Cold Tolerance in Pinewood Nematode Bursaphelenchus xylophilus Promoted Multiple Invasion Events in Mid-Temperate Zone of China

Round 1

Reviewer 1 Report

The reviewed manuscript presents data on cold tolerance in the pinewood nematode Bursaphelenchus xylophilus found in the mid-temperate zone of China.
In this study clear language is used throughout, literature is well referenced and relevant. Figures are relevant although Figure S2. should be changed as the codes are gard to distinguish from each other. In tables S10. and S14. spaces are needed between "21" and "pinewood" in both cases.
The manuscript presents original research which is within a scope of the journal. Research question is well defined and relevant. The incestigation was performed to well-known standards. The authors described the methods they used with sufficient detail and information that can be used to replicate the study. Conclusions are well stated and linked to original research question and limited to supporting results.

Author Response

Thanks for your comments. Figure S2, now Figure S4 is revised with lowercase letters to distinguish each part. Tables in supplementary materials are also corrected.

Reviewer 2 Report

The study is well designed in methodology and significant in conclusions. The results show that there are no genetic differences between groups of the PWN isolates originated from three different climatic zones, and there are no genetic markers of adaptation to cold conditions; as well no differences between the isolates themselves were revealed according to the results of the experiment. The results indicate a broad adaptation of all PWN isolates to temperature.

Four  notes.

1) Because the study was performed on two different phases of the life cycle, the dispersed larval DJ3 and the mixed population, please give the illustrations (photographs or BW drawings) of the anterior section (pharynx, medial bulb) and the genital primordium  region of the DJ3 from your experimental material to make sure that it belongs to third dispersal stage and exactly to the dormant form of the dispersed juvenile. Give the same photos of J3 from a mixed population (propogative generation) for a comparison.

2) Mortality was assessed by the absence of motility in response to mechanical stimulus. However, dispersed juvenile, as you indicated, have very slow motility, i.e., an error is possible to recognize the dead or live individual. In live mycophagous nematodes, the muscular part of the pharynx (median bulb) showing regular frequent pulsations, even if the individual itself is immobilized, it is a more reliable criterion for whether the nematode is alive or dead. Why is this criterion was not used?

3) L 264:

M3 solution (super-cooled unfrozen water?) – please give the reference, compounds and concentrations.

4) From the results however, it is not distinct, whether the nematodes are reproductive-functional, whether they  can multiply and produce eggs after your experimental procedures. Did you test the reproductive abilities of the population after your experiment? 

Author Response

1) Thanks for your comments. Pictures of DJ3 juveniles and J3 juveniles of propagative stages are provided in supplementary materials Figure S1 and S2.

2）Thanks for your advice. We employed the mechanical stimulus approach to assess mortality, which we think is commonly used in PWN researches, such as Wang et al. (2020, https://onlinelibrary.wiley.com/doi/10.1002/ps.5839). The survival rates of dispersal juveniles in our study were also at a high level of approximate 95%. Thus, we think it’s OK here. The pulsation of median bulb is a good criterion and we will employ it in future research.

3) A recipe and a reference is added (Line 103).

4) We agree that there might be difference on reproduction or virulence upon isolates after cold stress. However, as we mentioned in text, there were no genetic variance that could lead to phenotype difference. Thus, we didn’t test more characters. We observed that except the isolates in -10℃ for 24 h, the other survived nematodes in our experiments were able to mate and reproduce.