Identification of Suitable Reference Genes for RT-qPCR Normalization in Amylostereum areolatum Cultured on Pinus sylvestris var. mongholica Wood Powder

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

There is an interesting study on the symbiosis between Eurasian woodwasp and Amylostereum areolatum based on gene expression. In this study, the authors use the culture of Mongolian Scots pine wood powder that is closer to the natural growth conditions of Mongolian pine.

All the sections of the manuscript are well-written, which I overall accept. The methods chosen for the assessment of gene expression are widely used and scientifically appropriate. However, there are certain things which need a clarification.

The description of the result section 2.4. “Validation of reference genes” needs to be written based on statistical analysis. In lines 214-215, there is written “increasing trend in the expression level of AaLac1 in 14-day samples compared to 7-day samples”. However, it is not written about the errors, which probably using T-test display non-significant differences between the expression level of AaLac1 in 14- and 7-day samples. “Almost negligible upward trend” is another phrase that cannot be used in a scientific report.

In all the descriptions of figures and tables, where you write “of these candidates”, I recommend you change it to “of analyzed candidates” since a reader should understand a figure by reading only its description, and all the demonstratives, such as it, these, those, etc., should be avoided.

Line 31. Need space after “RT-qPCR”. Instead “analisis” it should be “analysis”.

Line 44. There is enough to write the full name of the fungus Amylostereum areolatum once in the introduction section, as you did in the previous sentence.

Line 75. Unnecessary dot after “analysis”.

Line 83. Unnecessary dot after “outcomes”.

Line 87. Unnecessary dot after “contexts”.

Line 136. “Cycle” should be in a lowercase.

Line 229. Need a comma before “and”.

Line 239. Double “That”.

Line 241. “Unpublished”. I do not recommend comparing any results in this manuscript with unpublished results. Unless you could write that “By comparing the growth curves of A. areolatum on this medium with on PDA, performed as a pilot, we did not find significant differences. However, a higher growth rate was determined on the Mongolian Scots pine wood powder medium.”

Line 409. Need space after “method”.

 

Line 416. Double space.

Author Response

Comments 1: The description of the result section 2.4. “Validation of reference genes” needs to be written based on statistical analysis. In lines 214-215, there is written “increasing trend in the expression level of AaLac1 in 14-day samples compared to 7-day samples”. However, it is not written about the errors, which probably using T-test display non-significant differences between the expression level of AaLac1 in 14- and 7-day samples. “Almost negligible upward trend” is another phrase that cannot be used in a scientific report.

Response 1: Thank you for pointing this out. I agree with this comment. Therefore, I have changed this part to read “When α-TUB, P450, and UBC were used for normalization, the expression patterns of AaLac1 were similar to transcriptomic findings (Figure 4). When normalized by the unstable reference genes, the expression patterns of AaLac1 were not compatible, with the expression levels in 14-day and 7-day samples being nearly identical”.

Comments 2: In all the descriptions of figures and tables, where you write “of these candidates”, I recommend you change it to “of analyzed candidates” since a reader should understand a figure by reading only its description, and all the demonstratives, such as it, these, those, etc., should be avoided.

Response 2: Agree. I have completed the revision according to the comments.

Comments 3: [Line 31. Need space after “RT-qPCR”. Instead “analisis” it should be “analysis”.]

Response 3: Complete revision.

Comments 4: Line 44. There is enough to write the full name of the fungus Amylostereum areolatum once in the introduction section, as you did in the previous sentence.

Response 4: Complete revision.

Comments 5: Line 75. Unnecessary dot after “analysis”.

Response 5: Complete revision.

Comments 6: Line 83. Unnecessary dot after “outcomes”.

Response 6: Complete revision.

Comments 7: Line 87. Unnecessary dot after “contexts”.

Response 7: Complete revision.

Comments 8: Line 136. “Cycle” should be in a lowercase.

Response 8: Complete revision.

Comments 9: Line 229. Need a comma before “and”.

Response 9: Complete revision.

Comments 10: Line 239. Double “That”.

Response 10: Complete revision.

Comments 11: Line 241. “Unpublished”. I do not recommend comparing any results in this manuscript with unpublished results. Unless you could write that “By comparing the growth curves of A. areolatum on this medium with on PDA, performed as a pilot, we did not find significant differences. However, a higher growth rate was determined on the Mongolian Scots pine wood powder medium.”

Response 11: I have changed this part to read “By comparing the growth curves of A. areolatum on this medium with on PDA, performed as a pilot, we did not find significant differences. However, a higher growth rate was determined on the Mongolian Scots pine wood powder medium”.

Comments 12: Line 409. Need space after “method”.

Response 12: Complete revision.

Comments 13: Line 416. Double space.

Response 13: Complete revision.

Reviewer 2 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The authors have seriously revised the manuscript. I just have one small comment. Table 2 in Note add Rank

Author Response

Comments 1: The authors have seriously revised the manuscript. I just have one small comment. Table 2 in Note add Rank

Response 1: Thank you for pointing this out. I agree with this comment. Therefore, I have added the note of Rank.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

The article is interesting and can be published after the revisions. The M&M section needs to be explained in detail. Please review the entire paper before submission to avoid any errors.

 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Author Response

Comments 1: The article is interesting and can be published after the revisions. The M&M section needs to be explained in detail. Please review the entire paper before submission to avoid any errors.

Response 1: Thank you for pointing this out. I agree with this comment. Therefore, I have completed the revision according to the reviewer's comments.

Comments 2: Line 25: Please correct RT-qPCRanalisis

Response 2: Complete revision.

Comments 3: 2.4 section: please provide more information

Response 3: Complete revision.

Comments 4: 4.2 section: RNAprep Rure Plant Kit (Tiangen) please correct with PURE

Response 4: Complete revision.

Comments 5: Line 298: All experimental procedures were followed in accordance with the manufacturers’ recommended, please correct with Recommendations.

Response 5: Complete revision.

Comments 6: The 4.3 section is not well described, please provide a detailed description

Response 6: I have added the description “The 25 μL reactions contained 12.5 μL of 2× Es Taq MasterMix (Dye) (Cwbio, China); the usage of primers, cDNA, and water was the same as that in RT-qPCR reactions). The amplification conditions were as follows: 95°C for 30 seconds, 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds.”

Comments 7: Why did you put the M&M after the results and discussion? After checking other articles, it seems it is not a rule of the Journal, please check!

Response 7: Complete revision.

Reviewer 4 Report (New Reviewer)

Comments and Suggestions for Authors

Review of the article “Selection and validation of reference genes for RT-qPCR normalization in Amylostereum areolatum cultured on Pinus sylvestris var. mongholica wood powder ”

Sirex noctilio is a pest of pine trees (Pinus spp.). Sirex noctilio, also called the purple horntail or Sirex wood wasp, is a native species to Eurasia and North Africa, where it is not considered a pest. However, it is an invasive pest of pine trees in many other parts of the world. It was discovered in New Zealand in the early 1900s, and in Tasmania and Australia in the 1950s and 1960s. By the 1980s it had moved to South America, by the 1990s to South Africa, and in 2004 it was discovered in North America. In the Southern Hemisphere, approximately 8 million hectares of exotic pine plantations are at risk of extinction. In Australia, it is estimated that uncontrolled horntail could cost $16-60 million per year, and in Brazil, $6.6 million per year could be a loss in the absence of pest control programs. In the southern United States, pine production could be at risk of losing $8 billion a year if Sirex noctilio gains a foothold in the region. Sirex noctilio is a powerful parasite that kills pine trees with the help of a symbiotic, tree-pathogenic fungus Amylostereum areolatum along with phytotoxic mucous, which is introduced into the tree along with eggs during oviposition. The mucilage interferes with the tree's ability to repel attacks from the fungus, which grows throughout the tree and causes fatal wilt. When a tree dies, the needles, especially on its crown, turn brown and fall off. A tree can die in just a year, as it rots from fungus. After laying eggs, the fungus causes the wood to rot, and the emerging pest larvae feed on the rotting wood. Sirex noctilio takes 1-2 years to develop into an adult, depending on the site of infection.

 

Studies related to the analysis of transcriptomic data and their changes at various stages of growth of Amylostereum areolatum are always of great scientific interest. In this work, an important result is the preliminary selection of 13 genes with a stable expression profile. Interestingly, the stability of the expression of these genes turned out to be dependent on the fertility of the nutrient media, which indicates the important influence of external conditions on the genetic profile of the organism. The results of the study indicate that they have not only practical, but also theoretical value.

 

To correct shortcomings in the design of the article in accordance with the requirements of the Forests Journal, you need to make the following changes. Firstly, the research results should be presented in accordance with the requirements of the journal and located in the appropriate paragraph - this will help facilitate the perception of information by readers. Materials and methods, in turn, should be highlighted in a separate section and placed before the results of the study, which is standard practice in scientific publications. You should also pay attention to the accuracy of the presentation of information, clarity of presentation and consistency of presentation of the material.

 

Author Response

Comments 1: To correct shortcomings in the design of the article in accordance with the requirements of the Forests Journal, you need to make the following changes. Firstly, the research results should be presented in accordance with the requirements of the journal and located in the appropriate paragraph - this will help facilitate the perception of information by readers. Materials and methods, in turn, should be highlighted in a separate section and placed before the results of the study, which is standard practice in scientific publications. You should also pay attention to the accuracy of the presentation of information, clarity of presentation and consistency of presentation of the material.

Response 1: Thank you for pointing this out. I agree with this comment. Therefore, I have completed the revision according to the reviewer's comments.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses a valuable topic in forest pathology and gene expression. It is well-known that Eurasian woodwasp together with symbiotic fungus Amylostereum areolatum causes massive damage to pines worldwide. However, the studies of reference genes in A. areolatum using as closer as possible to natural pine’s growth conditions are gaping. For this, the authors made an experimental design using the culture of Mongolian Scots pine wood powder.

The objective of the study was well and clearly formulated. The methods were correctly described. The sections of results, discussion, and conclusions were clear. It is a good scientific documentation with accurate interpretations. However, there are several points to be considered:

Lines 29-30. Do not use the same words in keywords as they are in the title. It will you allow to reach more readers.

Lines 57-59. Unpublished results should not be written in an introduction section.

Lines 98-104. Although Artificial Intelligence (AI) and AI-assisted technology in manuscript preparation is available, authors are fully responsible for the originality of their manuscript. The authors copied from ChatGPT or other large language models the nonsense paragraph about a general info how to write the section Materials and Methods and paste it in the results section.

Figure 3. The units M and V should be written in the same format – without brackets “M and V”. The legend before Figure 3a about stability of genes from the left to right is also unnecessary since it is explained in the text and in the figure’s description.

Table 2. Rank instead of “Rnak”.

Lines 183-185. Do the expression levels differ statistically significantly?

Lines 192-196. The explanation of the results should be written in a discussion section. Meanwhile, in the description of the results section I miss values with errors.

Figure 4. There is not shown statistical significance. To see bottom error bars, they should be in black instead of the same colors as charts.

Lines 204-227. All Latin species names must be Italic.

Line 221. Instead of “In” should be “in”.

Lines 235-267. The authors mainly explained the results they get without enough scientific justification and comparison with other studies. I would suggest expanding the discussion section with not basically only their team’s previous findings on gene expression in A. areolatum in PDA culture conditions, but also with another scientific knowledge.  

There is missing a statistical part (boxplots) of expression analysis of candidate reference genes in the section of Materials and Methods. Furthermore, I suggest adding “4.5. Statistical analysis” and then “4.5.1. Stability analysis of candidate reference genes”, and “4.5.2. Validation of reference genes.”

At the end of the discussion section, it would be useful to provide a remaining knowledge gap on A. areolatum for further studies.

Reviewer 2 Report

Comments and Suggestions for Authors

This study presents a highly interesting research problem concerning the specific symbiosis between a fungus and an insect, which is necessary for effective tree infection. Identifying genes conducive to this process may serve as a basis for future solutions to eliminate this issue. In my opinion, the paper is well-written and suitable for publication in a journal with minor revisions. I have two comments:

Keywords: Keywords should not repeat words used in the title. Additionally, it is advisable to arrange them alphabetically.

Line 36: Do not start the sentence with an abbreviation. Change to "Amylostereum areolatum".

Reviewer 3 Report

Comments and Suggestions for Authors

Studying symbiotic relationships is challenging. And the authors of the presented manuscript chose a complex of symbiotic relationships as the object of study: on the one hand, there are mutualistic mutually beneficial relationships between the fungus Amylostereum areolatum and Sirex noctilio, and on the other hand, a parasitic symbiosis between the fungus A. areolatum and Pinus sylvestris; active reproduction of the fungus can lead to the death of the pine tree. At the first stage, the authors began the study with the symbiosis of A. areolatum and Sirex noctilio. Although Mongolian pine is a unique species of pine and saving it from the parasite A.areolatum is also an important task. The use of pine wood powder allowed the authors to identify candidate reference genes for further study of the regulation of symbiotic relationship genes.

The manuscript is written accurately and carefully, there are some minor comments.

35 - space before the bracket and further in the text.

42- indicate the age of woodwasps larvae (days)

73- decipher PDA culture conditions

99- indicate the characteristics of the A. areolatum mycelium used (age) for RNA isolation

312- amplification occurs at 60°C, and at 95° Taq activation occurs.

341- decrypt Vn/Vn+1

4.5.2. - remove Laccase description