Relationships between Volatile Organic Compounds Released by Wheat Plants Following Artificial Stress and Their Potential Influence on Natural Pest Management
, Julia Aksoy 1, Jacek Łyczko 2, Jan Bocianowski 3, Bogusław Buszewski 4, Magdalena Piesik 5 and Chris A. Mayhew 6
Round 1
Reviewer 1 Report
This is a very interesting study that uses an innovative and exciting approach, which might be accepted after doing some corrections.
Comment 1: It will be better to repeat all test three times at least in order to validate the results.
Comment 2: The authors are requested to clarify how they treated the controls, if there is a difference between controls of each test.
Comment 3: The authors are requested to clarify how they did the MeJA vapourization test.
Comment 4: It will better to determine the amount of methyl jasmonate released by plants exposed to mechanical wounding in order to confirm the methyl jasmonate liberation in response to insect feeding. This will make the both tests (mechanical wounding and exposure to methyl jasmonate) complementary.
Comment 5: the authors are requested to improve discussion and conclusion parts.
Comment 6: The authors are requested to correct some typos and formatting in manuscript ( lines; 38, 87, 100, 121……. In line 344 (and not ond).
Author Response
Response to Reviewer 1 Comments
Comments and Suggestions for Authors
This is a very interesting study that uses an innovative and exciting approach, which might be accepted after doing some corrections.
Thank you very much.
Comment 1: It will be better to repeat all test three times at least in order to validate the results.
For each MeJA dosage three replications were performed. Both, the VOC and the four-way olfactory measurements were done in triplicates so that good statistics could be obtained. Please see lines: 164-165 and 213-214, respectively.
Comment 2: The authors are requested to clarify how they treated the controls, if there is a difference between controls of each test.
The controls or both artificial stress treatments were the same – untreated plants, which were placed for measurements in nalophan bags without scraping the leaves or exposing them to MeJa. We have an added appropriate description in Materials and Methods section. Please see lines 137-139.
Comment 3: The authors are requested to clarify how they did the MeJA vapourization test.
This is provided on lines 140-146 of the manuscript.
Comment 4: It will better to determine the amount of methyl jasmonate released by plants exposed to mechanical wounding in order to confirm the methyl jasmonate liberation in response to insect feeding. This will make the both tests (mechanical wounding and exposure to methyl jasmonate) complementary.
This would be an interesting experiment. However, that is the aim of the current study. Our objective was to us MeJa only as an activator of natural defense system.
Comment 5: the authors are requested to improve discussion and conclusion parts.
We have included more details as requested. We added: “Moreover, using GC/MS, Sendel et al. (2022) found that Aelia acuminata L. (1 or 2 adult pairs) significantly induced VOC emissions from wheat plants, where, as a result of biotic stress, larger amounts of the following VOCs were released: (Z)-3-hexenal, (E)-2-hexenal, (Z)-3-hexen-1-ol, (E)-2-hexen-1-ol, β-pinene, β-myrcene, (Z)-3-hexen-1-yl acetate, 1-hexyl acetate, 4-heptanone, (Z)-ocimene, linalool, linalool oxide, benzyl acetate, methyl salicylate, indole, β-caryophyllene, and (E)-β-farnesene. It was also reported by them that two pairs of insects caused a significantly stronger plant reaction.“; “Ameye et al. (2018) found that plants respond to stress by releasing biogenic volatile organic compounds (BVOCs). They used a meta-analysis approach and found that there is less variation in the GLV profile than we presumed, and that pathogens induce more GLVs than insects and wounding.” and in Conclusions: “In comparison to spraying and wounding, vaporization resulted in the largest VOC emissions Of the two tested cultivars, "Serenada" released larger amounts of VOCs. Taking into consideration the olfactory tests, an especially large negative reaction of Aelia acuminata was observed for cv. Serenada (vaporization).”
Comment 6: The authors are requested to correct some typos and formatting in manuscript ( lines; 38, 87, 100, 121……. In line 344 (and not ond).
We have corrected all typographical and formatting mistakes. Thank you for bringing these to our attention.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Piesik et al. investigated the release of VOC emissions from wheat plants following abiotic stress and their potential to be used as an indicator of plant health.
The manuscript is of interest and relevant for the field of application and it well fits with the aims and scope of the journal. The introduction refers to published literature within the field of research; the experimental design is appropriate and the results are clearly reported. Overall, it is a well put together manuscript, the graphical representations are suitable for the purpose and explanation of results were well addressed. Overall, the English language is quite good.
Although some minor points need to be addressed.
Line 153 – The use of n-decane as internal standard, how can you tell the difference from the response of n-decane in a sample from the response of the added n-decane? Can you guarantee that n-decane is never found in samples?
Line 170 – Is this study semi-quantitative or fully quantitative? Please indicate in the text. If fully quantitative then details of calibration are needed, calibration range, regression coefficients, etc.
Line 198 – The normality was tested but the result is not given in the text. Please indicate in the text that the population follows a normal distribution, if this is the case.
Line 208 – Outliers impact significantly on statistical results, were outliers taken into account and what was the adopted approach?
Line 214 – VOCs retention times need to be indicated – the reader will need this info for duplication purposes.
Table 1 – I’d rather read 1LF and 3LF as “L” can be mistaken with the unit of litre.
Table 2 and table 3 – The table headers are difficult to read.
Figure 1, 2 and 3 – The labels are barely visible.
Author Contributions and acknowledgments are missing.
Author Response
Response to Reviewer 2 Comments
Reviewer #2
Point 1: Piesik et al. investigated the release of VOC emissions from wheat plants following abiotic stress and their potential to be used as an indicator of plant health.
The manuscript is of interest and relevant for the field of application and it well fits with the aims and scope of the journal. The introduction refers to published literature within the field of research; the experimental design is appropriate and the results are clearly reported. Overall, it is a well put together manuscript, the graphical representations are suitable for the purpose and explanation of results were well addressed. Overall, the English language is quite good.
Response: Thank you very much.
Although some minor points need to be addressed.
Point 2: Line 153 – The use of n-decane as internal standard, how can you tell the difference from the response of n-decane in a sample from the response of the added n-decane? Can you guarantee that n-decane is never found in samples?
Response: n-decane even if present in the sample (rather sporadically) is recorded in very small amounts. After 20 years of my experience with volatile organic compounds I have never seen chromatogram "peaks" with repeatable amounts of n-decane. In parallel, all of the recorded by the GC-MS components act as attractants and repellents in the olfactory tests. I have never seen n-decane active in olfactory tests with insects.
Point 3: Line 170 – Is this study semi-quantitative or fully quantitative? Please indicate in the text. If fully quantitative then details of calibration are needed, calibration range, regression coefficients, etc.
Response: Yes, thank you. We can say semi-quantitative results.
Point 4: Line 198 – The normality was tested but the result is not given in the text. Please indicate in the text that the population follows a normal distribution, if this is the case.
Response: The empirical distribution of observations of the all VOCs was normal. We added this text in the manuscript.
Point 5: Line 208 – Outliers impact significantly on statistical results, were outliers taken into account and what was the adopted approach?
Response: We did not observe any outliers.
Point 6: Line 214 – VOCs retention times need to be indicated – the reader will need this info for duplication purposes.
Response: We corrected sentence to: "In total nine volatiles were identified, namely (Z)-3-hexenal (retention time: 5.59 min), (E)-2-hexenal (8.32 min), (Z)-3-hexen-1-ol (9.02 min), (E)-2-hexen-1-ol (9.16 min), (Z)-3-hexen-1-yl acetate (13.45 min), (E)-β-ocimene (15.36 min), linalool (17.35 min), β-caryophyllene (26.02 min) and (E)-β-farnesene (26.25 min).".
Point 7: Table 1 – I’d rather read 1LF and 3LF as “L” can be mistaken with the unit of litre.
Response: We corrected Table 1. Now is: “1SL” and “3SL”.
Point 8: Table 2 and table 3 – The table headers are difficult to read.
Response: We corrected Tables 2 and 3.
Point 9: Figure 1, 2 and 3 – The labels are barely visible.
Response: We corrected all figures.
Point 10: Author Contributions and acknowledgments are missing.
Response: We added: “Author Contributions: Conceptualization, D.P. and J.A.; methodology, D.P., J.Ł. and B.B.; software, J.B.; validation, B.B.; formal analysis, J.B. and M.P.; investigation, J.A.; resources, J.A. and J.Ł.; data curation, D.P., J.A. and J.Ł.; writing—original draft preparation, J.Ł.; writing—review and editing, D.P., J.B. and Ch.M.; visualization, J.B.; supervision, D.P.; project administration, D.P.; funding acquisition, D.P. All authors have read and agreed to the published version of the manuscript.”
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript “Relationships between volatile organic compounds released by wheat plants following abiotic stress and their potential influence on natural pest management” describes the green leaf volatiles and other compounds released by wheat plants after artificial wounding or exposure to methyl jasmonate. In addition, an olfactometer experiment was done with the pest insect, Aelia acuminate, to test its attraction or repellence to the volatile profiles from plants exposed to different concentrations of methyl jasmonate, either sprayed or vaporized.
Overall, the manuscript reads very well and presents informative results. To put the research into context, I feel that the manuscript could be improved by integrating more recent work on VOC production by plants. In particular, a quick literature search turned up a useful meta-analysis (Ameye et al., 2018. Green leaf volatile production by plants: a meta-analysis. New Phytologist 220(3): 666-683).
The best part of the research was that the volatiles were collected in vivo, from intact plants, which accurately describes the plants’ natural defence strategies. It was also discovered that many of the volatiles are correlated.
At the end of the introduction, the authors state: “We present here the first report on how Bishops’s Mitre shieldbugs could be managed using plant volatiles as inducers in natural defence mechanisms.” After reading the manuscript, it is not clear how this can be achieved. Do they refer to the repellent effects of the VOCs? The main conclusion is about improved monitoring and developing an analytical tool. I propose that the above sentence is altered to “We present here the first report on how Bishops’s Mitre shieldbugs could be detected using plant volatiles that are induced in natural defence mechanisms.”
The authors suggest that the observed volatiles can be used to monitor for this pest. However, the reasoning is a bit tenuous, because the methods to generate plant stress were artificial, i.e. artificial wounding and exposure to methyl jasmonate. As described in Ameye et al. (2018), other pests or pathogens can also induce the generation of the same or similar VOC’s, sometimes at higher levels. In addition, it has been shown that when insects feed on the plant, there is an effect of their salivary proteins that can induce a very specific response in the plant. This cannot be imitated by mechanical wounding alone. Some discussion on this is needed. I also expect that volatile monitoring would rather alert farmers to general stress in the crop, and that it is not possible to specify which organism (e.g. shieldbugs) is responsible, unless there is a unique compound or combination of compounds that is correlated with that organism.
I’m surprised that there is no mention of a recently published paper from the same authors that describes the VOCs released by actual feeding of this insect (Sendel et al., 2022. Journal of Applied Entomology 146(6): 710-724).
The insect olfactometer assay seems to be added to the manuscript later and the results are not well discussed beyond the mention of other studies that also showed attraction and/or repellence. The abstract doesn’t even mention this bioassay. It is important to know that green leaf volatiles are also involved in communication in this insect family (Pentatomidae) as alarm and sex pheromones. However, this is not discussed nor used to explain attraction or repellence. What is the value of this experiment to demonstrate that VOCs can be used to monitor for this pest?
Finally, I don’t think the use of the term “abiotic stress” is appropriate here. Especially because it is also mentioned in the title. Abiotic stress has environmental causes. The experiments here are clearly designed to imitate stress induced by a pest (which is a biotic stress). Maybe calling it artificial or simulated stress would be better.
More specific comments follow below:
Line 93-96: This hypothesis is not clear. What is meant by “comparable levels of their emission”?
Section 2.7: More details are needed on how the VOCs were obtained. In the results, two concentrations of methyl jasmonate are mentioned. Also, at what time point after induction were the volatiles collected?
Line 252: replace 4.05*(a) and 6.05*(a) by Chi2 = 4.05 and 6.05 (same for line 255).
Line 252-255: Describe the results in a bit more detail.
Line 275-279: This should be moved to the results.
Line 279-281: How is this explained by the common biosynthetic pathway?
Line 340: The word “stage” suggest that measuring 1 volatile can actually give information on the severity of the pest infestation. This has not been demonstrated in the research. Replace by pest presence. In addition, as mentioned above, other pests and diseases can also trigger the release of green leaf volatiles, so suggesting pest specificity is dubious.
Table 1: the stress codes 1L and 3L can easily be confused with the use of L for “litre”
Table 2, 3, 4: all tables need better formatting.
Author Response
Response to Reviewer 3 Comments
Comments and Suggestions for Authors
The manuscript “Relationships between volatile organic compounds released by wheat plants following abiotic stress and their potential influence on natural pest management” describes the green leaf volatiles and other compounds released by wheat plants after artificial wounding or exposure to methyl jasmonate. In addition, an olfactometer experiment was done with the pest insect, Aelia acuminate, to test its attraction or repellence to the volatile profiles from plants exposed to different concentrations of methyl jasmonate, either sprayed or vaporized.
Overall, the manuscript reads very well and presents informative results. To put the research into context, I feel that the manuscript could be improved by integrating more recent work on VOC production by plants. In particular, a quick literature search turned up a useful meta-analysis (Ameye et al., 2018. Green leaf volatile production by plants: a meta-analysis. New Phytologist 220(3): 666-683).
The best part of the research was that the volatiles were collected in vivo, from intact plants, which accurately describes the plants’ natural defence strategies. It was also discovered that many of the volatiles are correlated.
Thank you very much for your comments.
At the end of the introduction, the authors state: “We present here the first report on how Bishops’s Mitre shieldbugs could be managed using plant volatiles as inducers in natural defence mechanisms.” After reading the manuscript, it is not clear how this can be achieved. Do they refer to the repellent effects of the VOCs? The main conclusion is about improved monitoring and developing an analytical tool. I propose that the above sentence is altered to “We present here the first report on how Bishops’s Mitre shieldbugs could be detected using plant volatiles that are induced in natural defence mechanisms.”
Thank you for this suggestion, which we have taken into account, and hence modified the text. Please see lines 100-104.
The authors suggest that the observed volatiles can be used to monitor for this pest. However, the reasoning is a bit tenuous, because the methods to generate plant stress were artificial, i.e. artificial wounding and exposure to methyl jasmonate. As described in Ameye et al. (2018), other pests or pathogens can also induce the generation of the same or similar VOC’s, sometimes at higher levels. In addition, it has been shown that when insects feed on the plant, there is an effect of their salivary proteins that can induce a very specific response in the plant. This cannot be imitated by mechanical wounding alone. Some discussion on this is needed. I also expect that volatile monitoring would rather alert farmers to general stress in the crop, and that it is not possible to specify which organism (e.g. shieldbugs) is responsible, unless there is a unique compound or combination of compounds that is correlated with that organism.
We agree with the reviewer’s point. Therefore, we rephrased all relevant manuscript parts, to avoid incorrectly suggesting that GLVs emissions are specific for shieldbugs. Please see lines 100-107, and the conclusions section. We have referenced Ameye et al. (2018).
I’m surprised that there is no mention of a recently published paper from the same authors that describes the VOCs released by actual feeding of this insect (Sendel et al., 2022. Journal of Applied Entomology 146(6): 710-724).
Agreed. Sendel et al. (2022) has now been added.
The insect olfactometer assay seems to be added to the manuscript later and the results are not well discussed beyond the mention of other studies that also showed attraction and/or repellence. The abstract doesn’t even mention this bioassay. It is important to know that green leaf volatiles are also involved in communication in this insect family (Pentatomidae) as alarm and sex pheromones. However, this is not discussed nor used to explain attraction or repellence. What is the value of this experiment to demonstrate that VOCs can be used to monitor for this pest?
Corrected. A sentence has been added at the end of Introduction– “Moreover, the main goal of this manuscript was to demonstrate that MeJa can be used to activate the natural defence mechanisms of plants and prepare spring wheat for an insect attack”.
Finally, I don’t think the use of the term “abiotic stress” is appropriate here. Especially because it is also mentioned in the title. Abiotic stress has environmental causes. The experiments here are clearly designed to imitate stress induced by a pest (which is a biotic stress). Maybe calling it artificial or simulated stress would be better.
Thank you for this suggestion. The term “abiotic stress” has been replaced with “artificial stress” in all appropriate places.
More specific comments follow below:
Line 93-96: This hypothesis is not clear. What is meant by “comparable levels of their emission”?
We agree that the hypothesis was not clear. By using the term “comparable levels”, we intended to highlight the correlation phenomenon amongst emitted VOCs. The hypothesis has been rephrased to avoid any misleading statements.
Section 2.7: More details are needed on how the VOCs were obtained. In the results, two concentrations of methyl jasmonate are mentioned. Also, at what time point after induction were the volatiles collected?
Section 2.7 concerns the olfactometer, not the collection of volatile organic compounds. VOCs collection is described in section 2.4.
Line 252: replace 4.05*(a) and 6.05*(a) by Chi2 = 4.05 and 6.05 (same for line 255).
We corrected these sentences.
Line 252-255: Describe the results in a bit more detail.
We are not certain why the reviewer is requesting more details on these lines. The details on the correlation coefficients are in our opinion adequately described. Therefore, we have made no changes to these lines.
Line 275-279: This should be moved to the results.
The lines have been moved.
Line 279-281: How is this explained by the common biosynthetic pathway?
The common biosynthetic pathway was referring only to C6 aldehydes and alcohols corresponding to them. We meant that if the pathway was upregulated by the plant exposure to methyl jasmonate, synthesis of all those compounds would be increased and correlated. We have rephrased the paragraph in the discussion to cover this issue.
Line 340: The word “stage” suggest that measuring 1 volatile can actually give information on the severity of the pest infestation. This has not been demonstrated in the research. Replace by pest presence. In addition, as mentioned above, other pests and diseases can also trigger the release of green leaf volatiles, so suggesting pest specificity is dubious.
The term “stage of pest invasion” has been replaced with “pest presence”.
Regarding the GLVs increased emission, we agree that this may not only be caused by A. acuminata, but also other factors. Our intention was not to suggest that the GLVs increased emission is a specific indicator for A. acuminata presence. We did not emphasize that sufficiently in the original text. Therefore, the conclusion’s section has been modified to bring out this point.
Table 1: the stress codes 1L and 3L can easily be confused with the use of L for “litre”
We agree that just the use of L is confusing. Therefore, we have replaced it with with 1SL or 3SL, which refer to one scraping or three scrapings per leaf, respectively.
Table 2, 3, 4: all tables need better formatting.
The tables have been formatted according to the journal guidelines.
Author Response File: Author Response.pdf
Reviewer 4 Report
I feel the manuscript is in primitive stage and need lot of refinement before it can be accepted. The main aim of the manuscript is to focus on “Relationships between volatile organic compounds released 2 by wheat plants following abiotic stress and their potential 3 Influence on natural pest management” but very little information was incorporated in the manuscript and moreover as quoted in the abstract it is not a new technique, it is very well established from the past one decade. Despite of that, no sufficient data related to various parameters involved in the process, advancement taken place in the equipment or various accessories involved, troubleshooting etc. As such there is no new or at least sufficient data to support the title.
Author Response
Response to Reviewer 4 Comments
Comments and Suggestions for Authors
I feel the manuscript is in primitive stage and need lot of refinement before it can be accepted. The main aim of the manuscript is to focus on “Relationships between volatile organic compounds released 2 by wheat plants following abiotic stress and their potential 3 Influence on natural pest management” but very little information was incorporated in the manuscript and moreover as quoted in the abstract it is not a new technique, it is very well established from the past one decade. Despite of that, no sufficient data related to various parameters involved in the process, advancement taken place in the equipment or various accessories involved, troubleshooting etc. As such there is no new or at least sufficient data to support the title.
Thank you for the comments, but we do not agree with these comments. We have investigated two spring wheats (“Kandela” and “Serenada”), in two types of application (vapourization and spraying) in context of 250 µg of MeJA per 100 mL. We are unaware of any other study that has undertaken such investigations. Perhaps, MeJa, spring wheat associated with vaporization and spraying were tested separately not in this combination.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments have been addressed appropriately. Some minor English editing is still required.
Author Response
Dear Reviewer,
thank you. Minor English editing has been attached to the manuscript. One of the author is an English native speaker.
Author Response File: Author Response.pdf
Reviewer 4 Report
The author had corrected the manuscript according to my comments.
Author Response
Dear Reviewer,
thank you. Yes, we have corrected the manuscript according to your comments. Minor English editing has been attached to the manuscript. One of the author is an English native speaker.
Author Response File: Author Response.pdf
