Volatile Profiling of Fifteen Willow Species and Hybrids and Their Responses to Giant Willow Aphid Infestation

Round 1

Reviewer 1 Report

Valid questions about VOCs from willows and giant willow aphid, with valid approaches to answering them. Study seems appropriate for this journal. Lots of data and results. Too bad that VOCs didn’t match up well with aphid infestation levels. Overall, fairly well written.

I have A) a few major points about the study, and then B) several minor points—mainly stylistic—that should all be addressed.

A) Questions about the study and its implications.

1. The term “cultivar” has been used too loosely to designate the experimental plants in this study. “Cultivar” is defined as a plant variety of a particular species that has been produced in cultivation by selective breeding. The definition of cultivar in this paper is unclear, and not all of the experimental plants may be cultivars. The term “code” in Table 1 is not defined, but perhaps that is functionally equivalent to cultivar in this study?
   In lines 72-73, the authors state that the study aims to explore “intra-genus (Salix) variation in VOCs.” The authors call these cultivars but simply use species names (e.g. S. candida) throughout the manuscript, except for NZ 1040 and NZ 1184. This is imprecise and inconsistent. As such, the authors need to revise the manuscript in the following ways.
   
   a) First, in general, it would seem best to refer to the experimental plants as willow species (and maybe hybrids), with obvious exceptions for cultivars(?) NZ 1040 and NZ 1184. Thus, general term(s) need to be substituted for “cultivar” throughout the manuscript.
   
   b) Second, the reader has no introduction about the nature of the diversity of the willow genus Salix and what constitutes cultivated vs. wild species within the genus. Thus, it is imperative to have a background paragraph in the Introduction that describes the variety within Salix, and the concepts and definitions of hybrids, cultivars (for NZ 1040 & 1184), code, ramets, etc. This could also include discussions about the heteroecity of Salix, as only one sex of tree was used for some of the 15 plant types.
   
   c) Third, no source is cited for the information given in Table 1, and so readers know virtually nothing about the origin of the willow plants used in this study. Please add citation(s) here.
   
   d) Finally, results and discussions about the VOCs need to be presented basically by species (and maybe hybrid). Because single cultivars of Salix species were generally used in this study, “cultivar” is confounded with species / hybrid. Thus, one cannot know if the VOC levels would differ within that species due to intraspecific variability, and whether testing a different “cultivar” of each species would have produced substantially different results. Thus, it’s appropriate to discuss results by species (and perhaps hybrid)—again, with the exceptions of NZ 1040 & 1184.
   
   
2. Perhaps I missed them, but sample sizes and the number of trees that made up experimental units are unclear. It seems that it may have differed between samples for aphid density (lines 95-100) and samples for VOCs (section 2.3), no? In line 220, it’s unclear what n=? Is it n=15 or 45 or what? Similarly, the determination of df is also unclear and seems inconsistent among results presented in lines 184, 194, 195, 198 and 202. A brief explanation will help. Please explain (and perhaps correct).
3. Lines 165-167. Why were aphid population levels compared only with total VOCs? Seems like some relationship might have been detected by comparing within each (or at least some) of the four major classes of VOCs.
4. Line 287. But what about the potential for (and value of) attracting generalist natural enemies with VOCs?
5. Line 293-295. This statement seems contradictory to the results shown in Figure 4, in which VOCs differed with respect to infested vs. control trees. Perhaps the authors mean the degree of infestation, but this needs to be clarified.
   
   B) Minor points.
6. Line 35. “honey bees” (two words).
7. Line 35. Use a more precise term than “affected.” How are willows affected?
8. Line 36. Delete “large.” It’s been stated that the insect is the giant willow aphid, and though large for an aphid, there are many much larger insects.
9. The phrase “these secondary metabolites” appears suddenly without precedent and hasn’t been adequately defined. Please revise.
10. Line 61. Use the acronym GLV since it’s already been defined.
11. Line 72. Change to “Therefore, in this study, we explored the….”
12. Line 73. No comma after cultivar (or whatever term might be substituted for ‘cultivar’).
13. Line 96. I think the authors mean “ December 6-7 2018.”
14. Line 105. Insert “of” between total and six
15. Line 116. Semi-colon, not comma, after “72 hours.” Same for line 286 after “New Zealand.”
16. Line 145. Change to “The VOC data were square-root transformed….”
17. Line 218. The reason for running a Spearman correlation test was already given in line 165. Redundant.
18. Line 233. Change wording: “There were also similarities in the VOC composition was also similar among….”
19. Line 237. Change simply to “Both compounds….”
20. Line 266. Change to “the aphids do not directly damage….”
21. Table S2. (1) and (2) need to be defined here.
22. Fig. S1. “…(NZ 1040) and (NZ 1184).” Add “, respectively” to the end of this sentence, as in the footnotes of Tables S3 and S4.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

In their study, Tun et al. characterized the volatile emissions of fifteen different willow cultivars with and without infestation by an invasive species, the giant willow aphid Tuberolachnus salignus. The manuscript is well written, the experimental design looks solid and the volatile collection was made state-of-the-art. The data collection of such a big number of cultivars, compared to most other studies, is very interesting from an ecological or biochemical perspective, but I doubt this study falls within the scope of agronomy; I rather would see it published in MDPI forests or plants. The main reason for this is that economically interesting parameters, such as biomass production or occurrence of natural enemies, were not investigated. Instead, the partly reduced emission of volatiles in response to aphid infestation is a good starting point to study the molecular regulation of defenses in willows: why do some willows reduce VOC emission while others do not? What distinguishes them in stress recognition, defense hormone levels or other signaling paths? Are there any ecological consequences from the reduced emission?

I therefore would recommend to delete the few parts (line 28, 75, 310, 322) that aim to add an agronomic side to the study. Instead, the differences and relations between cultivars, and speculations why they differ in VOC emission (e.g. shrub vs. tree willows) and aphid response, could be extended.

Following a few more detailed comments and questions:

Abstract:

- line 14 – 18: what was the motivation to study VOC emission and not any other parameter? This doesn't become very clear.

Introduction:

- the introduction should be re-structured a little to reduce content-related 'jumps', for example with the following order: volatiles (HIPVs) in general – volatiles in willows – study system of willows and aphids – aims of study.

- line 44: replace 'green leaf volatiles (GLVs)' by ' fatty acid derivatives'; or add 'and other fatty acid derivatives' after '(GLVs)'

- line 46 – 49: VOCs can also be attractants to herbivores and are therefore not always beneficial for the plant. Please add this aspect to the introduction.

- line 62: delete 'chemical'

- line 64 – 67: phytohormones should either be introduced properly or not be mentioned namely at all (instead write 'different signaling pathways'). As no phytohormone measurements are presented and they are not part of the discussion, I would suggest the latter.

Materials and Methods:

- line 95 – 97: were the experiments done in two blocks (2018/ 2019)? Why were aphids added once one month after the first infestation, and the other almost a year before? Maybe there is some mistake in the dates?

- line 110: do you mean 'L/ min'?

- line 133: a maximum GC oven temperature of 95°C seems very low to me. Maybe the authors could provide an examplatory chromatogram to get a better impression of how well the compounds were separated?

- line 139: I guess the units should be same as in the axes of the graphs (delete 'µl-1')

Results:

- line 180 – 183: switch these two sentences, so that the occurrence is sorted better: all cultivars – 50% of them – 25% of them – one cultivar

Figures and Tables:

- Figure S1 is quite informative and important for this study, I suggest making it a main figure.

- Table 2: it might useful to sort the cultivars by their growth form (shrub/ tree), or their degree of relation to another, instead of alphabetically. Please add some more information to the heading and table: are the compounds found in all or only some of the replicates of each cultivar? Which of the compounds were identified with authentic standards? Please add (E, E) to alpha-farnesene. Replace the M in 'beta-famesene' by RN.

- Figure 2: would it be possible to depict shrub vs. tree willows in this graph? For example by using the same symbol or shades of the same color for each of the groups?

- Figure 4 and 5: the asterisks next to the species names is confusing, please place them next to the bars

Discussion:

- line 236 – 259: these paragraphs focus too much on chewing herbivores, which are not part of this study. I suggest shortening this part to at least half the length.

- line 246 – 247: even the opposite pattern was observed compared to the literature cited: a decrease instead of increase upon herbivore infestation. What does that mean for the biotic environment? Why is the reaction different? Please discuss this in more detail.

- line 262 – 264/ 268 – 270: there are some interesting studies on galling aphids in poplar (Pemphigus spyrothecae), which was shown to manipulate its hosts physiology and affect hormone signaling pathways and volatile emissions. I guess some of the references would be good to mention here, e.g. Ye et al. 2019 (https://doi.org/10.1007/s00468-018-1756-2)

- line 279 – 280: the lack of correlation between aphid numbers and volatile release actually speaks against this hypothesis.

- line 285 – 288: this hypothesis assumes, that the willows 1) recognize that it is an invasive species that feeds on them, and 2) have adapted to this aphid species, which I doubt is possible when T. salignus was introduced not even 10 years ago. Please delete or change this speculation.

- line 303 – 305: why should the two compounds that are present in all cultivars be good candidates for testing natural enemy attraction? Shouldn't be compounds that are differently emitted by resistant and susceptible cultivars be more promising?

- line 311, 'same sex': this is confusing: table 1 shows that the cultivars have different sexes, so what was actually used? Also: why should it be interesting to test different sexes? Either cut this sentence out or explain why this would be worth studying.

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Dear Authors,
you have carried out an interesting and original research in order to assess volatile profiling of fifteen willow cultivars and  their responses to giant willow aphid.

This work is of great importance because it detects the differences in the emission of VOCs directly in the field before and after inoculation of the aphids. The only flaw is the low number of repetitions, but as the authors say it is an exploratory work.

I think the your manuscript in the present form can be accepted for publication in the Agronomy Journal

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Adequately revised.