Effect of Species, Fertilization and Harvest Date on Microbial Composition and Mycotoxin Content in Forage
Round 1
Reviewer 1 Report
This paper describes an interesting study to see the effect of forage species, fertilization, and harvest date on mycotoxins on forage. I think it is a valuable study; however, significant revisions need to be made before it can be published. Hopefully, my recommendations below can help in this process.
General recommendations:
In general, the authors need to be careful to use the same terms, significant digits, display of data, etc. throughout the paper. They also need to make sure that their methods sections line up with the rest of the paper. For example, sometimes 100 kg N/ha is the high listed and sometimes it is 150 kg N/ha. Finally, if not done already, it would be very helpful if the authors had someone help revise the English grammar throughout the paper.
Specific recommendations by line number:
L26 and 28: Write out “ZEN” and “DON” or use the abbreviation with the original mention of deoxynivalenol and zearalenone (I assume this is what don and zen are).
L53: Change “For food and feed safety are the most notable ones” to “For food and feed safety the most notable mycotoxins are”
L68: I assume a.s.l. means above sea level, but I don’t think this is a common enough acronym to use without description. I suggest simply writing “above sea level” instead.
L74: It is good to have Table 1 to show the chemical composition of the digestate. However, what was the digestate itself? Specifically, what was the feedstock used at the biogas station from which the digestate was produced?
L77-78: How are Variants B and C both equal to 1/3 annual dose, but end up being 2-fold difference in N (50 vs. 100 kg N/ha). Is Variant C actually 2/3 dose?
L95-96: This sentence is not clear as to how CFUs were determined. What does it mean that “colonies on Petri dishes were subtracted”—subtracted from what? Please describe in greater detail.
L99: DON and ZEA should be written out on first use of the acronym. Does this mean ZEN in the abstract should have been ZEA as well?
L106-107: This sentence is unclear. I am unable to understand exactly what happens when the substrate is added to the well.
L116-117: Should keep the verb tenses the same in most of the manuscript. In this case, the present tense is used whereas in most other cases (and understandably so) the past tense is used.
L118-119: Either this sentence about TMC is incorrect or Table 2 is incorrect. Table 2 shows exactly the opposite of the sentence.
L124-125: As with L118-119, Table 2 is opposite of this statement.
L135—Table 2: The significant digits shown in this table really don’t make sense. They vary from the ones to the 1/10,000ths. With the size of these numbers I have a hard time seeing that going beyond the ones units is necessary or even possible. Also, scientific notation is used in the text of the manuscript but not in the table. The results should be reported the same in both.
L137—Table 2: Is showing significance in a red font the approved method for this journal? I have not seen this done in other journals nor do I think it should be used here. People who are color blind will not be able to distinguish between the black and red font colors. Therefore, superscripts, letters, LSD, or some other mean separation technique should be used.
L138-142—Figure 2: It is nearly impossible to read the scales or any other words/numbers on this figure.
L145-161: Based on the fact, as stated in the first sentence, that there was no effect of species on microbial communities, I think it is probably unwise to show the high and low for some of them. My suggestions is to delete L148-161 or too speak more generally about the data.
L167-168: It is unclear to me what this “mixed sample” is. Does this mean that a combination of 0, 50, and 100 kg N fertilizer applied or something else.
Figure 2: The high N rate shown in this figure is 150 kg/ha; however in the methods section, 100 kg N/ha is listed as the high N rate. Which is it?
Table 3: I have similar issues with Table 3 as I did with Table 2. Specifically, be consistent with significant numbers and use another method other than red font to denote statistical significance.
Figure 3: I suggest using something besides color to denote ZEN and DON to avoid issues with people with color blindness. Also, I’m not sure the legend for this figure is accurate, at least based on other similar figures.
L215-218: Can we really make this conclusion based on this one study? It seems to me that further work would need to be done in other locations and other species to make this broad a conclusion.
L229: Include year for Karlsson et al. immediately after the citation rather than at the end.
L231-234: Variant B was 50 kg N/ha not 100.
L265-266: This may be the case in this one environment, but it may be different elsewhere.
Figures: Make sure the figures have legible (large enough) fonts. Avoid use of color if possible.
Tables: Avoid the use of colors. Pay attention to the numbers used. Also, the headings should reflect what is in them. The heading with the concentrations should indicate this rather than in the heading with the title of the microorganism.
Author Response
Response to Reviewer 1 Comments
Point 1: L26 and 28: Write out “ZEN” and “DON” or use the abbreviation with the original mention of deoxynivalenol and zearalenone (I assume this is what don and zen are).
Response 1: Yes, DON and ZEN were abbreviations used for deoxynivalenol and zearalenone, full names of toxins have been added.
Point 2: L53: Change “For food and feed safety are the most notable ones” to “For food and feed safety the most notable mycotoxins are”
Response 2: Formulation has been corrected for better understanding.
Point 3: L68: I assume a.s.l. means above sea level, but I don’t think this is a common enough acronym to use without description. I suggest simply writing “above sea level” instead.
Response 3: Acronym a.s.l. has been rewritten to above sea level.
Point 4: L74: It is good to have Table 1 to show the chemical composition of the digestate. However, what was the digestate itself? Specifically, what was the feedstock used at the biogas station from which the digestate was produced?
Response 4: Input material for digestate production was maize silage (13210 tons were processed in biogas station per year 2018), cow manure (3000 t/year), cereal silage (2000 t/year), grass silage (1000 t/year) and fresh grass forage (500 t/year).
Point 5: L77-78: How are Variants B and C both equal to 1/3 annual dose, but end up being 2-fold difference in N (50 vs. 100 kg N/ha). Is Variant C actually 2/3 dose?
Response 5: Variant A was non-fertilized control. Variant B (150 kg N/ha/year): Total amount of nitrogen was applied in 3 doses, therefore each application dose contained 1/3 of annual dose = 9.43 kg digestate per 10m2 (equal to 50 kg N/ha/application). Variant C (300 kg N/ha/year): Total amount of nitrogen was applied in 3 doses, therefore each application dose contained 1/3 of annual dose = 18.87 kg digestate per 10m2 (equal to 100 kg N/ha/application).
Point 6: L95-96: This sentence is not clear as to how CFUs were determined. What does it mean that “colonies on Petri dishes were subtracted”—subtracted from what? Please describe in greater detail.
Response 6: CFUs were counted on ColonyStar colony counter (Funke Gerber, Germany) equipped with pressure-sensitive automatic counter and illuminated counting plate.
Point 7: L99: DON and ZEA should be written out on first use of the acronym. Does this mean ZEN in the abstract should have been ZEA as well?
Response 7: Acronyms have been corrected. ZEN and ZEA were used as an acronym for the same mycotoxin – zearalenone.
Point 8: L106-107: This sentence is unclear. I am unable to understand exactly what happens when the substrate is added to the well.
Response 8: Samples were prepared by drying fresh biomass at 60 °C. Dried samples were milled to 1 mm particles and supernatant was created for further testing by ELISA method.
Point 9: L116-117: Should keep the verb tenses the same in most of the manuscript. In this case, the present tense is used whereas in most other cases (and understandably so) the past tense is used.
Response 9: Present tens of verbs has been corrected to past tense.
Point 10: L118-119: Either this sentence about TMC is incorrect or Table 2 is incorrect. Table 2 shows exactly the opposite of the sentence.
Response 10: There have been a mistake in table description. In case of TMC samples showed a trend of decreasing microbial counts later in season.
Point 11: L124-125: As with L118-119, Table 2 is opposite of this statement.
Response 11: There have been a mistake in table description. Significantly higher amount of LAB was present on the plant mass in May in comparison to July.
Point 12: L135—Table 2: The significant digits shown in this table really don’t make sense. They vary from the ones to the 1/10,000ths. With the size of these numbers I have a hard time seeing that going beyond the ones units is necessary or even possible. Also, scientific notation is used in the text of the manuscript but not in the table. The results should be reported the same in both.
Response 12: Table 2 has been corrected in terms of structure and measurements.
Point 13: L137—Table 2: Is showing significance in a red font the approved method for this journal? I have not seen this done in other journals nor do I think it should be used here. People who are color blind will not be able to distinguish between the black and red font colors. Therefore, superscripts, letters, LSD, or some other mean separation technique should be used.
Response 13: Significant values have been differentiated from others by marking in bold italics.
Point 14: L138-142—Figure 2: It is nearly impossible to read the scales or any other words/numbers on this figure.
Response 14: Figures have been removed as per suggestion of Reviewer 2. Tables are serving the purpose of concise presentation of data.
Point 15: L145-161: Based on the fact, as stated in the first sentence, that there was no effect of species on microbial communities, I think it is probably unwise to show the high and low for some of them. My suggestions is to delete L148-161 or too speak more generally about the data.
Response 15: Paragraph has been updated.
Point 16: L167-168: It is unclear to me what this “mixed sample” is. Does this mean that a combination of 0, 50, and 100 kg N fertilizer applied or something else.
Response 16: Mixed samples were created by blending of samples gathered from fertilization variants A, B and C for purposes of creating reference sample.
Point 17: Figure 2: The high N rate shown in this figure is 150 kg/ha; however in the methods section, 100 kg N/ha is listed as the high N rate. Which is it?
Response 17: The highest fertilization was indeed 100 kg N/ha. Figures have been removed as per suggestion of Reviewer 3. Tables are serving the purpose of concise presentation of data.
Point 18: Table 3: I have similar issues with Table 3 as I did with Table 2. Specifically, be consistent with significant numbers and use another method other than red font to denote statistical significance.
Response 18: Table 3 have been corrected in terms of typography, structure and measurements.
Point 19: Figure 3: I suggest using something besides color to denote ZEN and DON to avoid issues with people with color blindness. Also, I’m not sure the legend for this figure is accurate, at least based on other similar figures.
Response 19: Figures have been removed as per suggestion of Reviewer 3. Tables are serving the purpose of concise presentation of data.
Point 20: L215-218: Can we really make this conclusion based on this one study? It seems to me that further work would need to be done in other locations and other species to make this broad a conclusion.
Response 20: From results gathered in this experimental study it is possible to conclude that botanical species does not affect microbial composition of plant’s phyllosphere. Species variants with the same climatic and soil factors were observed to have similar CFU counts, which is probably true for the unmonitored weed species as well. However, this conclusion is only applicable in terms of the experimental location of this study and similar weather conditions.
Point 21: L229: Include year for Karlsson et al. immediately after the citation rather than at the end.
Response 21: Citation in text has been corrected.
Point 22: L231-234: Variant B was 50 kg N/ha, not 100.
Response 22: We found no significant difference between microbial counts in control and low digestate fertilization, so from economical perspective it is more convenient to use 50 kg N/ha/year (variant B of fertilization) to achieve lower microbial contamination and also sufficient forage yields and quality.
Point 23: L265-266: This may be the case in this one environment, but it may be different elsewhere.
Response 23: Conclusions of this paragraph have been reformulated.
Point 24: Figures: Make sure the figures have legible (large enough) fonts. Avoid use of color if possible.
Tables: Avoid the use of colors. Pay attention to the numbers used. Also, the headings should reflect what is in them. The heading with the concentrations should indicate this rather than in the heading with the title of the microorganism.
Response 24: Figures have been removed as per suggestion of Reviewer 3. Tables have been corrected in several aspects as per reviewers’ suggestions.
Reviewer 2 Report
Agriculture-481154
General comments:
English usage is poor, and makes for a very confusing read. I request a full re-examination of the manuscript by a native English speaker before the paper can be considered for publication.
Some specific comments (after English is addressed) should also be considered at this time:
Abstract:
Should state problem/question immediately (feed safety)
Define ZEN and DON
Introduction:
Define phyllosphere
Line 48, define “poor agricultural practices”
Why is performing the work in the Czech Republic important? Is this area important for EU agriculture in general? That is an earnest question, there is no way to know, and there is probably a really good reason for doing this work here, but some context on the economic importance of the area would be more compelling.
Methods:
What is the digestate? Is this like distillers grain? Need more information (liquid, solid, etc)
Line 99, again, what are ZEN and DON?
Line 112: were any homogeneity of variance tests performed?
Table 2 and 3: please scale these values with scientific notation so they are more readable.
All figures need to be enlarged, font on the axes greatly enlarged. The arrows and paragraph symbols are very confusing.
Table 4, be consistent with using decimal or comma to separate 0’s from numbers to the right
Author Response
Response to Reviewer 2 Comment
Point 1: English usage is poor, and makes for a very confusing read. I request a full re-examination of the manuscript by a native English speaker before the paper can be considered for publication.
Response 1: Article has been checked by an English speaker.
Point 2: Abstract: Should state problem/question immediately (feed safety).
Response 2: Abstract has been corrected. The aim of the project was to evaluate the potential of microbial threat to feed safety.
Point 3: Define ZEN and DON.
Response 3: DON and ZEN were abbreviations used for deoxynivalenol and zearalenone, full names of toxins have been added.
Point 4: Define phyllosphere.
Response 4: Phyllosphere is the above ground part of the plant which is the home to epiphytes (Thapa et al. 2017).
Point 5: Line 48, define “poor agricultural practices”.
Response 5: Mycotoxins are produced by molds under specific conditions, such as high humidity, poor agricultural practices (e.g. inadequate fertilization, disuse of crop rotation, contaminated seeds) or damaged and contaminated crops.
Point 6: Why is performing the work in the Czech Republic important? Is this area important for EU agriculture in general? That is an earnest question, there is no way to know, and there is probably a really good reason for doing this work here, but some context on the economic importance of the area would be more compelling.
Response 6: Electricity consumption is increasing rapidly in Czech Republic and decarbonization of its production is under way. One of the more sustainable production methods includes biogas stations. With more than 50% of land being agricultural, possible energy sources for biogas stations are abundant (Ministry of Agriculture of Czech Republic). This however presents a challenge of utilization of biogas secondary product referred to as digestate. (Simeckova et al. 2018).
Point 7: What is the digestate? Is this like distillers grain? Need more information (liquid, solid, etc).
Response 7: Digestate has potential of becoming a new sustainable form of semi-liquid fertilizer. It is produced as a secondary product in anaerobic digestion of biomass (Simeckova et al. 2018).
Point 8: Line 99, again, what are ZEN and DON?
Response 8: DON and ZEN were abbreviations used for deoxynivalenol and zearalenone, full names of toxins have been added.
Point 9: Line 112: were any homogeneity of variance tests performed?
Response 9: Data was tested for normality of distribution by Shapiro-Wilk test. Single-factor variance analysis ANOVA and post-hoc Scheffé test were conducted.
Point 10: Table 2 and 3: please scale these values with scientific notation so they are more readable.
Response 10: Tables have been corrected in several aspects as per reviewers’ suggestions.
Point 11: All figures need to be enlarged, font on the axes greatly enlarged. The arrows and paragraph symbols are very confusing.
Response 11: Figures have been removed as per suggestion of Reviewer 3. Tables are serving the purpose of concise presentation of data
Point 11: Table 4, be consistent with using decimal or comma to separate 0’s from numbers to the right.
Response 11: Table 4 have been corrected, 0’s have been separated with decimal.
Reviewer 3 Report
The paper investigates the effect of fertilization and harvest date on the mycotoxin concentration in various grass species. Differences were found between the grass species as well as between the fertilization regimes, however these were not significant.
The entire paper should be improved in terms of sentence structure, transition between paragraphs and wording. My suggestion is to have it checked by a native speaker.
The authors present the same data in tables and figures. My suggestion is to only use tables and remove the figures, also they have a very low resolution and are hard to read.
Details on the plot design (2.1) are missing, such as what was the plot design (randomized complete block, latin square,...), in addition the authors did not mention the number of treatment repetitions or whether the entire experiment was repeated at all. Other information in this section can be removed (line 73: "which was..."). Furthermore, some abbreviations are not explained and unclear to me such as "ie" in line 77/78.
What are the content(%) calculations in Table 1 based on (100g) ? This is important information and needs to be added.
Please provide information what kind of shaker you used (orbital, vertical ?) line 85.
Please provide the full name of all used media and not only the abbreviation (line 88-92).
Please provide more information in the statistical analysis section, such as the testing procedure for normalisation of residuals and heterogeneity of data.
In table 4 & 5 you can limit mean and SE to two digits after the comma.
In line 233/234, the authors state that 100 kg N/ha/year are sufficient to achieve good yields and quality. Based on the data provided, no measurements of yield and/or quality were taken during this study. Hence, a evaluation can not be done.
Please check the entire references for nomenclature that needs to be in italics. such as Fusarium, Aspergillus etc.
Author Response
Response to Reviewer 3 Comments
Point 1: The entire paper should be improved in terms of sentence structure, transition between paragraphs and wording. My suggestion is to have it checked by a native speaker.
Response 1: Article has been checked by an English speaker.
Point 2: The authors present the same data in tables and figures. My suggestion is to only use tables and remove the figures, also they have a very low resolution and are hard to read.
Response 2: Figures have been removed for clearer orientation in the article.
Point 3: Details on the plot design (2.1) are missing, such as what was the plot design (randomized complete block, latin square,...), in addition the authors did not mention the number of treatment repetitions or whether the entire experiment was repeated at all. Other information in this section can be removed (line 73: "which was..."). Furthermore, some abbreviations are not explained and unclear to me such as "ie" in line 77/78.
Response 3: Randomized plot design was used in experiment, each variant was sown on small plots (1.25 × 8 m) in three repetitions. Line 73 have been corrected. Descriptions of fertilization variants have been corrected as per recommendation of Reviewer 1.
Point 4: What are the content (%) calculations in Table 1 based on (100g)? This is important information and needs to be added.
Response 4: Chemical composition described in Table 1 was based on 100 g of digestate
Point 5: Please provide information what kind of shaker you used (orbital, vertical ?) line 85.
Response 5: Orbital shaker PSU-10i by company Biosan was used in the experiment.
Point 6: Please provide the full name of all used media and not only the abbreviation (line 88-92).
Response 6: Total microbial count (TMC) was cultivated on Plate Count Agar, lactic acid bacteria (LAB) on De Man, Rogosa and Sharpe Agar, Enterococcus sp. on COMPASS Enterococcus agar, Enterobacteriaceae on Violet Red Bile Glucose Agar and micromycetes (yeasts and molds) on Chloramphenicol Glucose Agar. All media used were purchased from Biokar Diagnostics, France.
Point 7: Please provide more information in the statistical analysis section, such as the testing procedure for normalisation of residuals and heterogeneity of data.
Response 7: Data was tested for normality of distribution by Shapiro-Wilk test.
Point 8: In table 4 & 5 you can limit mean and SE to two digits after the comma.
Response 8: SE values in Table 4 and Table 5 have been limited to 2 digits after the comma.
Point 9: In line 233/234, the authors state that 100 kg N/ha/year are sufficient to achieve good yields and quality. Based on the data provided, no measurements of yield and/or quality were taken during this study. Hence, an evaluation can not be done.
Response 9: Statement has been removed to avoid imprecise evaluation.
Point 10: Please check the entire references for nomenclature that needs to be in italics, such as Fusarium, Aspergillus etc.
Response 10: Nomenclature typography in references has been corrected.
Round 2
Reviewer 1 Report
The authors have done a good job addressing the specific recommendations I made in my review. However, I still recommend that a thorough review of the grammar be completed before publication.
Author Response
Point 1: The authors have done a good job addressing the specific recommendations I made in my review. However, I still recommend that a thorough review of the grammar be completed before publication.
Response 1: Thank you for your recommendations. English has been checked.
Reviewer 2 Report
The authors have made some progress on improving the clarity of the manuscript. I am still uncomfortable with using parametric statistics without a homogeneity of variance test. The authors include a NORMALITY test, not an HOV test. ANOVAs are robust agains non-normality (although the authors do not state the results of the Shapiro-Wilk test...). I suggest a Levene's test or Fligner-Kileen test for HOV. If those tests are significant, ANOVA is NOT APPROPRIATE.
Please address this issue and report results from those critical tests, or change statistical methods as appropriate.
Author Response
Point 1: The authors have made some progress on improving the clarity of the manuscript. I am still uncomfortable with using parametric statistics without a homogeneity of variance test. The authors include a NORMALITY test, not an HOV test. ANOVAs are robust agains non-normality (although the authors do not state the results of the Shapiro-Wilk test...). I suggest a Levene's test or Fligner-Kileen test for HOV. If those tests are significant, ANOVA is NOT APPROPRIATE.
Response 1: As per request of Reviewers, statistical methods have been changed and results have been reevaluated.
Reviewer 3 Report
The manuscript is improved compared to the first version. However, there is still room for improvement and I hope that my comments might help by doing so:
Major revisions:
My biggest concern with this manuscript is still that the study was not repeated. One year of data is not sufficient to draw conclusions, especially in terms of mycotoxins which vary from year to year. Hence, I encourage the authors to repeat the study minimum once before resubmitting the manuscript. In addition laboratory or greenhouse/climate chamber experiments to support their hypothesis might be necessary.
The part about statistical analysis needs to be written in more detail. How was the model for the ANOVA chosen? Which parameters were set as fixed or random factors? Why did the authors not check for interactions between grass species and fertilization? These are important information.
Line 55 – 57: The statement here is partially wrong. Agricultural practices as well as damaged or contaminated crops might enhance the risk for mycotoxin contamination but they are not needed for molds to produce mycotoxins.
Minor revisions:
Also the manuscript was reviewed by a native speaker, it still needs to be improved. I only highly some of the main points I found by reading.
Line 5: Comma missing after Kalhotka
Line 22: consider using ‘senescence’ instead of ‘death’
Line 42: consider using ‘biomass’ instead of ‘plant parts’
Line 43: remove ‘to’ before ‘ phyllosphere’
Line 44: ‘are’ not ‘is’
Line 48: put ‘an’ after ‘is’
Line 50: put ‘a’ after ‘play’
Line 58: either write does exist or exists
Line 64: ‘are’ not ‘is’
Line 64/65: Consider rephrasing: “Frequently, more than one mycotoxin contaminates feed.”
Line 69: consider using ‘biomass’ instead of ‘surface’
Line 81/82: Consider rephrasing: “A randomized plot design was used and three repetitions of each variant was sown in small plots.”
Line 83 and 88: consider joining these sentences: ‘…digestate received from biogas…’
Line 108: consider using: ‘Subsequently chopeed into 3 cm….’
Line 113: which kind of dilutions did you use? 10 fold?
Line 132-133: italics is missing
Line 134: please specify which mill was used
Line 135: what was used to create the supernatant? Water, methanol?
Line 137-146: This is a lot of basic information. You can shorten this and just refer to the manufactures’ protocol.
Line 155: ‘did not affect’ + ‘influenced’
Line 157: you could remove ‘in case of’
Line 158: you could remove ‘mean values obtained were’ and refer to Table 2 at the end of the sentence
Line 160: remove ‘respectively’
Line 165: remove ‘was’
Line 170: consider using ‘significant’ rather than ‘demonstrable’
Line 228/229: there was only a slight increase of ZEA concentration and this should be mentioned; refer to Table 4 at the end of the sentence
Line 270: was it 50 kg of N or 100 kg ?
Line 279: ‘the toxic’ is redundant
Line 283 – 285: Consider rewriting this sentence, it is very hard to read
Line 291: refer to your own results here – how much ZEA did you find, how does this compare
Line 295 – 298: I These sentences do not make sense to me. Consider rephrasing.
Line 301: Why October? The authors wrote that samples were taken in May and July and fertilization occurred in October. No statement about mycotoxin contamination in October can be done here.
Line 309-310: Consider deleting this last sentence or put it in a more useful context
Line 349, 386, 420: put ‘fusarium’ in italics
Line 430: Fusarium is not written in italics in this particular case
Author Response
Point 1: My biggest concern with this manuscript is still that the study was not repeated. One year of data is not sufficient to draw conclusions, especially in terms of mycotoxins which vary from year to year. Hence, I encourage the authors to repeat the study minimum once before resubmitting the manuscript. In addition laboratory or greenhouse/climate chamber experiments to support their hypothesis might be necessary.
Response 1: The aim of this study was not a long-term observation, but evaluation of data in the concrete year (2018). It was an extreme year in terms of weather conditions and due to the lack of other publications with similar points of interest we wanted to inform public about our unique findings as quickly as possible. Information on other work done on mycotoxin occurrence has been added to Discussion section.
Point 2: The part about statistical analysis needs to be written in more detail. How was the model for the ANOVA chosen? Which parameters were set as fixed or random factors? Why did the authors not check for interactions between grass species and fertilization? These are important information.
Response 2: Statistical methods were revaluated as per Reviewers’ suggestion.
Point 3: The statement here is partially wrong. Agricultural practices as well as damaged or contaminated crops might enhance the risk for mycotoxin contamination but they are not needed for molds to produce mycotoxins.
Response 3: The statement has been corrected.
Point 4: Also the manuscript was reviewed by a native speaker, it still needs to be improved. I only highly some of the main points I found by reading.
Response 4: Minor revisions have been addressed.
