A New Double-Stranded RNA Mycovirus in Cryphonectria naterciae Is Able to Cross the Species Barrier and Is Deleterious to a New Host
, Helena Bragança 3,4, Nobuhiro Suzuki 2 and Daniel Rigling 1Round 1
Reviewer 1 Report
The reviewed manuscript presents biological and molecular characterization of the novel RNA mycovirus from Cryphonectria naterciae host. The most valuable and interesting part of this work, from my point of view, is that describing (inter)species virus transmission and influence of the virus on the physiology of the host, together with the fact that CnFGV1 showed hypovirulence feature in the non-original host C. carpinicola. The MS is clearly and thoroughly written with a lot of documentation material, corresponding methods were used in the experiments.
In future submission, please correct typos in References (journal abbreviations) No. 15, 26, 39, 41, 72.
Author Response
Reviewer 1
The reviewed manuscript presents biological and molecular characterization of the novel RNA mycovirus from Cryphonectria naterciae host. The most valuable and interesting part of this work, from my point of view, is that describing (inter)species virus transmission and influence of the virus on the physiology of the host, together with the fact that CnFGV1 showed hypovirulence feature in the non-original host C. carpinicola. The MS is clearly and thoroughly written with a lot of documentation material, corresponding methods were used in the experiments.
We highly appreciated your comments on this manuscript.
In future submission, please correct typos in References (journal abbreviations) No. 15, 26, 39, 41, 72.
Answer to Reviewer 1:
All these references have been corrected.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Carolina Cornejo and colleagues present an article on the identification and partial characterization of a novel RNA virus from the fungus Cryphonectria naterciae. In addition they show that co-cultivation results in within-species and cross-species transmissions of the virus to virus-free strains. Eventual benefits at the level of biocontrol are also discussed. In general the manuscript is well and comprehensibly written, the experiments presented appear to be carefully conducted and contains original and interesting information for people in the field.
The hypovirulence-associated traits shown for C. carpinicola and eventually also for other infected species are interesting and important. In a very recent publication the authors provided evidence that this fungus is associated with hornbeam decline. Given this, and also in order to increase the accessibility of your manuscript, potential differences compared with uninfected strains, for example in conidiation, specific fungal structures, hyphal appearance etc., should also be highlighted with morphological analyses at the microscopy level. In addition, in order to support the conclusions and strengthen the possibility of this virus acting as a biocontrol agent, at least some initial results in plants should be shown for C. carpinicola, along with the ones from C. naterciae.
The discussion may benefit from some compacting.
Please verify that the indicated numbering of figures in the text corresponds to the correct Figure. For example, Figure S4 should be Figure S5 in Lines 541-542.
Authors may want to consider moving Table 1 to the supplement. In this context, Figures S4-S9, or some of those, could be part of existing figures in the main text.
Author Response
Reviewer 2
Carolina Cornejo and colleagues present an article on the identification and partial characterization of a novel RNA virus from the fungus Cryphonectria naterciae. In addition, they show that co-cultivation results in within-species and cross-species transmissions of the virus to virus-free strains. Eventual benefits at the level of biocontrol are also discussed. In general, the manuscript is well and comprehensibly written, the experiments presented appear to be carefully conducted and contains original and interesting information for people in the field.
The hypovirulence-associated traits shown for C. carpinicola and eventually also for other infected species are interesting and important. In a very recent publication, the authors provided evidence that this fungus is associated with hornbeam decline. Given this, and also in order to increase the accessibility of your manuscript, potential differences compared with uninfected strains, for example in conidiation, specific fungal structures, hyphal appearance etc., should also be highlighted with morphological analyses at the microscopy level. In addition, in order to support the conclusions and strengthen the possibility of this virus acting as a biocontrol agent, at least some initial results in plants should be shown for C. carpinicola, along with the ones from C. naterciae.
We thank you for your interest and comments on this study. In what follow we would like to answer to all concerns you raised.
Answer to Reviewer 2:
We agree with Reviewer 2 that further elaborating on the morphological differences between infected and uninfected isogenic strains of C. carpinicola as well as inoculation experiments on trees would be helpful and worth attempting. However, expanding the experiments at this moment is neither feasible, given the time needed for suggested experiments, nor would they significantly support our arguments – that is, (i) the description of a novel mycovirus, (ii) its ability to infect closely related species within the genus Cryphonectria, and (iii) phenotypic alterations in new host fungi. For this reason, we would rather not perform these experiments at this time.
However, we agree with Reviewer 2 that the fact that CnFGV1 reduces conidiation on new host only partially indicates a suitable biocontrol agent - as it must be shown that it reduces the ecological fitness of host fungi under natural conditions. This subject will be addressed in a separate research project exclusively dedicated to all effects of CnFGV1 on C. carpinicola, including possible changes in hyphal morphology, conidiation levels, and most importantly virulence on trees etc.
To emphasize this issue, we have changed the final sentence in Discussion (line 620-623).
The discussion may benefit from some compacting.
Answer to Reviewer 2:
We have reworked the Discussion and shortened some parts that were somewhat redundant.
Additionally, we used some terms more stringent. For example, we avoided “wild type” (since it refers to a genotype independent of the virus infection). We use now “field-collected” in connection with “virus-free” or “virus-positive”.
Please verify that the indicated numbering of figures in the text corresponds to the correct Figure. For example, Figure S4 should be Figure S5 in Lines 541-542.
Answer to Reviewer 2:
We have checked all table and figure numbers in the manuscript and corrected if necessary.
Authors may want to consider moving Table 1 to the supplement. In this context, Figures S4-S9, or some of those, could be part of existing figures in the main text.
Answer to Reviewer 2:
We tried to move Table 1 to the supplement and to add some of the supplementary figures to the main text, but the layout did not fit in all changes we made. That’s why we have retained Table 1 in the main text. However, we reduced the information on Table 1 to make it fit better to JoF’s layout, and we have separated Figure 8A and 8B into 2 independent figures (Figure 8 and Figure 9) to better represent both graphs (line 389; line 402).
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
I respect the decision of the authors not to provide additional data to fully support the conclusions on the biocontrol potential at this stage, and also not to highlight eventual differences in morphology at the microscopy level, although I disagree.
In Figure 10, please include an extra panel, or an inset panel, with a higher magnification/resolution of wt pycnidia structures. Please also provide a quantification of the number of pycnidia in the conditions tested, for example per cm2 per petri dish.
Author Response
I respect the decision of the authors not to provide additional data to fully support the conclusions on the biocontrol potential at this stage, and also not to highlight eventual differences in morphology at the microscopy level, although I disagree.
Answer to Reviewer 2: We thank you for your understanding.
In Figure 10, please include an extra panel, or an inset panel, with a higher magnification/resolution of wt pycnidia structures. Please also provide a quantification of the number of pycnidia in the conditions tested, for example per cm2 per petri dish.
Answer to Reviewer 2: We included a box with magnification of the mycelium surface in Figure 10 (lines 459-460), and also added the average number of pycnidia per cm2 (line 453).
