Phytophthora ramorum and Phytophthora gonapodyides Differently Colonize and Contribute to the Decomposition of Green and Senesced Umbellularia californica Leaves in a Simulated Stream Environment

Round 1

Reviewer 1 Report

Dear Authors,

now I have finished reading and reviewing of your manuscript under the title "Phytophthora ramorum and Phytophthora gonapodyides differently colonize and contribute to the decomposition of green and senesced Umbellularia californica leaves in a simulated stream environment". The topic is really relevant and interesting and I was really enjoying reading the manuscript. Introduction, results, discussion and conclusions are well written and well presented in my opinion and also well supported by supplementary data. However, material and methods could be maybe  a little bit shorter, but I must admit that all the steps and procedures during these experiments were described in detail, what is giving additional value to the paper. Although I really appreciate this kind of detail description, some color figures of the experiments could support presentation of material and methods and results in better way.

In discussion part maybe you can more stress possible influence of other organisms colonizing leaf tissue and their possible interaction with Phytophthora species in leaf decomposition. One sentence about possible future studies could be enough. Also, you presented very interesting data about infection and persistence of Phytophthora species in fallen floating leaves in streams. Beside of well known pathogen P. ramorum, P. gonapodyides seems to be a bit underestimated and always assigned as saprotrophic or opportunistic pathogen due to ubiquitous presence, particularly in wet ecosystems. However, this species was proven to be highly aggressive to several plant species, and it would be good to relate your results in one sentence to this matter.

Bellow are some small technical mistakes that should be corrected.

Good luck with your future work!

Line 62, please explain SOD when you first mention it;

Line 118, please check the name of the species in Pseudotsuga menziesii?

line 119, please check the name of the species in Notholithocarpus densiflorus; also in Arbutus menziesii.

Line 158, please change Phytophthora into italic;

Lines 380 and 384, please change Phytophthora into italic;

Line 389, please change P. ramorum into italic in the table caption;

Line 395, please change Phytophthora into italic;

Line 402, please change Phytophthora into italic;

Line 437, please change Phytophthora into italic;

Author Response

Author responses follow “>>>”; notes in brackets (e.g.,[see below])

Dear Authors,

now I have finished reading and reviewing of your manuscript under the title "Phytophthora ramorum and Phytophthora gonapodyides differently colonize and contribute to the decomposition of green and senesced Umbellularia californica leaves in a simulated stream environment". The topic is really relevant and interesting and I was really enjoying reading the manuscript. Introduction, results, discussion and conc    lusions are well written and well presented in my opinion and also well supported by supplementary data. However, material and methods could be maybe  a little bit shorter, but I must admit that all the steps and procedures during these experiments were described in detail, what is giving additional value to the paper. Although I really appreciate this kind of detail description, some color figures of the experiments could support presentation of material and methods and results in better way.

>>>We appreciate the suggestion that figures may be helpful in conceptualizing the complex experimental set-up. I have added some photographs in a new figure S1 in the supplemental material that should give a more concrete sense of the experimental set-up. More substantive figures would require additional time that may not be worthwhile and will likely not substitute for the detailed description of the methods in the text.  We feel that the reference to Medeiros et al (2009) and to our Phytopathology article (Aram and Rizzo 2018), both of which include some diagrammatic presentations of methods, complement the elaborate description of the methods in attempting to balance detail and efficiency for interested readers to reconstruct the experiment conceptually or practically.

In discussion part maybe you can more stress possible influence of other organisms colonizing leaf tissue and their possible interaction with Phytophthora species in leaf decomposition. One sentence about possible future studies could be enough [see 1 below]. Also, you presented very interesting data about infection and persistence of Phytophthora species in fallen floating leaves in streams. Beside of well known pathogen P. ramorum, P. gonapodyides seems to be a bit underestimated and always assigned as saprotrophic or opportunistic pathogen due to ubiquitous presence, particularly in wet ecosystems. However, this species was proven to be highly aggressive to several plant species, and it would be good to relate your results in one sentence to this matter. [see 2 below]

>>> 1. More detailed mention of this consideration has been added at lines 470-473 and 599-  601.

>>> 2. Additional detail has been added about this at lines 567-572.

Bellow are some small technical mistakes that should be corrected.

Good luck with your future work!

            >>> Thank you!

Line 62, please explain SOD when you first mention it;

>>> Corrected. I believe that a previous version of the abstract introduced the disease,          hence the omission.

Line 118, please check the name of the species in Pseudotsuga menziesii?

>>> Corrected.

line 119, please check the name of the species in Notholithocarpus densiflorus; also in Arbutus menziesii.

>>> Corrected. Thank you for catching the misspellings.

Line 158, please change Phytophthora into italic;

>>> Corrected

Lines 380 and 384, please change Phytophthora into italic;

>>> Corrected

Line 389, please change P. ramorum into italic in the table caption;

>>> Corrected

Line 395, please change Phytophthora into italic;

Line 402, please change Phytophthora into italic;

Line 437, please change Phytophthora into italic;

>>> All corrected

Thank you for the helpful suggestions.

Reviewer 2 Report

In “Phytophthora ramorum and Phytophthora gonapodyides differently colonize and contribute to the decomposition of green and senesced Umbellularia californica leaves in a simulated stream environment” the Authors address an interesting topic related to the ecological role played by P. ramorum and P. gonapodyides in an environment still largely uninvestigated. The experimental design is convincing, the paper is well written and the discussion looks appropriate and consistent with the results obtained. However, I would suggest some minor revisions to improve the readability of the paper prior to publication.

L33: “have definitively indicated the genus for invasive species that can threaten natural ecosystems”: this sentence is unclear, please reformulate.

L39: Does “similar to” mean that they belong to known Phytophthora species or not? I guess such isolates were somehow identified. Please clarify.

L40: Maybe “visible” rather than “obvious”.

L62: Not all readers might know about Sudden Oak Death, please add a couple of sentences about SOD and clarify the meaning of the acronym the first time it is reported.

L64: Here one sentence about which are the symptoms on bay laurel should be included.

L79: I agree, P. ramorum is aggressive but its effects are largely dependent on the host, as before mentioned. I suggest to remove this consideration and move it before, when SOD and symptoms on bay will be shortly illustrated in the revised version of the manuscript.

L158, L280, L380, L384, L389, L395, L402…..: use italics for Phytophthora. Check throughout the text

L206: please, include these data in the supplementary material.

L251-252: this is a result, please move it in that section.

L284-286: I would reframe this sentence to clarify that -0.005 was assigned to 1, while + 0.005 to 0 values.

L289-290: which week was set as reference level when week variable was categorized?

fig. 1: the figure looks quite confused and inconsistent. Symbols and colors do not always match (e.g. symbol used for P. ramorum is a grey circle, but in the figure there are also grey triangles and squares. I understand that the color rather than the symbol refers to the fungal species, but a better legend could be presented). No letters for P-values comparison are reported. The lines and symbols look often too near. I suggest to use barplot or boxplots to present these results.

Tables S1 and S2: please, add in the tables the meaning of the abbreviations you present. (e.g. d.f. num and d.f. den). I understand they refer to degrees of freedom, but a clear statement of acronyms is recommendable. More importantly, I see that a full set of interactions was tested, yet I could not see any mention of the fact that interactions were tested too. This should be reported in the main text. Since many covariates and interactions were tested, I guess why a stepwise procedure was not implemented for model selection. This issue should be addressed. In addition, significance is good to assess the statistical evidence against the null hypothesis, but what about the magnitude of the effect? No information about the model coefficients is reported. This issue should be addressed too.

L389: Maybe “count” rather than “frequency” would be more appropriate.

Table 1: define acronyms and add acronyms when suitable to improve the readability (e.g. sw for sterile water). Add information to allow the reader to understand immediately the difference between - and 0.

figure 2: please, see my comment about figure 1.

Discussion

 I would suggest to include in the discussion some sentences stressing that a system including only 2 species might not fully mirror the complexity of the interspecific interactions in a real-world ecosystem, although it is scientifically fully justified as experimental approach. In addition, some speculations could be added about what outcome might be expected if bay leaves were already colonized by one of the two Phytophthora spp. prior to the colonization by the other one, since combined inoculations performed contemporarily cannot unravel such a temporal effect. I suggest to include in the references the following paper, that could be relevant in this regard Widmer, T.L., Tooley, P.W. & Camp, M.J., Recovery of Phytophthora ramorum in plant tissue with mixed infections, Eur J Plant Pathol (2018) 150: 253. https://doi.org/10.1007/s10658-017-1260-3.

Do the Authors think that in natural conditions their results could have been influenced by climate conditions? For instance, an interesting recent paper highlights the role of climate in Phytophthora species distribution patterns (Miguel A. Redondo, Johanna Boberg, Jan Stenlid & Jonàs Oliva, Contrasting distribution patterns between aquatic and terrestrial Phytophthora species along a climatic gradient are linked to functional traits. The ISME Journal volume 12, pages 2967–2980 (2018)). Maybe some more sentence about the potential role of climate might improve the discussion.  Moreover, recent paper (Lione G., Gonthier P., Garbelotto M., Environmental Factors Driving the Recovery of Bay Laurels from Phytophthora ramorum Infections: An Application of Numerical Ecology to Citizen Science, Forests 2017, 8(8), 293; https://doi.org/10.3390/f8080293) showed that bay laurel can also recover in the field from P. ramorum infection, especially under certain climatic conditions, a fact that seems to further support the results presented here by the Authors, especially around line 586. The displacement of P. ramorum might be related to decreased bay laurel infections documented in the field by the above paper. I would suggest the Authors to include some considerations about the above points, with emphasis on the potential effect of climate.

Finally, considering that P. ramorum is an invasive species, can the results presented in this work be related to the invasiveness/potential spread of P. ramorum?

L630-631: use italics for Salix and Populus.

Author Response

Author responses to the Reviewer 2 follow “>>>”; notes in brackets (e.g.,[see below])

In “Phytophthora ramorum and Phytophthora gonapodyides differently colonize and contribute to the decomposition of green and senesced Umbellularia californica leaves in a simulated stream environment” the Authors address an interesting topic related to the ecological role played by P. ramorum and P. gonapodyides in an environment still largely uninvestigated. The experimental design is convincing, the paper is well written and the discussion looks appropriate and consistent with the results obtained. However, I would suggest some minor revisions to improve the readability of the paper prior to publication.

 L33: “have definitively indicated the genus for invasive species that can threaten natural ecosystems”: this sentence is unclear, please reformulate.

>>> Revised for simpler expression

L39: Does “similar to” mean that they belong to known Phytophthora species or not? I guess such isolates were somehow identified. Please clarify.

>>> I agree that the word use is confusing and revised it for greater clarity. There is increasing recognition that not all isolates recovered from natural environments match perfectly with species genotypes known to cause disease in agricultural contexts. As Phytophthora species such as P. cactorum, P. citricola and P. cryptogea, to mention a few, are increasingly recognized to represent species complexes, it becomes uncertain whether isolates recovered from natural or agricultural environments have the same characteristics. I think the revised text acknowledges this sufficiently for this introduction.

L40: Maybe “visible” rather than “obvious”.

>>> Revised as “discernible”

L62: Not all readers might know about Sudden Oak Death, please add a couple of sentences about SOD and clarify the meaning of the acronym the first time it is reported.

>>> The acronym has been written out and a brief description added (now lines 64-66).

L64: Here one sentence about which are the symptoms on bay laurel should be included.

            >>> Added a description of symptoms (line 67).

L79: I agree, P. ramorum is aggressive but its effects are largely dependent on the host, as before mentioned. I suggest to remove this consideration and move it before, when SOD and symptoms on bay will be shortly illustrated in the revised version of the manuscript.

>>> The intention of the sentence (now lines 83-85) is to introduce the paragraph that highlights the differences in trophic specialization of the taxa being compared and is therefore indispensable. Though most Phytophthora species typically show some host-specificity and tissue specialization (foliar, crown, roots), they nevertheless are generally considered aggressive pathogens—and P. ramorum has a wider host and tissue range than most—notwithstanding that not all plants are susceptible to a given species to the same degree or in the same tissue. Clade 6 Phytophthora are a recently recognized exception to this pathogen “rule”, along with several other species historically considered opportunistic pathogens on a wide range of hosts. Nevertheless, the qualification, “on many species” has been added for increased clarity, and an additional citations added to reference the basis of the characterization. 

L158, L280, L380, L384, L389, L395, L402…..: use italics for Phytophthora. Check throughout the text

            >>> Corrected 

L206: please, include these data in the supplementary material.

            >>> Sequences were registered with GenBank and accession numbers are provided (lines                  211-212)

L251-252: this is a result, please move it in that section.

>>> While technically this information is a result, this information about sample handling and its effects fits better in the methods section. A reader may wonder about the effect of sample handling while reading this section, and would have to refer forward to the results for the answer, while at the same time presenting this information in the results would require repetition of the details about process handling where the focus is experiment effects. We believe that the text flows better as is, but if the senior editor prefers, we can follow this suggestion and revise accordingly. We have added a clarification that this factor was excluded from the final analysis (line 258).

L284-286: I would reframe this sentence to clarify that -0.005 was assigned to 1, while + 0.005 to 0 values.

            >>> Revised as suggested (line 291)

L289-290: which week was set as reference level when week variable was categorized?

>>>Added which week served as reference level as a footnote in the supplemental tables.

fig. 1: the figure looks quite confused and inconsistent. Symbols and colors do not always match (e.g. symbol used for P. ramorum is a grey circle, but in the figure there are also grey triangles and squares. I understand that the color rather than the symbol refers to the fungal species, but a better legend could be presented). No letters for P-values comparison are reported. The lines and symbols look often too near. I suggest to use barplot or boxplots to present these results.

>>> The figure intends to show trends over time, and we believe that the figure does this efficiently and clearly as composed. A version of the figure with slightly staggered positions of elements where overlapping and with more distinct difference in line and element shade has been substituted that should improve readability, as should added clarification in the figure title to identify how organism species and leaf type are represented by figure elements. A separate representation of each combination in the legend would result in unnecessary use of much more space and text, likely causing the figure to be more difficult to decipher. Box plots or bars would require packing many elements into a small area, or require a greater figure footprint overall. The figure as it is composed allows the results to be efficiently stacked and easily allows the viewer to compare levels of colonization at different time points. Regarding p-values, given the standard error bars and clear differences or overlap in the values, we think that additional indications of significant difference are not necessary. We have added the number of observations summarized by each point (n) to the figure title which should give additional confidence to the standard error.

Tables S1 and S2: please, add in the tables the meaning of the abbreviations you present. (e.g. d.f. num and d.f. den). I understand they refer to degrees of freedom, but a clear statement of acronyms is recommendable. More importantly, I see that a full set of interactions was tested, yet I could not see any mention of the fact that interactions were tested too. This should be reported in the main text. Since many covariates and interactions were tested, I guess why a stepwise procedure was not implemented for model selection. This issue should be addressed. In addition, significance is good to assess the statistical evidence against the null hypothesis, but what about the magnitude of the effect? No information about the model coefficients is reported. This issue should be addressed too.

 >>> A statement that the full set of interactions were included in the analysis for each experiment has been added to the methods (lines 305-306). Footnotes indicating the meaning of abbreviations have been added to the supplemental tables. I feel that the magnitude of effects is clear from the figures. Listing coefficients would seem to take up much space, even in the supplemental materials, without adding additional information. If the senior editor prefers, we can add greater detail on the statistical approach and results.

L389: Maybe “count” rather than “frequency” would be more appropriate.

>>> Made recommended change

 Table 1: define acronyms and add acronyms when suitable to improve the readability (e.g. sw for sterile water). Add information to allow the reader to understand immediately the difference between - and 0.

>>> Made recommended changes

figure 2: please, see my comment about figure 1.

>>> We think that the figure is justified based on the same arguments put forth for figure 1. Additionally, where the levels differ for a treatment, they stand out clearly. We believe it is clear that the fact that most of the other elements are superimposed on one another and cannot be distinguished demonstrates that they are not different.

Discussion

 I would suggest to include in the discussion some sentences stressing that a system including only 2 species might not fully mirror the complexity of the interspecific interactions in a real-world ecosystem, although it is scientifically fully justified as experimental approach [see 1 below]. In addition, some speculations could be added about what outcome might be expected if bay leaves were already colonized by one of the two Phytophthora spp. prior to the colonization by the other one, since combined inoculations performed contemporarily cannot unravel such a temporal effect [see 2 below]. I suggest to include in the references the following paper, that could be relevant in this regard Widmer, T.L., Tooley, P.W. & Camp, M.J., Recovery of Phytophthora ramorum in plant tissue with mixed infections, Eur J Plant Pathol (2018) 150: 253. https://doi.org/10.1007/s10658-017-1260-3. [see 3 below]

>>> 1. The limited scope of this experimental work is stated in the first paragraph of the discussion (esp. lines 461-470). However, more specific references to other possible organisms make sense and have been added at lines 471-473 and 599-601.

>>> 2. The question of preemptive colonization is an interesting one. Our 2018 article in Phytopathology addressed the question of partial colonization by P. ramorum prior to exposure to natural stream-borne inoculum of clade 6 Phytophthora spp. The reverse situation, where a leaf might be completely colonized by clade 6 Phytophthora spp. and then exposed to P. ramorum inoculum, is not one likely to occur in nature, but, again based on results published in our Phytopathology article, we would expect that P. ramorum could not colonize such leaves effectively. The aim of this work was to elucidate processes observed under natural conditions, and while hypothetical successional sequences are interesting and potentially important, they are outside of the scope of this study.

>>> 3. The paper by Widmer et al. (2018) is interesting for diagnostic approaches but focuses on recovery and does not directly evaluate relative colonization by the different taxa. They also do not give detail about how inoculations were done (vaguely “500 zoospores”), so it is not clear what kind of competition may have been observed. The context of that study is under very specific controlled conditions and I don’t see an appropriate way to bring it up in this discussion. It is interesting to consider competition between pathogenic species, but it does not seem to fit into the discussion of this experimental work, as we focused on species that occur at high inoculum levels in California forest streams, with known distinct trophic specializations. A sentence clarifying this has been added at lines 81-82.

Do the Authors think that in natural conditions their results could have been influenced by climate conditions? For instance, an interesting recent paper highlights the role of climate in Phytophthora species distribution patterns (Miguel A. Redondo, Johanna Boberg, Jan Stenlid & Jonàs Oliva, Contrasting distribution patterns between aquatic and terrestrial Phytophthora species along a climatic gradient are linked to functional traits. The ISME Journal volume 12, pages 2967–2980 (2018)). Maybe some more sentence about the potential role of climate might improve the discussion. [see 4 below] Moreover, recent paper (Lione G., Gonthier P., Garbelotto M., Environmental Factors Driving the Recovery of Bay Laurels from Phytophthora ramorum Infections: An Application of Numerical Ecology to Citizen Science, Forests 2017, 8(8), 293; https://doi.org/10.3390/f8080293) showed that bay laurel can also recover in the field from P. ramorum infection, especially under certain climatic conditions, a fact that seems to further support the results presented here by the Authors, especially around line 586. The displacement of P. ramorum might be related to decreased bay laurel infections documented in the field by the above paper. [see 5 below] I would suggest the Authors to include some considerations about the above points, with emphasis on the potential effect of climate.

>>> 4. The intrinsic role of California’s climate in this system is inherent in the introduction, discussed specifically in lines 58-74. The significance of temperature as an influence on these organisms and this system is discussed in more detail in our Phytopathology article, dealing with experimental work in the natural streams, but is also mentioned in the last sentence of the penultimate paragraph of the discussion in this article (lines 634-636). Nevertheless, I have added one more sentence to that paragraph (lines 636-638) to highlight the relevance of temperature to the results in this study.

The paper by Redondo and colleagues discusses interesting considerations that may affect the prominence of certain taxa over a range of environments. The Phytophthora spp. that we have focused on here, namely P. ramorum and P. gonapodyides, do differ in many of the characteristics that they considered: one being primarily associated with aquatic environments, the other mainly as a foliar pathogen; one producing many chlamydospores, the other having generally not well characterized hyphal swellings; one strongly pathogenic/parasitic, the other having saprotrophic ability in addition to facultative pathogenicity. These considerations may have been more relevant to our Phytopathology article which first explored these differences in a more natural, field context. Though the mechanisms and traits that we examine in these studies (trophic specialization, leaf quality) address broad concepts, both studies are framed by a very specific context, namely streams in northern California forests. The article by Redondo et al., on the other hand, focuses on comparing different kinds environments, something we have not really any basis to discuss based on our studies. While a broader discussion of the relevance of the characteristics of this system to other environmental contexts is worthy of exploration, it seems to me to require a tangential departure from the main points of the study.

>>> 5. The paper by Lione and colleagues deals with California bay leaves collected from trees, an environment very distinct from and essentially not relevant to the stream environment which we have addressed in our work. Furthermore, they have defined “recovery” simply as a tree once having tested positive subsequently testing negative. No mechanism, and certainly not any observation, is discussed that would suggest that an infected leaf could become not infected. The authors acknowledge that the so-called “recovery” is explainable by trees simply shedding infected leaves in one season without there being subsequently suitable conditions for new infections to occur. Another possibility is that public participants gathering samples may simply have missed the infected leaves when not all leaves on a tree are necessarily infected, especially under arid conditions. For these reasons, and many more, we do not believe that the article is relevant to this work.

Finally, considering that P. ramorum is an invasive species, can the results presented in this work be related to the invasiveness/potential spread of P. ramorum?

>>> We do address this question substantially in our Phytopathology article and therefore it is less elaborated in this manuscript, though it is acknowledged, for example in that clade 6 Phytophthora spp. appear to mitigate the persistence of the invasive P. ramorum in streams (lines 510-512) and that vegetative litter should be considered as a source of P. ramorum (lines 639-654). The consideration of how introduced and resident species interact is in fact a premise of our work, but in this work, we focus more on the importance of pathogenic compared with saprotrophic specialization, which, may be more broadly considered. It is an interesting question whether any aggressively plant pathogenic Phytophthora can be considered other than invasive in a natural environment. These are important general considerations, worthy of interpretation and speculation, but we feel that between our Phytopathology article and this one, the topic is fairly well addressed within the scope of these experiments focused on a specific system and on particular mechanisms.

L630-631: use italics for Salix and Populus.

>>> Corrected

>>> Thank you for your suggestions.

Reviewer 3 Report

I found this paper to be well written and presented. The results describe interesting interactions between Phytophthora species found in streams associated with leaf litter. They help us to interpret the findings that plant pathogens like Phytophthora ramorum are found in streams in many regions, including those where the pathogen is not yet significantly affecting the forest ecoystem outside the stream. The paper offers better understanding of the infection biology of P. ramorum (e.g. it's ability to infect yellow leaves and survive in the stream environment).

A part of me would like to hear more about the authors' thoughts about the evolution of the pathogenic versus saprophobic properties of Phytophthora species. If I understand the statements in the introduction correctly, Phytophthora is still considered mostly a clade of plant pathogens with a few lineages migrating to a mostly saprophobic life, but the results seem to support the idea that many species exist in conditions where they mostly live as saprobes. Is this transition somehow facilitated by aspects of the biology of Phytophthora? And should we still consider this a lineage of plant pathogens with a few saprobes evolving from them? Or should we turn this on its head and view them as mostly saprobes that somehow are preadapted to form close interactions with living host plants that they infect?

I agree with the authors that seasonal changes in bay leaves did not outweigh other factors influencing experimental results (li 503-507) but I am not sure I agree fully with statements in the premise to that statement. I think that bay leaves change a lot over the course of the season, and leaves that are newly flushed are very different chemically and with respect to susceptibility than leaves that are one or more years old. The seasonal changes might be especially pronounced for young leaves, and my guess is that the leaves sampled for the experiment (and most leaves that would be found as litter in streams) were mostly one or more years old. So the lack of difference in results from experiments initiated at different times of year might more have to do with the lower fluctuations found in chemistry and susceptibility for older leaves than with stability of the leaves across a year.

I have two small corrections to suggest for the authors. First, please change the name to 'Fairfield Osborn Preserve (without the e at the end, line 121). Second, it looks to me like Table 2 is misformatted a bit, so that it's hard to follow the labels across the table, they are slightly stepped upwards. Perhaps this is a production problem, but it should be fixed.

Author Response

Author responses to the Reviewer 3 follow “>>>”; notes in brackets (e.g.,[see below])

Comments and Suggestions for Authors

I found this paper to be well written and presented. The results describe interesting interactions between Phytophthora species found in streams associated with leaf litter. They help us to interpret the findings that plant pathogens like Phytophthora ramorum are found in streams in many regions, including those where the pathogen is not yet significantly affecting the forest ecoystem outside the stream. The paper offers better understanding of the infection biology of P. ramorum (e.g. it's ability to infect yellow leaves and survive in the stream environment). 

A part of me would like to hear more about the authors' thoughts about the evolution of the pathogenic versus saprophobic [assuming ‘saprotrophic’ or ‘saprophytic’ was intended] properties of Phytophthora species. If I understand the statements in the introduction correctly, Phytophthora is still considered mostly a clade of plant pathogens with a few lineages migrating to a mostly saprophobic life, but the results seem to support the idea that many species exist in conditions where they mostly live as saprobes. Is this transition somehow facilitated by aspects of the biology of Phytophthora? And should we still consider this a lineage of plant pathogens with a few saprobes evolving from them? Or should we turn this on its head and view them as mostly saprobes that somehow are preadapted to form close interactions with living host plants that they infect?

>>> These are interesting questions, indeed, and they were discussed to some extent in our Phytopathology article (Aram and Rizzo 2018) which is extensively cited in this one. We added more detailed discussion of this at lines 563-572. Hopefully our articles will inspire further study and discussions of the topic.

I agree with the authors that seasonal changes in bay leaves did not outweigh other factors influencing experimental results (li 503-507) but I am not sure I agree fully with statements in the premise to that statement. I think that bay leaves change a lot over the course of the season, and leaves that are newly flushed are very different chemically and with respect to susceptibility than leaves that are one or more years old. The seasonal changes might be especially pronounced for young leaves, and my guess is that the leaves sampled for the experiment (and most leaves that would be found as litter in streams) were mostly one or more years old. So the lack of difference in results from experiments initiated at different times of year might more have to do with the lower fluctuations found in chemistry and susceptibility for older leaves than with stability of the leaves across a year.

>>> I am not sure what is exactly referred to here, but I take the point that we are indeed talking about older leaves. I have added the qualifier “of mature cuticle” at relevant points (lines 129, 513, 518) to clarify this. That was an important detail and I appreciate that it was pointed out.

I have two small corrections to suggest for the authors. First, please change the name to 'Fairfield Osborn Preserve (without the e at the end, line 121). Second, it looks to me like Table 2 is misformatted a bit, so that it's hard to follow the labels across the table, they are slightly stepped upwards. Perhaps this is a production problem, but it should be fixed.

>>> I am grateful for the detection of the misspelling and formatting issue. I have corrected them.

>>>Thank you for the helpful suggestions.