Review Reports

Round 1

Reviewer 1 Report

Report on manuscript forests-485996 Cleary et.al.

 

In this manuscript, the authors examine the fungal communities of the seed of 14 different Pinus species and 23 seed lots obtained from seed banks (seed orchards and seed stands) and online retail sources from Europe and North America.  The aim of the paper is to determine the fungal communities present in seeds of Pinus species to assess the level of risk that movement and trade of seeds for planting poses for the introduction of invasive pathogenic fungi into unaffected regions.

 

Each seed lot was tested using high throughput sequencing (HTS). By extracting DNA of 100 randomly chosen seeds three replicate PCRs were conducted and their products sequenced. Output data was analysed and annotated. OTUs with identity and coverage ≥ 97% were assigned to a species level and below that threshold, to a genus or family level. After sequence processing, a total of 1,997 unique fungal OTUs were recorded from the 23 seed lots. The authors detected that about 23% of these fungal OTUs were classified as plant pathogens. Although these results may be highly relevant to inform future policies for seed trade, it is unclear to me what are the consequences in real terms of the results found by the authors.

 

According to the authors, 23% of the OTUs were classified as plant pathogens and according to figure 5, at least a third of the seed lots tested contained the four most common plant pathogens (S. polyspora, D. intermedia, L. theobromae and D. sapinea) mentioned by the authors. The authors also mention that Pinus species and country of origin were not related to the fungal communities discovered. This raises the following questions: what does this mean in terms of trade given that these pathogenic species are present in samples from North America, Europe and Asia? Does this mean that the pathogens are already present in these 3 continents and in a large proportion of seeds? If this is the case, why would it matter to move seed from one continent to another? What does the presence of these pathogenic communities in such high proportions mean in terms of other traded plant products? Why is it not considered riskier to trade seed than live plants?

 

The authors need to clarify what is the meaning of their results to back the claim that seed trade poses a considerable risk for the introduction of diseases. I can clearly see that the authors discovered fungal communities present in seed lots of a variety of Pinus species obtained, including several pathogenic communities. In some cases, common sense and a number of studies back the claim that infected seeds are the source of introduction of invasive pathogens. The authors provide good examples of this. However, it is not clear to me how do the results presented here prove that seed trade is a risk for the introduction of invasive disease as the pathogenic communities found in the samples are discovered across a large proportion of the seed lots sampled, independently of their host or origin.

 

Specific comments:

 

1.     There are too many keywords, the authors may consider keeping a maximum of 8 specific and relevant keywords to make the search for their article more relevant.

 

2.     As the authors focus on Pinus species, it may be worth focusing on pathogenic fungus affecting Pinus species. I would suggest that lines 52-61 are removed and lines 272-310 are moved to the introduction. Note that lines 305-310 can also help strengthening the impact of the results found later by the authors.

 

3.     Please spell out what OTUs are in the first instance (abstract).

 

4.     Elaborate on lines 87-91. Why is it the case that live plants pose a bigger risk than seed? Why is seed trade not seen as risky as live plant trade? How does the increase in seed trade increase the risk of disease introduction? Are there any available data to back this up? (e.g. the increase in seed trade has resulted in an increase of the introduction of plant pathogens in “x” percentage in “y” region).

 

5.     Explain what do your results mean. For example, what do the richness and the Shannon diversity mean for your results; what does Chao1 richness disparity mean? How do you explain it? What does it mean to have a low/high Shannon index? What does it tell you that it is not possible to define the ecological function of 47% OTUs? What life strategies had the 212 OTUs found in other guilds? More importantly, what does it tell you that 23% of the total number of OTUs were classified as plant pathogens?

 

6.      Explain the importance and relevance of your figures to the study. For example, I don’t see what the impact and relevance of Figure 3 to the study is. Or, how does figure 4 explain the fungal communities structures? Does it tell you that the communities of PS6 and PS24 are more similar than for example PS12 and PS11?

 

7.     The argument presented in lines 305-310 has to be highlighted and reinforced by results found by the authors in order for the hypothesis that the seed trade is a risk for invasive disease introduction be valid in this case scenario.

 

8.     Lines 349-350 are not validated through this study, at least not in its’ present form.

 

9.     The supplementary information lacks captions and explanations. It has no use to the reader in its’ present format.

Author Response

We thank the Reviewer for her/his comments to the manuscript which we found to be very useful and insightful. Accordingly, we have carefully considered all comments when revising the manuscript. Our responses to the general and specific comments are given in the attached document.  

Author Response File: Author Response.docx

Reviewer 2 Report

General

 The authors used pyrosequencing to screen 25 seedlots (100/lot) sources from four countries to document presence of fungal DNA associated with the seeds (internally and externally-borne). Furthermore, they categorized fungus identities according to ecological function and trophic level using FUNGuild. As one would expect, large data sets were obtained. The OTU’s obtained did reveal “the full diversity of the fungal communities” of the seedlots assayed and fungal DNA detected using fungal primers fITS7 and ITS4 in the ITS2 region. Of 53% OTUs for which ecological functions could be determined, 10% were classified as known “plant pathogens” and 12% as “pathogens-saprotrophs-symbionts.” Conspicuously absent from the pathogens detected [and noted by authors] was Fusarium circinatum.  Low levels of other Fusarium species (e.g. F. solani) was also curious.  Overall, the manuscript is a succinct and clear summary of the results. The presentation of the results in the figures is clear and complete. The main weaknesses of the manuscript concern the interpretation and discussion of the findings.  For example, what taxa may be “missed” or “swamped” due to use of ITS2 region primers? More specifically, did the authors consider Pinus pathogens of known concern and previously known to be carried on/in seed and whether ITS2 region primers would perform well or not?  For example, F. circinatum is known to common occur on P. radiata seed (e.g. Dwinnell 1999) and sensitive detection of the pathogen in pine seed has resulted through sequencing using primers from the IGS region of the nuclear ribosomal DNA (Ioos et al. 2009. Phytopathology). The low frequency of F.solani detection may also “suffer” from relatively poor ability of ITS2 primers to detect it. In addition and as universally known, detection of DNA only reflects presence of non-viable and viable fungus presence. The reviewer recommends that the authors state the limitations of the methodology they used. In addition, a brief paragraph on the example of F. circinatum could be used to demonstrate primer limitations.

 

Specific Comments

1.      Did the authors compare fungal communities (esp. pathogens) on seed by source category, i.e. commercial vs. seed banks/seed orchards?  One might hypothesize that a difference may be found.

2.      Percentages are used to state a number of the results, but the sample size (n) that was the basis for each was not always given or not clearly evident. For example, line 229 “10% of all SpOTUs” left the reviewer wondering whether this is 10% of the 23% of all SpOTUs that were categorized as plant pathogens. In context, one would assume so, but inclusion of “n = X” would be helpful here.

Author Response

We thank the reviewer for their thorough review and for providing insightful comments to improve the quality of this manuscript. Our responses to specific comments are given in the attached document and the appropriate changes made to the manuscript are marked with trackchanges

Author Response File: Author Response.docx

Reviewer 3 Report

This work represents an interesting aspect of biosecurity management. Seeds do have much lower regulation than other plant products, and the authors present a highly pertinent question. The work is well written and well presented.

One point I would like to see briefly addressed is the potential to import/move pathogens of other plants with seed. In other words how many of the other microbes present are not pine pathogens but pathogens of other plants? Is there potential for introducing a major cereal crop pathogen via seed of some highly unrelated ornamental species or vice versa? Again no extensive discussion is needed, but a brief paragraph addressing this question would strengthen the overall warning of the paper.

While I think this work is sufficient for a paper introducing the question, I would suggest the authors consider single seed analyses in future work. Homogenizing seed within a lot is a standard protocol to stretch resources, but much data is lost in the process. Percentages of seed carrying pathogens would clarify the extent of the threat and provide a basis for determining what is acceptable for movement in various states based on current pathogen distributions.

Author Response

We thank the reviewer for her/his comments. Our responses to specific comments are given as follows and the appropriate changes made to the manuscript are marked with trackchanges

Author Response File: Author Response.docx