Einbock and Conen present a well-written manuscript on measurements of ice nucleating particles (INPs) in washing water of leaves from a number of trees and air samples collected in Switzerland. The study appears to be well-conceived and methodologically sound. The results provide valuable insights into INPs from plant surfaces and provide a solid basis for further research. It is a valuable contribution to the literature on potential sources of atmospheric INPs. Some issues still need to be addressed before publication.

Specific comments:

L15: Please clarify what is meant by "exposed leaves" with respect to the type of exposure.

L37-38: “..and living as well as decaying vegetation (Lindemann et al., 1982; Lindow et al., 1978a; Schnell and Vali, 1976) are major sources of biological INPs.” The statement needs to be modified and references added, because vegetation is defined as the plant cover in a given area. However, the references cited are for bacterial INPs. While these studies link bacteria to living or decaying vegetation, they don’t directly address the vegetation itself as source of INP. Given that this study analyzed INPs from leaf washing water, the possible contribution of plants themselves as sources of biological INPs should be mentioned (e.g., Pummer et al., 2012, Hiranuma at al., 2015, Felgitsch et al., 2018., Seifried et al., 2020)

L38-40: Please also consider the recent study by Wieland et al., 2024 that found birch INP to be active above -10°C.

L112: While I would expect that any counting error would be detected when two independent observers count by eye, I wonder about a potential error when only one observer counts by eye. How often has it been counted?

L116: Leaves were initially collected in polyethylene zip bags and were transferred into 50 mL tubes after colour assessment. The authors should add information on the handling of the leaves for colour assessment. Were the leaves removed from the zip bags? Where were they placed? For how long? How were the leaves handled? Both, the material used and the handling of the leaves could have introduced INP contamination. Did the authors perform INP tests on the zip bag and tubes used, e.g., by washing them with MilliQ to exclude such contamination?

L126: Were all standards used in all calibrations? Can the authors add more details about the standards used, e.g., company, reference?

L135: “Usually” implies that some samples were collected differently. No number is given, but does this refer to “The remaining four samples” mentioned later? Consider rephrasing for more clarity.

L138: Was this also done after the last 5-min sampling interval? If yes, I suggest to write “ ..was replenished after each 5-min sampling interval”

L162: The authors found a correlation between INP concentration and relative humidity (RH). Given that RH and rainfall are related, I wonder about the potential influence of rainfall events on this observed correlation. For example, Bigg et al. (2015) have reported a persistent effect of rainfall on INP concentrations. It would be interesting to explore this relationship for this dataset by plotting INP concentration against rainfall frequency, if such data are available.

L172-174: The statement regarding the contribution of pollen to INP concentrations should be explained. The authors do not present data to support the absence of pollen, pollen fragments, or pollen-derived INP in their samples. While the four tree species studied do not appear to pollinate during the seasons studied, other tree species, flowers, and shrubs - including some for which IN activity may not be known - may do so. Pollen could also come from long-distance transport from other locations.

L184/185: Consider including results on temperature exposure of different tree species in the main text or supplementary section of the manuscript.

L185/186: Clarify what "different exposure of the trees" refers to. Exposure to what?

L211: Clarify what the asterisk and the slash mean in this context

L215: Please clarify what “this position” refers to

L253: For completeness, it may be helpful to provide a brief explanation of what Type II INPs are. Also, the reference provided supports this classification for bacterial INPs, but not for biological INPs, which include fungal or plant INPs.

L258: Please add specification of what "leaf habitat properties" might involve (e.g., microclimate, leaf morphology, etc.).

L275: It may be useful to clarify that "radiation" refers to solar radiation or UV exposure to avoid ambiguity.

L280: The statement “The more efficient an INP, the more sensitive it is to stress (Govindarajan and Lindow, 1988).” does not seem to apply universally to all types of INPs and stresses (e.g., Kunert et al., 2019, Eufemio et al., 2023).

L283/284: This is rather speculative. Fagus sylvatica could simply harbor different INPs, such as those associated with specific plant pathogens. Certain plant pathogens can contribute to the diversity of biological INPs (e.g., Morris et al., 2008, 2013, Kunert et al., 2019).

Plant pathogens are typically host-specific, meaning that they are adapted to infect particular plant species. While they can occasionally be found on non-host plants due to factors like accidental contamination or environmental conditions, they do not cause disease or reproduce effectively on these plants. This host specificity suggests that different plant species might host distinct INP-producing microorganisms or pathogens that are not present or are less prevalent on other species.

L286/287: Please provide an explanation or context for how these factors influence INP behaviour?

Figure 3: Clarify what is meant by “dark backgrounds” when all backgrounds are in light-colors.

L334-336: Please specify what dynamics refers to (e.g., distribution, activity, concentration).

Technical corrections/typos:

L36: remove the period after “thereof”

L76: Form-> From

L85: remove space before “100”

L91: NSC->NCS

L113/115: I assume the authors mean “dilutions” instead of “dilution series” in both instances

L114: were -> where

L118: concentration -> concentrations

L123: was -> were

L127: analyszed -> analysed

L131: Consider rephrasing to “ JFJ is at a 3 km higher elevation than the foilage sampling sites”

L159/160: I think it should read “per cm²” of leaf area

L236: Consider rephrasing the last part of the sentence to “ leaves that are more exposed to sunlight in the canopy”

L247: „warmer“ instead of „colder“ temperatures; the values given afterwards are higher than the values given in the sentence before

L332: from -> form

Table S1/S2 captions: Add a definition for LMA

Table S2 caption: leaf -> Leaf

References:

Bigg, E. K., Soubeyrand, S., and Morris, C. E.: Persistent after-effects of heavy rain on concentrations of ice nuclei and rainfall suggest a biological cause, Atmos. Chem. Phys., 15, 2313–2326, https://doi.org/10.5194/acp-15-2313-2015, 2015.

Eufemio, R. J., de Almeida Ribeiro, I., Sformo, T. L., Laursen, G. A., Molinero, V., Fröhlich-Nowoisky, J., Bonn, M., and Meister, K.: Lichen species across Alaska produce highly active and stable ice nucleators, Biogeosciences, 20, 2805–2812, https://doi.org/10.5194/bg-20-2805-2023, 2023.

Felgitsch, L., Baloh, P., Burkart, J., Mayr, M., Momken, M. E., Seifried, T. M., Winkler, P., Schmale III, D. G., and Grothe, H.: Birch leaves and branches as a source of ice-nucleating macromolecules, Atmos. Chem. Phys., 18, 16063–16079, https://doi.org/10.5194/acp-18-16063-2018, 2018.

Hiranuma, N., Möhler, O., Yamashita, K., Tajiri, T., Saito, A., Kiselev, A., Hoffmann, N., Hoose, C., Jantsch, E., Koop, T., and Murakami, M.: Ice nucleation by cellulose and its potential contribution to ice formation in clouds, Nat. Geosci., 8, 273–277, https://doi.org/10.1038/ngeo2374, 2015.

Kunert, A. T., Pöhlker, M. L., Tang, K., Krevert, C. S., Wieder, C., Speth, K. R., Hanson, L. E., Morris, C. E., Schmale III, D. G., Pöschl, U., and Fröhlich-Nowoisky, J.: Macromolecular fungal ice nuclei in Fusarium: effects of physical and chemical processing, Biogeosciences, 16, 4647–4659, https://doi.org/10.5194/bg-16-4647-2019, 2019.

Morris CE, Sands DC, Vinatzer BA, Glaux C, Guilbaud C, Buffière A, Yan S, Dominguez H, Thompson BM. The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle. ISME J. 2008 Mar;2(3):321-34. doi: 10.1038/ismej.2007.113. Epub 2008 Jan 10. PMID: 18185595.

Morris, C. E., Sands, D. C., Glaux, C., Samsatly, J., Asaad, S., Moukahel, A. R., Gonçalves, F. L. T., and Bigg, E. K.: Urediospores of rust fungi are ice nucleation active at > −10 °C and harbor ice nucleation active bacteria, Atmos. Chem. Phys., 13, 4223–4233, https://doi.org/10.5194/acp-13-4223-2013, 2013.

Pummer, B. G., Bauer, H., Bernardi, J., Bleicher, S., and Grothe, H.: Suspendable macromolecules are responsible for ice nucleation activity of birch and conifer pollen, Atmos. Chem. Phys., 12, 2541–2550, https://doi.org/10.5194/acp-12-2541-2012, 2012.

Seifried, T. M., Bieber, P., Felgitsch, L., Vlasich, J., Reyzek, F., Schmale III, D. G., and Grothe, H.: Surfaces of silver birch (Betula pendula) are sources of biological ice nuclei: in vivo and in situ investigations, Biogeosciences, 17, 5655–5667, https://doi.org/10.5194/bg-17-5655-2020, 2020.

Wieland, F., Bothen, N., Schwidetzky, R., Seifried, T. M., Bieber, P., Pöschl, U., Meister, K., Bonn, M., Fröhlich-Nowoisky, J., and Grothe, H.: Aggregation of ice-nucleating macromolecules from Betula pendula pollen determines ice nucleation efficiency, EGUsphere [preprint], https://doi.org/10.5194/egusphere-2024-752, 2024.

Review: Similar freezing spectra of particles on plant canopies as in air at high-altitude site

Summary:

In this manuscript, Einbock and Conen investigate tree surfaces as sources of atmospheric ice-nucleating particles (INPs) active above -10C. Specifically, they analyzed the freezing spectra of washing water from leaves of four different tree species that are abundant in Switzerland. The authors identified recurring freezing patterns and compare these with air INP samples collected at a high-altitude mountain station in the Alps (Jungfraujoch). Their findings reveal a correlation between the freezing patterns of leaf washing water and the air samples, suggesting a potential link between plant-derived INPs and those found at high altitudes.

This study is of interest to the INP community, offering valuable insights into the potential sources of biological INPs. The connection between the plant surface INPs and INPs collected at high altitude is particularly intriguing, contributing to ongoing discussions about the origins of biological INPs. The manuscript is well-structured and merits publication in Biogeosciences after addressing some minor revisions.

GENERAL COMMENTS:

While the manuscript offers a compelling analysis, a discussion on the transport mechanisms of INPs from leaf surfaces to high altitudes (Jungfraujoch station) is notably absent. Although this topic may not be the primary focus of the manuscript, it is crucial to address how these INPs could be transported to such altitudes, especially if the claim is that they originated from plants at lower elevations. 

Furthermore, did the authors consider analyzing the back trajectory of air masses during the field campaign? This would help clarify whether the INPs sampled at the station could indeed have originated from local vegetation, or if they were transported from distant sources.

SPECIFIC COMMENTS:

Title:

• Consider changing ‘as’ to ‘and’ for better grammar: ‘Similar Freezing Spectra of Particles on Plant Canopies and in Air at a High-Altitude Site.’

Abstract:

• Line 8: The phrase "efficiently freeze" is vague. Consider rephrasing to specify whether you mean freezing at higher temperatures or some other criterion.

Introduction:

• The introduction is well-written and provides a comprehensive background on the topic.
• Line 77: typo ‘form’ should be ‘from’

Methods:

• Line 106: Sonicating leaves may release INPs from within the leaf structure, not just the surface. This could lead to an overestimation of surface-derived INPs. Did the authors compare results with and without sonication to assess its impact on the data?
• Line 127: typo ‘analyszed’ should be ‘analyzed’
• Line 144- 146: The explanation of INP corrections is somewhat vague. It would be helpful to expand on how the sodium concentration correlates with INP concentration, possibly with an additional equation for clarity.

Results & Discussion:

• Line 211: Was there any rainfall during the field campaign? If so, could rain have washed INPs from the top of the canopy to the bottom, leading to their accumulation in the lower parts of the trees, similar to the findings of Seifried et al. 2020?
• Figure 1: To improve clarity, consider labeling the individual graphs as (a), (b), (c), and (d). This would allow for a more structured caption and make it easier to refer to specific graphs in the text. Additionally, using "hollow" and "filled" symbols instead of "dark" and "bright" could enhance differentiation. Assign the corresponding Spearman correlation analysis to each labeled panel.
• Figure 3 and 4: The connection between the last pattern in Figure 4 and those in Figure 3 is unclear. Is it the blue pattern? Could you in general provide more explanation on how the patterns were selected?
• Line 297: What does "16INP_-10" refer to? Please clarify.

Conclusion:

• Line 334: typo ‘form’ should be ‘from’

References:

Seifried, T. M., Bieber, P., Felgitsch, L., Vlasich, J., Reyzek, F., Schmale III, D. G., & Grothe, H. (2020). Surfaces of silver birch (Betula pendula) are sources of biological ice nuclei: in vivo and in situ investigations. Biogeosciences, 17(22), 5655-5667.