the author investigated the fungicide properties of 20 native plants from the central zone of Argentina against Penicillium italicum, P. digitatum, and G. citri-aurantii. To screen for natural fungicides that can be used for citrus disease control. This study shows interesting and relatively novel results. But there are several points to take into consideration.
1. The authors describe in detail the types of citrus fruit diseases, and the main chemical fungicides currently used in the Introduction, but the presentation of 20 natural plants, which are important materials for this study, is lacking.
2. The authors used two solutions dichloromethane (DCM) and methanol (MeOH) to extract the natural products and performed activity assays, why did you choose these two extraction methods?
3. For the “Curative effect of S. pilcomayense methanolic extract over oranges infected with P. digitatum”, the experimental design should present certain data and not just the results in the form of photos.
4. In this work, natural plant components with significant control of citrus fruit diseases were screened, but safety evaluation is lacking. Since the properties of these plants are not known, it is important to consider whether their extracts are toxic or bad for human health.
5. Some formatting errors need to be corrected, for example, “4” should be superscripted on line135, authors should check similar issues carefully and modify them.
