Chemical Composition of Healthy and Raspberry Leaf Blotch Emaravirus-Infected Red Raspberry ‘Willamette’ Fruits
, Marko Petković 1, Bojana Vasilijević 4, Vera Katanić 4 and Darko Jevremović 4Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript compares the phenolic profiles of healthy raspberry plants to those of the same raspberry cultivar infected with raspberry leaf blotch virus (RLBV) at three different locations in Serbia. The authors present data supporting dominant roles for climatic conditions and soil chemistry in the differences in phenolic profiles. It seems more difficult to see the effect of RLBV infection on phenolic profiles, and the authors address this issue. Some other comments about the manuscript follow.
Lines 126-127. What is the molarity of the HCl in the ethanol-HCl 85:15? Is it 12 M?
Results and Discussion: Given the importance of climatic conditions to this study and the reference to differences in climatic conditions among the three collection sites, I think it would be helpful to summarize the results of Table 2 in a short paragraph. On line 228, it is stated that the differences in weather conditions are “clearly seen” in Table 2, but I think that the single sentence following that statement does not say enough about something so important to the study.
Table 3: Why is there a capital A in the table, when all the other letters are lowercase?
Table 3 footnote: Change “raw” to “row”.
Line 174-175. Why could the influence of harvest year and locality actually be understood as the influence of weather conditions and soil composition? Please elaborate on this statement with a brief description of differences in weather conditions among the years, and a brief description of the differences in soil composition among the localities.
Line 181. By Appendix, do you mean the supplementary data (Table S1)?
Line 210. Change this Figure 1 to Figure 2.
Lines 213-214. I recommend putting “Table S1” in parentheses at the end of the first sentence in this paragraph so that readers can immediately refer to that table as they follow the paragraph.
Line 216. If you have “Table S1” at the end of the first sentence in the paragraph, you can delete “Appendix” here.
Line 255. Very little is ever proven in science. Hypotheses are not proven; they are either supported or rejected. I recommend changing “proved” to “demonstrated” or “determined.”
Line 270-271. I recommend changing “such as” to “including,” and deleting the “etc.” at the end of this sentence. I think that “etc.” is too vague for a scientific manuscript.
Line 284. Do you mean “CY3-GLU” or “Q3-RHA”?
Lines 285-286. Many authors have found a relationship between increased hydroxycinnamic acid production and response to pathogens. There are many studies on the relationship between lignification (involves hydroxycinnamic acids) and disease resistance. The following paper has some good results on the effects of hydroxycinnamic acid inhibition and some possibly helpful references: Moerschbacher BM, Noll U, Gorrichon L, Reisener H-J. 1990. Specific inhibition of lignification breaks hypersensitive resistance of wheat to stem rust. Plant Physiology 93:465-470.
Lines 291-295. I think that tolerance is typically assessed from symptoms, not from phenolic profiles. Can you define tolerance in this paragraph and cite a couple of papers to support your definition?
Lines 295-296. Why was the effect of RLBV indisputable? Please support this statement by referring to the appropriate table or figure.
Lines 329-330. I do not think that tolerance would be assessed from phenolic profiles, but if you define tolerance earlier, as suggested above, that issue will be resolved.
Lines 335-336. Can you determine, based on only 2 years of data, that influences of soil and weather will always overpower the RLBV effect on the chemical composition?
Table S1. Please insert a footnote explaining the meaning of the superscripted lowercase letters within the columns. Without that information, I cannot follow the results described within the text of the manuscript.
Comments on the Quality of English LanguageThe English was quite easy to follow, with minor errors.
Author Response
Comment 1: Lines 126-127. What is the molarity of the HCl in the ethanol-HCl 85:15? Is it 12 M?
Response: This data was missing and is added now in the manuscript. It is 1.5 M HCl.
Comment 2: Given the importance of climatic conditions to this study and the reference to differences in climatic conditions among the three collection sites, I think it would be helpful to summarize the results of Table 2 in a short paragraph. On line 228, it is stated that the differences in weather conditions are “clearly seen” in Table 2, but I think that the single sentence following that statement does not say enough about something so important to the study.
Response: The reviewer raised a good point. The new paragraph is added in the manuscript, regarding the weather conditions.
Comment 3: Table 3: Why is there a capital A in the table, when all the other letters are lowercase?
Response: It is a typo, and is corrected.
Comment 4: Table 3 footnote: Change “raw” to “row”.
Response: It is corrected.
Comment 5: Line 174-175. Why could the influence of harvest year and locality actually be understood as the influence of weather conditions and soil composition? Please elaborate on this statement with a brief description of differences in weather conditions among the years, and a brief description of the differences in soil composition among the localities.
Response: A description is added in the manuscript.
Comment 6: Line 181. By Appendix, do you mean the supplementary data (Table S1)?
Response: Indeed! It is corrected in the manuscript.
Comment 7: Line 210. Change this Figure 1 to Figure 2.
Response: It is corrected.
Comment 8: Lines 213-214. I recommend putting “Table S1” in parentheses at the end of the first sentence in this paragraph so that readers can immediately refer to that table as they follow the paragraph.
Response: It is corrected.
Comment 9: Line 216. If you have “Table S1” at the end of the first sentence in the paragraph, you can delete “Appendix” here.
Response: It is corrected.
Comment 10: Line 255. Very little is ever proven in science. Hypotheses are not proven; they are either supported or rejected. I recommend changing “proved” to “demonstrated” or “determined.”
Response: Great comment. It is corrected.
Comment 11: Line 270-271. I recommend changing “such as” to “including,” and deleting the “etc.” at the end of this sentence. I think that “etc.” is too vague for a scientific manuscript.
Response: It is corrected.
Comment 12: Line 284. Do you mean “CY3-GLU” or “Q3-RHA”?
Response: This was a mistake. It should be ISO-Q, and thus it is corrected.
Comment 13: Lines 285-286. Many authors have found a relationship between increased hydroxycinnamic acid production and response to pathogens. There are many studies on the relationship between lignification (involves hydroxycinnamic acids) and disease resistance. The following paper has some good results on the effects of hydroxycinnamic acid inhibition and some possibly helpful references: Moerschbacher BM, Noll U, Gorrichon L, Reisener H-J. 1990. Specific inhibition of lignification breaks hypersensitive resistance of wheat to stem rust. Plant Physiology 93:465-470.
Response: This is great article. It is added in the manuscript as a reference.
Comment 14: Lines 291-295. I think that tolerance is typically assessed from symptoms, not from phenolic profiles. Can you define tolerance in this paragraph and cite a couple of papers to support your definition?
Response: You are indeed right. According to the literature, tolerance is the ability to lessen the effects of a viral infection, regardless of the pathogen load. The plant's ability to grow, yield, or reproduce is very slightly impacted, even when a significant viral load persists, and any obvious symptoms are either absent or mild. From the pomologists and phyto-pathologists point of view the cultivar is considered tolerant in the case when it can be infected with the virus, the symptoms are present on the leaves (mild or severe), but there are no significant changes on the fruits (physical properties and basic chemical properties) and no significant decrease of yield. Here, we wanted to strengthen the tolerance level of examined plum cultivar with these additional properties (phenolic profile).
We added this explanation to the paper supported with adequate literature and corrected this in the manuscript.
Also, often in the papers, researchers from other disciplines misinterpret resistance and tolerance. Resistant cultivar cannot be infected – it is immune to the virus.
Comment 15: Lines 295-296. Why was the effect of RLBV indisputable? Please support this statement by referring to the appropriate table or figure.
Response: It is added in the manuscript.
Comment 16: Lines 329-330. I do not think that tolerance would be assessed from phenolic profiles, but if you define tolerance earlier, as suggested above, that issue will be resolved.
Response: As given above in the previous comment we corrected this in the manuscript.
Comment 17: Lines 335-336. Can you determine, based on only 2 years of data, that influences of soil and weather will always overpower the RLBV effect on the chemical composition?
Response: Indeed, two years of examination is too short for such strong conclusion. Thus, such conclusion is "softened" in the manuscript.
Comment 18: Table S1. Please insert a footnote explaining the meaning of the superscripted lowercase letters within the columns. Without that information, I cannot follow the results described within the text of the manuscript.
Response: The footnote is added in the Supplementary Table 1.
Reviewer 2 Report
Comments and Suggestions for AuthorsDear,
Upon reading your manuascript here are my comments/concerns:
Introduction is proper no comments.
Material and methods section is lacking:
Line 118-128:
- samples (~ 150 g) were poured into liquid nitrogen: This is probably a tipo, samples were not poured?
- A powdered sample (10 g) was mixed with 50 mL of 96% ethanol and ultrasonicated: Why use 96% ethanol. In fruit there are a lot of phenolic acids, using so much ethanol and that little water will not shop up phenolic acids?
Furthermore what is the remaining 4%, bidistiled water or something else??
You say the samples were ultrasonicated, on which temperature?
Moving on:
After 30 min of extraction, the mixture was centrifuged two sequential times for 15 min at 3500 rpm, and the supernatant was filtered through a 0.45 mm Minisart filter before analysis
Why were they centrifuged 2 times? what is the point in that? Furthermore express the rpm in g which is correct and comonly used, so other researchers can know what speed it was, as it is depended on the radious of the machine... Furthermore: Add suplier of the filters.
Three repetitions is not enough, you have such a smal sample only 6 extractions?? why not go for at least 10?
All these determinations were performed in triplicate, and results were presented as the mean value of three measurements ± standard deviation.
Standard error or deviation?? Rewrite this sentence, poor english.
Line 129:134 Major problem!
2.5. HPLC-DAD Analysis
As previously published [18], specific phenolic components were quantified using reversed phase HPLC analysis (Agilent Technologies, Santa Clara, CA, USA). The retention times and spectra of phenolic compounds were compared to those of the standards, and quantification was based on the calibration curves and peak areas. The results were obtained in mg/mL and then reported as mg/100 g or mg/kg of fresh weight.
You canot identify phenolic compounds without confirming the compounds on mass spectrometer. This data is therefore not correct, peaking is not being done for some time now, and where it was the results are not correct in majority of cases.
Unfortunately due to the absence of mass spectrometer to confirm the compounds (if used you would see many of the compounds you detected are not even present), this article canot be accepted. I suggest you find an institution that has a mass spectrometer to identify the compounds (peaks in your chromatogram). Without that this can not and should not be published as it has serious flaws in experimental design, to be precise compound identification. Once corrected this would prove an interesting articlet. I also suggest having more than 3 replicates (at least 5 if you have so little samples).
Kind regards
Comments on the Quality of English LanguageMinor speel check in material and methods section is required
Author Response
Comment 1: Line 118-128: samples (~ 150 g) were poured into liquid nitrogen: This is probably a typo, samples were not poured?
Response: Indeed, it is a typo. It is corrected.
Comment 2: A powdered sample (10 g) was mixed with 50 mL of 96% ethanol and ultrasonicated: Why use 96% ethanol. In fruit there are a lot of phenolic acids, using so much ethanol and that little water will not shop up phenolic acids? Furthermore what is the remaining 4%, bidistiled water or something else??
Response: Ethanol is a usual solvent for fruits/vegetables/herbs extraction (as well as methanol, hexanol, acetone). Sometimes is even pure ethanol used, but usually it is an aqueous ethanol solution, and the fraction of ethanol varies in extraction procedures. For instance:
Puganen et al. (2018) used 92% ethanol (DOI: 10.1021/acs.jafc.8b00177);
Unal et al. (2022) used 80% ethanol (DOI: 10.3390/horticulturae8030267);
Zengin et al. (2023) used 70% and 100% ethanol (DOI: 10.3390/antiox12101842);
Neimkhum et al. (2021) used pure hexanol, pure EtOAc, and 95% ethanol (DOI: 10.3390/antiox10091345). In the Conclusion it is stated: In contrast, ethanol could extract the greatest amount of phenolics and flavonoids from C. carandas.
Apparently using 96% ethanol yields in significant amount of extracted polyphenolic. Utilization of some other solvent would probably yield a higher level of polyphenolics, but the aim of this manuscript is not extraction optimization.
Comment 3: You say the samples were ultrasonicated, on which temperature?
Response: At 20 C. It is added in the manuscript.
Comment 4: After 30 min of extraction, the mixture was centrifuged two sequential times for 15 min at 3500 rpm, and the supernatant was filtered through a 0.45 mm Minisart filter before analysis.
Why were they centrifuged 2 times? what is the point in that? Furthermore express the rpm in g which is correct and comonly used, so other researchers can know what speed it was, as it is depended on the radious of the machine... Furthermore: Add suplier of the filters.
Response: Centrifugation was performed in order to remove all solid particles present in the extract. These particles may prompt problems in subsequent analyses (especially HPLC) and give incorrect results.
RPM is expressed in g and added in the manuscript. Supplier of the filters is added in the manuscript.
Comment 5: Three repetitions is not enough, you have such a smal sample only 6 extractions?? why not go for at least 10?
Response: 150 g of raspberry samples was frozen in liquid nitrogen, and subsequently grounded in the stainless-steal blender, so very fine raspberry powder was obtained. Ultrasonification/extraction of ethanol/powder system is very efficient, and we believe that 3 extraction is quite sufficient in order to obtain representative sample for further analyses.
Comment 6: All these determinations were performed in triplicate, and results were presented as the mean value of three measurements ± standard deviation.
Standard error or deviation?? Rewrite this sentence, poor english.
Response: Results are expressed as mean ± standard deviation (not standard error). The sentence is rewritten.
Comment 7: Line 129:134 Major problem! 2.5. HPLC-DAD Analysis: As previously published [18], specific phenolic components were quantified using reversed phase HPLC analysis (Agilent Technologies, Santa Clara, CA, USA). The retention times and spectra of phenolic compounds were compared to those of the standards, and quantification was based on the calibration curves and peak areas. The results were obtained in mg/mL and then reported as mg/100 g or mg/kg of fresh weight.
You canot identify phenolic compounds without confirming the compounds on mass spectrometer. This data is therefore not correct, peaking is not being done for some time now, and where it was the results are not correct in majority of cases.
Response: We are fully aware that MS is the most precise and the most sensitive detector, but this absolutely does not exclude HPLC as reliable method for identification and quantification of, in this articular case, polyphenolic compounds. No one pronounce HPLC-MS as the only standard method for this purpose. HPLC is being widely used elsewhere for separation, identification and quantification of compounds in complex mixtures.
Comment 8: Unfortunately due to the absence of mass spectrometer to confirm the compounds (if used you would see many of the compounds you detected are not even present), this article canot be accepted. I suggest you find an institution that has a mass spectrometer to identify the compounds (peaks in your chromatogram). Without that this can not and should not be published as it has serious flaws in experimental design, to be precise compound identification. Once corrected this would prove an interesting articlet. I also suggest having more than 3 replicates (at least 5 if you have so little samples).
Response: The use of the MS detector would result in a greater number of detected polyphenolic compounds, but certainly not to the non-detection of compounds that were already identified by HPLC. Furthermore, the Horticulturae Journal recently published so many manuscript where HPLC without MS was utilized:
Al-Ramamneh et al. (2024, DOI: 10.3390/horticulturae10010026)
Pereira Basilio et al. (2024, DOI: 10.3390/horticulturae10010018)
Araujo et al. (2023, DOI: 10.3390/horticulturae9121251)
Su et al. (2023, DOI: 10.3390/horticulturae9111233)
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript: "Chemical composition of healthy and raspberry leaf blotch emaravirus-infected red raspberry ‘Willamette’ fruits" describes the effects of viral status, harvest year and locality on the phytochemical composition of raspberry fruits. This study could have a practical application for ‘Willamette’ producers for the obtainment of high-quality fruits.
The following clarifications and changes are recommended and should be make:
Introduction
- The authors should provide a brief review from the previous data for the effect of viral infection on phytochemical composition in raspberry fruits.
- The aim of the study should be more clearly presented.
Materials and methods
- The RLBV abbreviation should be defined as a note in Table 1.
Results and Discussion
- The PCA analysis should be presented in detail. Which of tested parameters are related positively or negatively to PC1 and PC2?
PPV abbreviation should be defined: „On the other hand, Usenik et al. [32] found a lower content of hydroxycinnamic acids in PPV-infected plums compared to the healthy samples.“.
Comments on the Quality of English LanguageMinor editing of English language.
Author Response
Comment 1: Introduction: The authors should provide a brief review from the previous data for the effect of viral infection on phytochemical composition in raspberry fruits.
Response: The review on the data regarding the effect of viral infection on phytochemical composition in raspberry fruits was added to the manuscript.
Comment 2: Introduction: The aim of the study should be more clearly presented.
Response: Introduction is broadened, with the aim being clearly defined.
Comment 3: Materials and methods: The RLBV abbreviation should be defined as a note in Table 1.
Response: It is added as footnote of Table 1.
Comment 4: Results and Discussion: The PCA analysis should be presented in detail. Which of tested parameters are related positively or negatively to PC1 and PC2?
Response: The reviewer raised a great point. The entire paragraph based on PCA analysis is added in the manuscript.
Comment 5: Results and Discussion: PPV abbreviation should be defined: „On the other hand, Usenik et al. [32] found a lower content of hydroxycinnamic acids in PPV-infected plums compared to the healthy samples.“.
Response: Abbreviation is defined.
Reviewer 4 Report
Comments and Suggestions for Authorsline n. 137, 164: the references cited (18, 19) are wrong
line 138: the description of the analysis method is insufficient
line n. 139: indicate the source of the standards
lines n.159-164: by the HPLC, the 11 compounds are only putatively detected. Nowadays, hplc is an imprecise method (even if there are the standards) for the analysis of metabolites, especially when you evaluating the levels and presence of highly represented classes of compounds. It can be many metabolites under the same peak. Besides, UV-vis spectrum doesn't help to discriminate all metabolites of the same class, exactly. The references, reported in the work, about the biochemical and metabolomic analysis are unclear for the aim. Some chemicals have the same or very close retention time. Therefore, for the acceptance of the present work, it is necessary, at least, to have a more robust, recent references that can be comparable to the results obtained.
Author Response
Comment: line n. 137, 164: the references cited (18, 19) are wrong.
Response: The references have been corrected.
Comment: line 138: the description of the analysis method is insufficient.
Response: Description of the method has been broadened.
Comment: line n. 139: indicate the source of the standards.
Response: It has been added in the manuscript.
Comment: lines n.159-164: by the HPLC, the 11 compounds are only putatively detected. Nowadays, hplc is an imprecise method (even if there are the standards) for the analysis of metabolites, especially when you evaluating the levels and presence of highly represented classes of compounds. It can be many metabolites under the same peak. Besides, UV-vis spectrum doesn't help to discriminate all metabolites of the same class, exactly. The references, reported in the work, about the biochemical and metabolomic analysis are unclear for the aim. Some chemicals have the same or very close retention time. Therefore, for the acceptance of the present work, it is necessary, at least, to have a more robust, recent references that can be comparable to the results obtained.
Response: We are fully aware that hplc has its own limits, and that hplc-ms is the most accurate and the most precise method for separation, identification and quantification of complex extracts. Nevertheless, the polyphenolic profile of the raspberry fruit extracts is quite known and so many articles have been published on this topic. Our results are in great agreement with plenty of published data (identificationwise and quantificationwise), regardless the methods used (hplc, hplc-ms). We really appreciate a good intention of the reviewer who has given us a change to support our results with some recent, robust, comprehensive and trusted references. And therefore we added four recent references which are, in our opinion, quite representative and may sustain our results.
- Fotirić Akšić et al. Chemical fruit profiles of different raspberry cultivars grown in specific Norwegian agroclimatic conditions. Horticulturae2022, 8: 765. https://doi.org/10.3390/horticulturae8090765
- Kobori et al. Changes in the polyphenol content of red raspberry fruits during ripening. Horticulturae2021, 7: 569. https://doi.org/10.3390/horticulturae7120569
- Toshima et al. Comparison of anthocyanins, polyphenols, and antioxidant capacities among raspberry, blackberry, and Japanese wild Rubus species. Sci. Hortic.2021, 285: 110204. https://doi.org/10.1016/j.scienta.2021.110204
- Ponder & Hallmann. The effects of organic and conventional farm management and harvest time on the polyphenol content in different raspberry cultivars. Food Chem.2019, 301: 125295. https://doi.org/10.1016/j.foodchem.2019.125295
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsDear to anwser your comments in short:
We are fully aware that MS is the most precise and the most sensitive detector, but this absolutely does not exclude HPLC as reliable method for identification and quantification of, in this articular case, polyphenolic compounds. No one pronounce HPLC-MS as the only standard method for this purpose. HPLC is being widely used elsewhere for separation, identification and quantification of compounds in complex mixtures.
The use of the MS detector would result in a greater number of detected polyphenolic compounds, but certainly not to the non-detection of compounds that were already identified by HPLC. Furthermore, the Horticulturae Journal recently published so many manuscript where HPLC without MS was utilized:
Al-Ramamneh et al. (2024, DOI: 10.3390/horticulturae10010026)
Pereira Basilio et al. (2024, DOI: 10.3390/horticulturae10010018)
Araujo et al. (2023, DOI: 10.3390/horticulturae9121251)
Su et al. (2023, DOI: 10.3390/horticulturae9111233)
HPLC is not a toll to identify phenolic compounds. This can only be done with different type of mass spectrometers or similar instruments. This information is false. You can quantify and analyse all the compounds but they need to be confirmed using a mass spectrometer. This is not true at all. If other research was published in this yournal that is a problem with the journal standards as this is not correct and it should not be published as results if checked by a mass spectrometer will show that 90% of the compounds are falsly identified. This is a serious problem. In order to publish your study, ship your samples to one of the institutions that has a mass spectrometer. This should however not be published.
Comments on the Quality of English LanguageEnglish is fine
Author Response
Comment: HPLC is not a toll to identify phenolic compounds. This can only be done with different type of mass spectrometers or similar instruments. This information is false. You can quantify and analyse all the compounds but they need to be confirmed using a mass spectrometer. This is not true at all. If other research was published in this yournal that is a problem with the journal standards as this is not correct and it should not be published as results if checked by a mass spectrometer will show that 90% of the compounds are falsly identified. This is a serious problem. In order to publish your study, ship your samples to one of the institutions that has a mass spectrometer. This should however not be published.
Response: The chemical composition of raspberry fruits extract is well known and published elsewhere. Comparing our obtained results with plenty of previously published data reveals great match, in both identification and quantification. Hence, we are confident in the accuracy of our results, and we believe that hplc was an efficient tool for our intended objective.
Reviewer 4 Report
Comments and Suggestions for AuthorsIf I have a low metabolites separation capacity, previuosly, I have to use databases built with a more performing instrument or I have to use references that can strengthen my data. The new revisions made the data obtained clearer
