Diversity and Function of Endo-Bacteria in Bursaphelenchus xylophilus from Pinus massoniana Lamb. in Different Regions
Round 1
Reviewer 1 Report
I enjoyed reading this well executed, thorough study on the diversity, identification, and functional characterization of endosymbiotic bacteria of the pine wilt nematode. The paper is generally well written, but the English needs some attention. I started to copy-edit the introduction, but the work would have been too extensive on my part.
Notably, the first para of the intro is a hodgepodge of haphzardly concatenated concepts and needs some detailed attention to make it more readable.
Other than that, and specific comments, questions, and observations in the attached annotated PDF, I think the paper is well presented, statistically sound, and all figures and tables are necessary and sufficient.
Comments for author File: 
Comments.pdf
Author Response
Point 1: I enjoyed reading this well executed, thorough study on the diversity, identification, and functional characterization of endosymbiotic bacteria of the pine wilt nematode. The paper is generally well written, but the English needs some attention. I started to copy-edit the introduction, but the work would have been too extensive on my part.
Notably, the first para of the intro is a hodgepodge of haphzardly concatenated concepts and needs some detailed attention to make it more readable.
Other than that, and specific comments, questions, and observations in the attached annotated PDF, I think the paper is well presented, statistically sound, and all figures and tables are necessary and sufficient.
Response 1: Thank you very much for your recognition and comments to our manuscript. We have revised our manuscript, especially the first para of the intro, following your annotation in the PDF.
Point 2: Abstract: The inoculation experiments need to be mentioned in here, as they are very important in the functional characterization of the bacteria.
Response 2: We have mentioned the inoculation experiments in the Abstract section. It is that these strains also accelerated the development of PWD, and P. fluorescens had a more beneficial effect on PWN than S. maltophilia.
Point 3: Line 102. “A barcoded-tag including 6 nucleotide bases was added upstream of the primers for distinguishing among the different samples.” How?
Response 3: Nine different barcoded-tags can distinguish our nine samples in the sequencing process.
Point 4: Line 103. “After quantification and quality assurance,” How?
Response 4: The 16S rDNA PCR products were examined using electrophoresis on a 1% agarose gel and quantified using a Nanodrop2000C spectrophotometer (Thermo Fisher Scientific, Waltham, USA).
Point 5: “Determination of ROS Content and Antioxidant Enzyme Activity in PWN”. Details on nematode extraction and testing are needed. At the very least references should be provided.
Response 5: Revised according to the comment. Following oxidative stress treatment, the nematodes were washed five times with ddH2O and the ROS content and antioxidant enzyme activity in the nematodes were determined. According to Kampkotter et al. [45], after 100 nematodes were ground into a homogenate, 300 μL of homogenate was added with 100 μM fluorescent probe H2DCFH-DA and incubated at 130 rpm for 30 min in the absence of light at 37°C. Then, the fluorescence intensity was detected as the ROS content under the conditions of an excitation wavelength of 485 nm and a blocking wavelength of 528 nm using a fluorescence spectrophotometer (CARY-100 PTP-1 Fluorescence Peltier System; Varian, Atlanta, GA, USA). After the nematodes were ground into homogenate and centrifuged, the supernatant was taken for testing and partly used for protein determination using coomassie brilliant blue G-250. The activities of SOD, CAT, and GSH-Px were detected with 0.03mmol/L pyrogallol, H2O2, and 5,5′-dithiobis-(2-nitrobenzoic acid) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) at 325nm, 250nm, and 422nm wavelengths, respectively, using a spectrophotometer (Spectronic Helios Gamma; Thermo, UK).
Point 6: “An OTU represents a species.” Not necessarily. See https://en.wikipedia.org/wiki/Operational_taxonomic_unit
Response 6: OK. Thanks for your information.
Point 7: Line 222. “Biolog microbial identification of culturable endo-bacteria in different pine wood nematode isolates”. What is the minimum probability and maximum distance to assign ID? I am not familiar with Biolog and others may not be either, so some explanation would help.
Response 7: The minimum probability is not considered in the Biolog Microbial Identification System and a minimum similarity of 0.5 is used to assign ID. Usually a distance less than 5 is considered a good result. We have added “a minimum similarity of 0.5 was used to assign ID” to the Materials and Methods section.
Point 8: Line 332. “Chitinase plays an important role in the physiological processes of many organisms”. Too generic.
Response 8: Since chitinase is usually reported in the study of controlling fungal diseases, which has nothing to do with our study, we have deleted this content.
Reviewer 2 Report
This work describes two objectives. First one is to analyze diversity of endobacteria from PWN (Pine Wilt Nematode) collected in Pinus massoniana growing in different Chinese regions; and second goal is to study the effect of culturable endobacteria on fecundity of PWN, on PWN oxidative stress resistance and on PWN virulence in plants. Both goals are very interesting and meaningful.
The methods to fulfill both objectives are adequate. To study diversity they carry out High-Throughput Sequencing of Endo-bacteria from Nematodes. For the second objective, they identify first the culturable endobacteria, and later they determine the effects of two specific taxa, Pseudomonas fluorescens or Stenotrophomonas maltophilia, on PWN pathogenesis-related parameters.
Although the overall experimentation is correct, I miss some important points here:
Sampling to determine diversity of endobacteria. If authors are assuming that there is a regional effect, they must specify all other environmental variables that may affect this diversity, such as tree growth stage, seasonal variability, tree age, etc. All these variables need to be specified to compare effect of the region. Otherwise, they may be dealing with confounded effects.
Statistical analysis. This needs a clear explanation throughout the paper. This is really important since authors are comparing the presence effect of either Pseudomonas fluorescens or Stenotrophomonas maltophilia with sterile PWN. For each parameter measured they need to specify the experiment design and its corresponding anova. And consequently, in the results section, fig 4, 5, 6 and their corresponding text, information is required: they need to clearly state if comparison is among or within all treatments. For example, in fig 5b, do different letters mean significant differences among bacteria or among enzymes? And then, why the other treatment effect has not been tested?. Likewise, for endobacteria diversity analysis, have authors considered that diversity is nested in tree? Fig 1b requires further explanation. What is the legend meaning? Are differences in species frequencies statistically tested?
Finally, Introduction requires much information to be added
In summary, I found this article contains important information but these two points relating to sampling and statistical analysis should be specified or corrected.
Author Response
Point 1: This work describes two objectives. First one is to analyze diversity of endobacteria from PWN (Pine Wilt Nematode) collected in Pinus massoniana growing in different Chinese regions; and second goal is to study the effect of culturable endobacteria on fecundity of PWN, on PWN oxidative stress resistance and on PWN virulence in plants. Both goals are very interesting and meaningful.
The methods to fulfill both objectives are adequate. To study diversity they carry out High-Throughput Sequencing of Endo-bacteria from Nematodes. For the second objective, they identify first the culturable endobacteria, and later they determine the effects of two specific taxa, Pseudomonas fluorescens or Stenotrophomonas maltophilia, on PWN pathogenesis-related parameters.
Response 1: Thank you very much for your recognition of our manuscript.
Point 2: Sampling to determine diversity of endobacteria. If authors are assuming that there is a regional effect, they must specify all other environmental variables that may affect this diversity, such as tree growth stage, seasonal variability, tree age, etc. All these variables need to be specified to compare effect of the region. Otherwise, they may be dealing with confounded effects.
Response 2: All the pines used had wilted and were basically the same size, and were felled in September. We have added this to the Materials and Methods section.
Point 3: Statistical analysis. This needs a clear explanation throughout the paper. This is really important since authors are comparing the presence effect of either Pseudomonas fluorescens or Stenotrophomonas maltophilia with sterile PWN. For each parameter measured they need to specify the experiment design and its corresponding anova. And consequently, in the results section, fig 4, 5, 6 and their corresponding text, information is required: they need to clearly state if comparison is among or within all treatments. For example, in fig 5b, do different letters mean significant differences among bacteria or among enzymes? And then, why the other treatment effect has not been tested?. Likewise, for endobacteria diversity analysis, have authors considered that diversity is nested in tree? Fig 1b requires further explanation. What is the legend meaning? Are differences in species frequencies statistically tested?
Response 3: Revised according to the comment. In fig. 4 and 5, different letters above the bars indicate significant differences (P < 0.05) among the three treatments according to Tukey's HSD test. In fig. 6, different letters above the bars indicate significant differences (P < 0.05) among all data according to Tukey's HSD test. For endo-bacteria diversity analysis, we thought that diversity could be found in fig. 1 and the legend of fig. 1b should be heatmap cluster at the genus level and different colors represent the level of abundance. Statistical tests were performed when differences in species frequency among the three provinces were analyzed. We have revised these in our Results section.
Point 4: Finally, Introduction requires much information to be added.
Response 4: We have added some related information to the Introduction section.
Round 2
Reviewer 2 Report
Some questions are still not well explained. They are the following:
Sampling: then, each reference number represents a sample from a tree? Please specify it.
Line 216: These results indicate that the community structure of endo-bacteria in PWNs from different regions varies greatly, although there were no significant differences in the species of genera
- If authors stated what I pointed out in bold, they need to explain the statistical analysis they performed. And it is not explained if this apply for the species of genera within regions
- Please discuss in the Discussion section the scope and validity of these data.
Line 220: Figure 1. Diversity of endo-bacteria in the pine wood nematode (PWN) Bursaphelenchus xylophilus from different regions.
- there is not mention in this text what SC (01-03), JS (01-03) and GD(01-03) represents
- In fig 1 b, legend is still not explained. What is the meaning of -2, -1, ….., 2 values? What is the meaning of negative values in abundance?
Line 285: Data are the means ± standard deviation, and different letters above the bars for each enzyme determination indicate significant differences (P < 0.05) among the three treatments according to Tukey's HSD test.
- I would add what I wrote in bold type
Line 314 Data are the means ± standard deviation, and different letters above the bars indicate significant differences (P < 0.05) among all data according to Tukey's HSD test.
What does bold text mean?
Author Response
Point 1: Sampling: then, each reference number represents a sample from a tree? Please specify it.
Response 1: Yes. We have specified it in the Materials and Methods section.
Point 2: Line 216: These results indicate that the community structure of endo-bacteria in PWNs from different regions varies greatly, although there were no significant differences in the species of genera
If authors stated what I pointed out in bold, they need to explain the statistical analysis they performed. And it is not explained if this apply for the species of genera within regions
Please discuss in the Discussion section the scope and validity of these data.
Response 2: We have revised it to "although there was no difference in the ten dominant genera".
Point 3: Line 220: Figure 1. Diversity of endo-bacteria in the pine wood nematode (PWN) Bursaphelenchus xylophilus from different regions.
there is not mention in this text what SC (01-03), JS (01-03) and GD(01-03) represents
In fig 1 b, legend is still not explained. What is the meaning of -2, -1, ….., 2 values? What is the meaning of negative values in abundance?
Response 3: Nine reference numbers represent nine PWN isolates from different regions. In fig. 1b, these values represent relative abundance levels, not exact abundance values. In high-throughput sequencing analysis, a range of 2 to -2 is usually selected for cluster analysis. We have made the revision in the legend of Fig. 1.
Point 4: Line 285: Data are the means ± standard deviation, and different letters above the bars for each enzyme determination indicate significant differences (P < 0.05) among the three treatments according to Tukey's HSD test.
I would add what I wrote in bold type
Response 4: Thank you so much for your information.
Point 5: Line 314 Data are the means ± standard deviation, and different letters above the bars indicate significant differences (P < 0.05) among all data according to Tukey's HSD test.
What does bold text mean?
Response 5: It means “among all treatments”. We have made the revision.
