Volatile Organic Compounds (VOCs) in Mediterranean Oak Forests of Hungarian Oak (Quercus frainetto Ten) Affected by Dieback Phenomena

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors analysed by SPME how dieback and non-dieback trees differ in terms of VOCs present in headspace analysis of their cores. The paper reads well, however the evidence that trees die because of change in VOC composition inside of wood is not strong here. Couldn’t that be that trees die because of something else and therefore there is a change in VOC composition? Authors claim the first option, however did not provide any evidence for that- that would need deeper further investigation. So that I think that the aim of the paper should be changed, that you just detected different compounds in headspace analysis in D and ND trees. Moreover, the hypothesis in line 83-85 is not proven. How did you prove that it was the pests who caused the decline of the trees? Assuming that only from accumulation of anti-herbivore VOCs is not direct prove. It is not in the manuscript.

 

line 20: define SPME abbreviation

line 43: air pollution as nitrogen deposition? Sounds odd. Much more sever air pollution is tropospheric ozone pollution, which is phytotoxic and causes injuries to photosynthetic apparatus.

line 46: See paper 10.1093/treephys/tpad024 showing that elevated CO2 (which is currently increasing every year) could help trees to cope with drought conditions together with high N supply. You might add this paper here.

line 146: were vials coated with PTFE?

line 150, 289 etc: why you define VOCs again?

Fig 1: what does the overbars refers to?

fig 2: do not produce 3-D graphs, it is harder to read that. Why you add in Fig 1 horizontal lines and here not? be consistent. It is better to do not have lines. Add description and units to y-axis!

line 212: which aromatics do you mean? In here you write about aromatic aldehydes, which you already mentio in Aldehydes in line 203.

Table 1: be consistent with the naming according IUPAC. You cannot have once 4-Oxo-β-dihydroionone - which should be correctly 4-oxo-β-dihydroionone and another time with capitals elsewhere such as N-p-Bromophenylselenoacetamide. There are plenty of those incorrections. Ceck it

table 2: average peak area is useless unit. Calculate concentration based on standards or do not show it here.

Author Response

Dear Reviewer,
Thank you very much for taking the time to review this manuscript and thank you for the suggestions and requested changes, which have significantly improved our Paper. We have revised the manuscript, as suggested, point by point:

Authors analysed by SPME how dieback and non-dieback trees differ in terms of VOCs present in headspace analysis of their cores. The paper reads well, however the evidence that trees die because of change in VOC composition inside of wood is not strong here. Couldn’t that be that trees die because of something else and therefore there is a change in VOC composition? Authors claim the first option, however did not provide any evidence for that- that would need deeper further investigation. So that I think that the aim of the paper should be changed, that you just detected different compounds in headspace analysis in D and ND trees. Moreover, the hypothesis in line 83-85 is not proven. How did you prove that it was the pests who caused the decline of the trees? Assuming that only from accumulation of anti-herbivore VOCs is not direct prove. It is not in the manuscript.

• Reply: The general aim of the work was to analyse whether there are differences in VOC compounds between D and ND trees, in stands subjected to dieback phenomena linked to climate change and in particular to drought conditions. In this stand there are already several paper published in recent years by the same authors proving that these trees died because of severe drought stress occurred during the beginning of 2000^th (Colangelo et al. 2017). Drought and heatwaves are the main reason of poor healthy status in most of the trees vegetating in such stand. Although we cannot completely ruled out other possible causes, as pointed above, we are rather confident that the changes in VOC accumulation is related to the differences in drought stress impact among trees. By observing variations of these substances, at certain growth rings and thus at certain years and periods of stress, this could be useful in identifying a signal of dieback phenomena and guide towards a deeper analysis of forest dynamics in response to climate change. However, we agree with the Reviewer that we need further investigation. As we mentioned in the text, this paper can be considered a novelty in this topic. 

So we edited aim with “We hypothesize that declining trees, due to extreme drought conditions occurred during the last two decades [15,16], led to considerable variations in VOCs and PSCs that had made the trees more susceptible to the action of pests. Here, we assessed the presence or absence of metabolites (e.g., phenolic compounds and their derivatives) within growth rings useful for warding off biotic attacks or the presence or absence of substances useful for enabling the vegetation to cope with stress conditions. Furthermore, by observing variations of these substances, this could be useful in identifying a signal of dieback phenomena and guide towards a deeper analysis of forest dynamics in response to climate change.”

line 20: define SPME abbreviation

• Reply: We defined SPME abbreviation with Solid Phase Micro Extraction (SPME)-GC/MS

line 43: air pollution as nitrogen deposition? Sounds odd. Much more sever air pollution is tropospheric ozone pollution, which is phytotoxic and causes injuries to photosynthetic apparatus.

• Reply: We are sorry for the mistake, you are right. Ozone and SO2 are more sever then nitrogen deposition as they can cause damages to photosynthetic apparatus in particular to stomata opening. This was clarified in the text (Lines 45-46).

line 46: See paper 10.1093/treephys/tpad024 showing that elevated CO2 (which is currently increasing every year) could help trees to cope with drought conditions together with high N supply. You might add this paper here.

• Reply: Ok, as kindly suggested, we have included "In a recent study, exploring the interactive effects of [CO₂], nitrogen supply, and water availability on physiological (gas-exchange and chlorophyll fluorescence), morphological and stoichiometric traits of Norway spruce (Picea abies), it was shown that elevated CO₂ concentration (which is currently increasing every year) could help trees to cope with drought conditions together with high nitrogen supply [6]" and added the respective reference 6 in the bibliography.

line 146: were vials coated with PTFE?

• Reply: We used clear glass vial, O.D. × H 15mm × 45mm, with silicone septum.

 

line 150, 289 etc: why you define VOCs again?

• Reply: As suggested, we have deletedeliminated all definitions of VOCs.

Fig 1: what does the overbars refers to?

• Reply: Modified text, overbars refer to Standard Error.

Fig 2: do not produce 3-D graphs, it is harder to read that. Why you add in Fig 1 horizontal lines and here not? be consistent. It is better to do not have lines. Add description and units to y-axis!

• Reply: We replaced the 3D graphs with 2D histograms, without horizontal lines, and added description and units to y-axis.

 

line 212: which aromatics do you mean? In here you write about aromatic aldehydes, which you already mentio in Aldehydes in line 203.

• Reply: We meant aromatic compounds, like Xylene, 1,1,3-Trimethyl-3-phenyl-2H-indene, 2,6-Di-tert-butylphenol etc.

Table 1: be consistent with the naming according IUPAC. You cannot have once 4-Oxo-β-dihydroionone - which should be correctly 4-oxo-β-dihydroionone and another time with capitals elsewhere such as N-p-Bromophenylselenoacetamide. There are plenty of those incorrections. Ceck it

• Reply: We have corrected the naming according to IUPAC, leaving only the first letter or number in capitals, while the remaining no.

 

Table 2: average peak area is useless unit. Calculate concentration based on standards or do not show it here.

• Reply: Thanks for suggestion, we have added the average percent area (% ± 0.03), because, in the absence of an internal standard, it allows to better visualize the different presence and/or absence of the compound between the D and ND trees, being proportional to the quantity of each identified compound present in the sample mixture. Furthermore for each compound we added the Kovats Index (KI) to identify the VOCs.

Reviewer 2 Report

Comments and Suggestions for Authors

This study aims to measure volatile organic compounds as a result of climate changes in oak fields

It seems that the researchers made a great effort to write the manuscript well and describe it well.

There are just some notes:

101 Is there infestation of some pest or pathogen?

110 In addition to your work, why you did not collected the sample by setup the SPME directly on the tree? As using drill make more injury and present several not likely compounds?.

 Figure 1, 2 … Please add the statistic differences between mean.

Please report the Kovat index (KI) for each compound.

Author Response

Dear Reviewer,
Thank you very much for taking the time to review this manuscript and thank you for the suggestions and requested changes, which have significantly improved our Paper. We have revised the manuscript, as suggested, point by point:

This study aims to measure volatile organic compounds as a result of climate changes in oak fields

It seems that the researchers made a great effort to write the manuscript well and describe it well.

There are just some notes:

101 Is there infestation of some pest or pathogen?

• Since we started investigations in 2015 we did not record large and strong infestation of pests, although during the field surveys, trace of fungi, cerambycids and mistletoe were very evident on the D trees. However, although there no documented studies, likely infestation may have occurred at pick of dieback phenomena during the beginning of 2000^th.

110 In addition to your work, why you did not collected the sample by setup the SPME directly on the tree? As using drill make more injury and present several not likely compounds?.

• because the SPME fiber becomes saturated very quickly in contact with the air, adsorbing above all all the compounds present in greater quantities (even just CO2 for example), not just the VOCs of the trees. Furthermore, if it depended on the drill we would have found many not likely compounds common to trees, both D and ND.

 Figure 1, 2 … Please add the statistic differences between mean.

• For fig. 1, Thanks, we have entered a table 1 with average -BAI, SE and SD and for Fig. 2 we added statistic differences between mean (overbars indicate the standard error).

Please report the Kovats index (KI) for each compound

• Thanks for suggestion, for each compound we added the Kovats Index (KI) to identify the VOCs.

Reviewer 3 Report

Comments and Suggestions for Authors

This is my first review of the manuscript entitled "Volatile organic compounds (VOCs) in Mediterranean oak forests of Hungarian oak (Quercus frainetto Ten) affected by dieback phenomena" by Mecca et al.

 

The manuscript is focused on determining differences in VOCs profiles in dieback and non-dieback oak trees in Mediterranean, and the potential of these VOCs as an early indicators of stress occuring in oak in natural habitats under changing climate.

The text elicits two important questions, one is on the methodology and the other is on the discuttion of results.

 

The main question to the results and their discussion in this manuscript is to which extent the VOCs extracted from the wood, that consists of predominantly dead xylem cells, could be involved in current plant metabolism and responsible for actual photosynthesis in leaves and defense against pests. The discussion is fine if focused on metabolism of living cells and transport processes in phloem, however volatile organic compounds (VOCs) were extracted from the dead tissue up to 20 years old.

 

The other question is on the methodology of analysis. Authors use GC-MS as an analysis method for VOCs, but omit the information on their exact approach to the identification of VOCs and calculation of their content. Please see below within questions to M&M section.

 

In addition, authors must make supplementary figures available through MDPI web site. They are neigher included into downloadable manuscript file nor exist in the article page in peer-review system.

 

 

Some other points to improve include the following.

 

Materials and methods:

L144, L184: Actually, SPME was not the method for analysis of VOCs, but the method of their extraction and concentration prior to the analysis by gas chromatography. Analysis method was GC-MS. I would suggest to change section titles for 2.5 and 3.2 accordingly.

 

L148–L150: specify temperature for SPME fiber incubation in the vial headspace. Was it +50°C as for the previous step?

L156–L157: include electon impact ionization parameters here.

L157–L158: authors should specify which software and RI system (presumably, Kovats' RI system) did they applied for peak identification.

In addition, authors must describe the method for quantitative or semi-quantitative assessment of VOCs in the samples. Simple peak area in arbitrary units varies even if sample mass are within very narrow range. Also specify whether any internal standard was used for chromatogram peak area normalisation. In addition, it is important to indicate what was the source for quantitative estimation of identified compounds: total ion current (TIC), extracted ion current (XIC), or something else.

Without that information and proper quantitative analysis, the information given on fig. 2 could be meaningless.

 

L183 (Fig. 1 caption): I would suggest to expand the abbreviation 'BAI' here. Also, authors should specify what do error bars indicate, what was the sample size (n=?), and whether there are any statistically significant differences over these five-year periods.

 

L201 (Fig. 2 caption): specify units for Y axis.

 

L207: when discussing alkanes, authors state that these are components of cuticle. However, VOCs were extracted from wood rings (L131–L134), not from the trunk bark. Please comment whether alkanes from wood may be related to those which are synthesized in and secreted from epidermis cells.

 

 

Table 1 (L276): please explain what corresponds to 100%.

Table 2 (L280): please indicate sample size (n=?)

 

 

To conclude, since methodology needs clarification and potentially affect the results, my suggestion is to reconsider the manuscript after major revision.

Comments on the Quality of English Language

The manuscript language is fine and needs just subtle edits to punctuation and sentense structure (L84, L99, L171).

Author Response

Dear Reviewer,
Thank you very much for taking the time to review this manuscript and thank you for the suggestions and requested changes, which have significantly improved our Paper. We have revised the manuscript, as suggested, point by point:

Materials and methods:

L144, L184: Actually, SPME was not the method for analysis of VOCs, but the method of their extraction and concentration prior to the analysis by gas chromatography. Analysis method was GC-MS. I would suggest to change section titles for 2.5 and 3.2 accordingly.

• Thanks for suggesting, we edited with "Solid Phase Micro Extraction-GC/MS Analyses".

L148–L150: specify temperature for SPME fiber incubation in the vial headspace. Was it +50°C as for the previous step?

• Extractions were realized by immersing the fiber in the headspace at 36 °C for 30 min and then withdrawn into the needle and transferred to a GC/MS system.

L156–L157: include electon impact ionization parameters here.

• We added “An HP 5973 mass selective detector (mass range: 15-800 amu; scan rate: 1.9 scans/s) (Agilent) was utilized with helium at 0.8 mL min⁻¹ as the carrier gas. Ionization was performed by electronic impact (EI), setting the electron multiplier to 1435 eV.”.

L157–L158: authors should specify which software and RI system (presumably, Kovats' RI system) did they applied for peak identification.

In addition, authors must describe the method for quantitative or semi-quantitative assessment of VOCs in the samples. Simple peak area in arbitrary units varies even if sample mass are within very narrow range. Also specify whether any internal standard was used for chromatogram peak area normalisation. In addition, it is important to indicate what was the source for quantitative estimation of identified compounds: total ion current (TIC), extracted ion current (XIC), or something else.

Without that information and proper quantitative analysis, the information given on fig. 2 could be meaningless.

• Thanks for suggestion, we have added the average percent area (% ± 0.03), because, in the absence of an internal standard, it allows to better visualize the different presence and/or absence of the compound between the D and ND trees, being proportional to the quantity of each identified compound present in the sample mixture. Furthermore for each compound we added the Kovats Index (KI) to identify the VOCs (Table 2).
• We added that the chromatograms obtained from the total ion current (TIC) were integrated without any correction for coelutions and tentatively identification of components was based on mass spectra and NIST 11 library comparison.

L183 (Fig. 1 caption): I would suggest to expand the abbreviation 'BAI' here. Also, authors should specify what do error bars indicate, what was the sample size (n=?), and whether there are any statistically significant differences over these five-year periods.

• We edited Fig.1 caption with “Average of basal area increment (BAI) in dieback (D) and non-dieback (ND) trees over five-year periods (overbars indicate the standard error)”. For fig. 1, We have entered a table 1 with average -BAI, SE and SD and for Fig. 2 we added statistic differences between mean (overbars indicate the standard error).

L201 (Fig. 2 caption): specify units for Y axis.

• We replaced the 3D graphs with 2D histograms, without horizontal lines, and added description and units to y-axis.

L207: when discussing alkanes, authors state that these are components of cuticle. However, VOCs were extracted from wood rings (L131–L134), not from the trunk bark. Please comment whether alkanes from wood may be related to those which are synthesized in and secreted from epidermis cells.

• Reply: We really thank you for this question. In our manuscript, we stated that alkanes are typically part of the waxy cuticle, serving as a protective barrier against environmental stressors. We found that eicosane, detected in ND trees, is completely absent in all samples of D trees. However, despite our deep investigation of the scientific community, we are not able to elucidate whether alkanes from wood may be related to those secreted from epidermis cells, at least that was not the main focus of our manuscript. However, worthy of attention were some published papers:

Wu, L., Guo, S., Wang, C., & Yang, Z. (2009). Production of alkanes (C7–C29) from different part of poplar tree via direct deoxy-liquefaction. Bioresource Technology, 100(6), 2069-2076.

Deneyer, A., Renders, T., Van Aelst, J., Van den Bosch, S., Gabriels, D., & Sels, B. F. (2015). Alkane production from biomass: chemo-, bio-and integrated catalytic approaches. Current Opinion in Chemical Biology, 29, 40-48.

Table 1 (L276): please explain what corresponds to 100%.

• We added in section 2.5 “the percent area (% ± 0.03), proportional to the amount of each identified compound present in the sample mixture, was given”.

Table 2 (L280): please indicate sample size (n=?)

• We added the number of samples to Table 2 and n,sample size in the legend.

To conclude, since methodology needs clarification and potentially affect the results, my suggestion is to reconsider the manuscript after major revision.

The manuscript language is fine and needs just subtle edits to punctuation and sentense structure (L84, L99, L171).

We edited punctuation and sentense structure.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript # forests-3027512 deals with the evaluation of the presence or absence of volatile organic compounds (VOCs) useful for enabling the vegetation to cope with stress conditions in Mediterranean oak forests of Hungarian oak. The topic is interesting and the results are looking promising. I would recommend its publication in the journal after following issues are addressed. 

1) Lines 20-22

..."In this study, we analysed by SPME how dieback (D) and non-dieback (ND) Hungarian oak trees from the San Paolo Albanese site respond to these climatic dynamics, in terms of volatile organic compounds (VOCs)."..

The authors use GC-MS for the analysis of VOCs but nowhere is described. SPME should be defined.

2) Lines 78-81

It was not clear that VOCs are first time used in order to evaluate the effects of climate change. The related part needs revise.

3) Lines 88-90

"These processes are still poorly understood, so further studies are needed to fully understand which metabolites are involved in the response to dieback phenomena."

This sentence should be moved to conclusion.

4) Sections 2.2 and 2.3 need refs.

5) Lines 135-137 

SPME is not an analyse technique. Please revise.

6) Lines 152-154

More details should be given for the column (i.e. coating type)

7) Fig. 2 doesnt make sense. It is not clear what the axis describe ? number of compounds?

8) Lines 203-256 

This part needs completely revise and should be written according to results given in Tables 1-2 as well as including refs.   

 

 

    

 

Comments on the Quality of English Language

minor revision 

Author Response

Dear Reviewer,
Thank you very much for taking the time to review this manuscript and thank you for the suggestions and requested changes, which have significantly improved our Paper. We have revised the manuscript, as suggested, point by point:

The manuscript # forests-3027512 deals with the evaluation of the presence or absence of volatile organic compounds (VOCs) useful for enabling the vegetation to cope with stress conditions in Mediterranean oak forests of Hungarian oak. The topic is interesting and the results are looking promising. I would recommend its publication in the journal after following issues are addressed.

1) Lines 20-22

..."In this study, we analysed by SPME how dieback (D) and non-dieback (ND) Hungarian oak trees from the San Paolo Albanese site respond to these climatic dynamics, in terms of volatile organic compounds (VOCs)."..

The authors use GC-MS for the analysis of VOCs but nowhere is described. SPME should be defined.

• We defined SPME abbreviation with Solid Phase Micro Extraction (SPME) and added GC/MS. The SPME-GC/MS system used is described in paragraph 2.3 lines 157-162.

2) Lines 78-81

It was not clear that VOCs are first time used in order to evaluate the effects of climate change. The related part needs revise.

• Ok, we have revised this part highlighting the studies that have reported the VOCs application for the first time.

3) Lines 88-90

"These processes are still poorly understood, so further studies are needed to fully understand which metabolites are involved in the response to dieback phenomena."

This sentence should be moved to conclusion.

• We have deleted this sentence in the introduction and inserted it in the conclusions.

4) Sections 2.2 and 2.3 need refs.

       -   We have added more references [17-22] to section 2.2 and reference [23] to section 2.3 

5) Lines 135-137

SPME is not an analyse technique. Please revise.

• We defined SPME with SPME-GC/MS.

6) Lines 152-154

More details should be given for the column (i.e. coating type)

• We added more details “Phenomenex Zebron ZB-5 MS capillary fused-silica column (30-m × 0.25-mm I.D., film thickness 0.25 µm FT coated with non-polar 5% poly(dimethyl siloxane) stationary phases)”.

7) Fig. 2 doesnt make sense. It is not clear what the axis describe ? number of compounds?

       -     We replaced the 3D graphs with 2D histograms, without horizontal lines, and added  description and units to y-axis.

8) Lines 203-256

This part needs completely revise and should be written according to results given in Tables 1-2 as well as including refs. 

• Thanks for the suggestions for improvement, so we have rewritten part of the results and discussion based on the results provided in Tables 2-3, although it was not possible to extend the argument and references as there are very few studies on this topic or that best can detail the action of each single compound, this however encourages us on the novelty of this work and on future investigations.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I thank authors for the changes they did. They improved the manuscript and responded to my comments.

Author Response

Dear Reviewer,
Thank you very much for taking the time to review this manuscript and thank you for the suggestions and requested changes, which have significantly improved our Paper.
Kind Regards

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors 

Thank you for your response to all my comments 

All the best

Author Response

Dear Reviewer,
Thank you very much for taking the time to review this manuscript and thank you for the suggestions and requested changes, which have significantly improved our Paper.
Kind Regards

Reviewer 3 Report

Comments and Suggestions for Authors

This is my second review of the manuscript entitled "Volatile organic compounds (VOCs) in Mediterranean oak forests of Hungarian oak (Quercus frainetto Ten) affected by dieback phenomena" by Mecca et al.

 

Authors replied in detail to the majority of reviewers' questions and edited the manuscript accordingly. A few points still arise, as explained below. However, in my opinion, the manuscript may be published after addressing these questions.

 

L182: Incorrect, should be Kovats retention index (RI) hereinafter (L319, ...) as accepted abbreviation.

See, for instance:

Hummel J., Strehmel N., Bölling C., Schmidt S., Walther D., Kopka J. (2013) Mass Spectral Search and Analysis Using the Golm Metabolome Database. In: The Handbook of Plant Metabolomics, Wiley-VCH Verlag GmbH & Co. KGaA; 321-343; http://dx.doi.org/10.1002/9783527669882.ch18

Kopka, J., Schauer, N., Krueger, S., Birkemeyer, C., Usadel, B., Bergmuller, E., Dormann, P., Weckwerth, W., Gibon, Y., Stitt, M., Willmitzer, L., Fernie, A.R. and Steinhauser, D. (2005) [email protected]: the Golm Metabolome Database, Bioinformatics, 21, 1635-1638. http://dx.doi.org/10.1093/bioinformatics/bti236

L206, Fig. 1 caption: authors are expected to indicate the sample size (n=?) here as well.

 

Comments on the Quality of English Language

L165: Please split "theanalysis" word.

L346: the same applies to 'phenomenaand'.

Author Response

Dear Reviewer,
Thank you very much for taking the time to review this manuscript and thank you for the suggestions and requested changes, which have significantly improved our Paper. We have made further changes by responding point by point:
- L182: Incorrect, should be Kovats retention index (RI) hereinafter (L319, ...) as accepted abbreviation.
Reply: Thanks, as suggested, we have changed with "Kovats retention index (RI)".
- L206, Fig. 1 caption: authors are expected to indicate the sample size (n=?) here as well.
Reply: As suggested, we have edited with "Average of basal area increment (BAI) in (n=12) dieback (D) and (n=12) non-dieback (ND) trees over five-year periods (overbars indicate the standard error)".
- L165: Please split "theanalysis" word.
- L346: the same applies to 'phenomenaand'.
Reply: Thanks, we have corrected grammar.

Kind Regards.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors addressed all my concerns in the revised manuscript and I would recommend its publication in the journal.

Comments on the Quality of English Language

minor revision 

Author Response

Dear Reviewer,
Thank you very much for taking the time to review this manuscript and thank you for the suggestions and requested changes, which have significantly improved our Paper.
Kind Regards