Tissue Structure Changes of Aquilaria sinensis Xylem after Fungus Induction

Round 1

Reviewer 1 Report

• suggestion : need to include the impact of this research to other similar research and how the result will be beneficial for agarwood growers.
• the introduction should be more concise so that readers can follow the line
• methods need to be explained in more detail
• please include comparison of results obtained with previously other studies in the discussion
• figures need to be informative and improved

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 1 Comments

 

 

 

Dear Reviewers,

Thank you very much for your time involved in reviewing the manuscript and your very encouraging comments on the merits. We also appreciate your clear and detailed feedback and hope that the explanation has fully addressed all of your concerns.

In the remainder of this letter, we discussed each of your comments individually along with our corresponding responses. The parts that were modified in the manuscript were also organized below. For ease of reading, we used highlight words to mark the changes.

We have modified all of your suggestions in the manuscript using the Track Changes. Some of the figures may be misaligned in Track Changes, so a PDF version of the hidden Track Changes was submitted at the same time. The number of Line in the response indicates the number of line in Track Changes.

The ANOVA data is organized in the file The Data of ANOVA. Since there are more new pictures and the picture names are different from the previous manuscript, we have organized all the pictures in folder Figure.

 

Point 1: need to include the impact of this research to other similar research and how the result will be beneficial for agarwood growers.

 

Response 1: Thanks for your great suggestion on improving the accessibility of our manuscript. The impact of this experiment for subsequent studies have been included in the discussion section as Line 628, and guidance for those growing Aquilaria sinensis for agarwood production have been included in the discussion section as Line 652 and 671. The relevant contents are provided below as a screen dump for your quick reference.

Point 2: the introduction should be more concise so that readers can follow the line

 

Response 2: Thank you for your suggestions on the readability of the introduction. We have revised the introduction to make the line smoother.

 

Point 3: methods need to be explained in more detail

 

Response 3: Thank you for your suggestions regarding the integrity of the methods. We have added more details and schematics in the manuscript as Line 112.

 

Point 4: please include comparison of results obtained with previously other studies in the discussion

 

Response 4: Thank you for your valuable suggestions regarding the discussion. We have added a comparison with the results of the previous study in the discussion, mainly including a comparison with physical and chemical induction method.

 

Point 5: figures need to be informative and improved

 

Response 5: Thanks for your suggestions on the readability of the images, we have added labels to the images to make them more informative and easier to understand.

 

Point 6: Line 20 “Material transformation was concentrated in the interxylary phloem, providing a structural and material basis for the formation of agarwood.”

Material: specific of general?

 

Response 6: Thank you for the detailed review and we regret for the inconvenience caused by not explaining clearly in the manuscript. In this experiment, Material specifically refers to nutrients such as starch grains and soluble sugars, and secondary metabolites such as sesquiterpenes.

 

Point 7: Line 24 “Compared with the A. sinensis inoculated for 12 months, the xylem of the A. sinensis inoculated for 18 months was more vigorous.”

 

Response 7: Thank you for the detailed review, and we regret for the inconvenience caused by not explaining clearly in the manuscript. In this experiment, more vigorous expressed by the number of nuclei.

In the early stages of inoculation, the fungus decomposes the xylem of the agarwood to obtain the carbon source needed for its own reproduction, while activating the defense reaction of the A. sinensis and weakening the physiological growth, leading to the production of secondary metabolites such as 2-(2-phenylethyl) chromone and sesquiterpene. The secondary metabolites block the vessels and xylem rays, thus cutting off the nutritional supply of the fungus and acting as a barrier to protect the tree, while on the other hand, the chromone acts as an antifungal agent and protects the A. sinensis from damage. The effective reduction of fungal abundance by secondary metabolites leads to a weaker defense response in A. sinensis[1], while the active physiological growth of the trees leads to a longer inoculation time and a decrease in the content of secondary metabolites[2]. In the experiment it was expressed as an increase in the content of nuclei.

 

Point 8: Line 34 “Agarwood is a precious natural species which has medicinal properties for treating chest pains and asthma. ”

species: component?

 

Response 8: Thank you for your suggestions, we have changed species to component based on your suggestion. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 9: Line 57 “There are drawbacks with both methods, such as complicated operation processes, chemical residues and unstable quality.”

complicated: less compicated?

 

Response 9: Thank you for the detailed review, and we regret for the inconvenience caused by unclearly express in the manuscript. Ordinary chemical methods and whole-tree agarwood-inducing techniques both have the drawback of complicated operation processes. The composition of inoculants is complex and difficult to configure, and the inoculation time is long, usually taking 3 to 5 days, while fungal inoculation takes only 24 hours. furthermore, chemical methods can easily cause xylem decay or even tree death, and it may contaminate the environment with heavy metals.

 

Point 10: Line 69 “In recent years, many studies have been conducted on the structural and functional changes in the xylem of A. sinensis during fungal infection-induced aloe forming.”

aloe: in what form?

 

Response 10: Thank you for the detailed review, and we are very sorry for using the wrong words. We have replaced the correct vocabulary, changing aloe to agarwood, and added to the missing information. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 11: Line 78 “Although the interxylary phloem, xylem rays, and axial parenchyma are all parenchyma cells that can store and transport and capable of photosynthesis, respiration and metabolism, they have different physiological functions.”

photosynthesis: wood?

 

Response 11: Thank you for the detailed review, and we regret for the error in the manuscript. We have removed the wrong words. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 12: Line 85 “In the previous study we found that, the agarwood induced were extracted by solvents and the contents of alcohol-soluble extract and essential oil were analyzed.”

missing word?

 

Response 12: Thank you for the detailed review, and we regret for the error in the manuscript. The words we did miss have been added to the manuscript. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 13: Line 94 “Ultra-sonic extraction was used to extract agarwood, and its characteristic substances, such as agarotetraol, 2-(2-phenylethyl) chromone and 6,7-dimethoxy-2-(2-phenylethyl) chromone, were quantitatively analyzed by high-performance liquid chromatography (HPLC) to understand the specific content of characteristic substances in agarwood more intuitively, and ultraviolet-visible spectrophotometry (UV–vis) was used to analyze the total chromone content.”

total chromone content: why not in HPLC

 

Response 13: Thank you for your valuable suggestions. HPLC was used for the quantification of the chromone monomer, while the simpler UV–vis method was used for the measurement of the total chromone content. We will refer to your suggestion to use HPLC in subsequent experiments to determine total chromone content.

 

Point 14: Line 98 “And the clearance rates of DPPH and ABTS+ radicals, total reducing power, anti-acetylcholinesterase, and anti-α-glucosidase activity were determined, we found that the artificial agarwood had the same biological activity with the wild agarwood”

why suddenly mention this?

 

Response 14: Thank you for the detailed review and we regret for the inconvenience caused by inadequate description in the manuscript. The medicinal value of agarwood relies mainly on the biological activity. We determined clearance rates of DPPH and ABTS+ radicals, total reducing power, anti-acetylcholinesterase, and anti-α-glucosidase activity to demonstrate that fungus-induced agarwood has the similar medicinal value as natural agarwood, to demonstrate the value of our research in the production of agarwood.

 

Point 15: Line 108 “In this study, the microstructural characteristics of A. sinensis at 12 and 18 months of inoculation were observed to investigate the differences and changes in various types of parenchyma cells, as well as the diffusion trends of mycelium in the xylem.”

diffusion trends of mycelium: difusion or movement?

 

Response 15: Thank you for the detailed review, and we regret for incorrect use of the term. We have changed difusion to movement according to your suggestion. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 16: Line 121 “Subsequently, the materials were disinfected with sterile dry blot moisture on filter paper. ”

disinfected materials were dried?

 

Response 16: Thank you for the detailed review, and we regret for the inconvenience caused by inadequate description in the manuscript. We have changed the presentation in the manuscript. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 17: Line 124 “For the enrichment of fungi, the dried materials were cultured at 30 ℃ for 7 d in a shake flask that was placed in a sterile PDA liquid medium.”

in sterile PDA, incubated at 30℃?

 

Response 17: Thank you for the detailed review, and we regret for the inconvenience caused by not explaining clearly in the manuscript. The source of A. sinensis is concentrated in southern China, where the average annual temperature is around 27°C. In production practice we found that the fungus used in the inducers multiplies faster at 30°C than at 27°C. Therefore, we chose 30°C as the incubation temperature in our experiments.

 

Point 18: Line 126 “After 7 d, the enriched fungal liquid and PDA liquid medium were cultured at a ratio of 1:100 (V: V) and 30 ℃ for 7 d to obtain the fungal inducer.” 


1:100 enriched to PDA?

 

Response 18: Thank you for the detailed review, and we regret for the inconvenience caused by not explaining clearly in the manuscript. We performed twice culture of the fungus, while the first culture was to enrich the fungus in the sample into the culture solution, and the second culture was to expand the fungus in order to obtain the fungal inducer. In the the second culture, the fungal culture was diluted at a ratio of 1:100 for the second culture, which was to increase the yield of the fungal inducer.

 

Point 19: Line 133 “Healthy 10-year-old A. sinensis trees, with a uniform diameter (15.6±0.8cm) at breast height were induced with fungus, and holes were drilled in the trunk surface in a spiral arrangement.” 


drilled followed by fungus inoculation?

 

Response 19: Thank you for the detailed review, and we regret for the inconvenience caused by inadequate description in the manuscript. We drilled holes in the trunk surface and then injected fungal inducer into the holes. We have added a diagram of the borehole to the manuscript, as shown in Fig.2, to hopefully improve readability.

 

Point 20: Line 141 “After felling the trees, 3-cm thick wood slices were collected at 20 cm above the roots, the diameter at breast height, and the branching point, respectively.”

felling?

 

Response 20: Thank you for the detailed review, and we regret for the inconvenience caused by our wording. Felling means cutting down the trees, since we were sampled the trees at the branching points, we cut down the trees to obtain higher samples. This is for easier sampling.

 

Point 21: Line 161 “The sections preserved by FAA were stained acetocarmine to detect nuclei.” 


by

 

Response 21: Thank you for the detailed review, and we regret for tmissing a preposition. We have added the missing words as you suggested. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 22: Line 174 “The number of cell nuclei and starch grains in an arbitrarily selected square with 100 μm sides were determined from micrographs at 40× magnification, and the morphology and the distribution locations were recorded.”

 

Response 22: Thank you for the detailed review, and we regret for incorrect use of the term. The distribution locations here refers to the location of the nuclei and starch grains in the xylem. At the tissue level, it refers to the distribution in the xylem rays, interxylary phloem or axial parenchyma; at the cellular level, it refers to the center of the cell or close to the cell wall.

 

Point 23: Line 175 “The soluble sugars and the sesquiterpenoids were counted by relative area, and the areas of dark secondary metabolites were determined.”

of dark secondary metabolites: what dose it means?

 

Response 23: Thank you for the detailed review, and we regret for the inconvenience caused by inadequate description in the manuscript. Of dark secondary metabolites here means agarwood resin, which contains secondary metabolites such as sesquiterpenes and chromones. It is darker and can be observed with the naked eye under an optical microscope. Therefore, we used the content of incense resin as the content of secondary metabolites.

 

Point 24: Line 188 “The sapwood of A. sinensis was white and without an aromatic smell.”

 

Response 24: Thank you for the detailed review, and we regret for the inconvenience caused by inadequate description in the manuscript. Aromatic smell here means the characteristic odor of agarwood, which is not present in the white zone, while the xylem that begins to produce agarwood can be smelled distinctly.

 

Point 25: Line 215 “On the transverse surface, there were banded interxylary phloem and disconnected vessels, both of which were cavity-shaped.”

 

Response 25: Thank you for the detailed review, and we regret for the inconvenience caused by our wording. We have changed which to them according to your suggestion. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 26: Line 220 “At the breast diameter and the ground diameter, the agarwood zone moved outward, and the area of the decay zone increases.”

 

Response 26: Thank you for the detailed review, and we regret for the inconvenience caused by our wording. We have changed moved to expand according to your suggestion. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 27: Line 221 “It can be seen that the agarwood was first produced in the middle of the tree, combined with the wood of the xylem to form the agarwood zone, and spread outward as the agarwood formation.”

 

Response 27: Thank you for the detailed review, and we regret for the inconvenience caused by our wording. We have changed the middle of the tree to the pith of the tree according to your suggestion. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 28: Line 226 “…and the agarwood zone was narrower with a distinct transitional zone and a wider white zone.”

 

Response 28: Thank you for the detailed review, and we regret for the inconvenience caused by our wording. narrower here means the agarwood zone of A. sinensis inoculated for 12 months was narrower than which in A. sinensis inoculated for 18 months. After ANOVA, we found that this difference was not significant, so we concluded that there was no significant difference in the amount of agarwood production between the 12 month and 18 month inoculated A. sinensis. We have revised the presentation in the manuscript. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 29: Line 260 “No material was stained in the decay zone”

 

Response 29: Thank you for the detailed review, and we regret for the inconvenience caused by our wording. We have modified the statement according to your suggestion. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 30: Line 317 “…and both had the same distribution pattern in different zones.”

same： similar?

 

Response 30: Thank you for the detailed review, and we regret for the inconvenience caused by our wording. We have changed same to similar according to your suggestion. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 31: Line 330 “The soluble sugars in the white zone was mainly distributed in the axial parenchyma and the interxylary phloem, whereas there was less distribution in the xylem rays.”

 

Response 31: Thank you for the detailed review, and we regret for the inconvenience caused by our wording. We have modified the statement according to your suggestion. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 32: Line 344 “The colored was approximately 48.85% of the total area.”

 

Response 32: Thank you for the detailed review, and we regret for the inconvenience caused by our wording. We have added area in the manuscript according to your suggestion. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 33: Line 353 “This process accelerated when there were reduced amounts of unmetabolized starch grains in the parenchyma cells, which could not be metabolized to soluble sugars or transported and had to be temporarily retained in the resin-filled parenchyma cells.”

process accelerated: is there any time series observation

 

Response 33: Thanks for your great suggestion. We have experiments in subsequent studies that follow a time series of observations, which sampling at different times after inoculation with the fungus, to observe structural changes in the xylem. The breakdown of starch grains to soluble sugars increases the osmotic pressure of the cells, which increases the resistance of the cells to stress and thus slows down the formation of agarwood.

 

Point 34: Line 357 “These starch grains gradually decomposed into nutrients or fungi.”

starch decomposed into fungi?

 

Response 34: Thank you for the detailed review, and we regret for the inconvenience caused by our wording. We have changed or to for in the manuscript. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 35: Line 390 “The xylem rays showed the least color with 83.14%, and the total color area of parenchyma cells w 84.61%.”

 

Response 35: Thank you for the detailed review, and we regret for the inconvenience caused by our wording. We have changed w to with in the manuscript. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 36: Line 434 “(e, f) Xylem rays of transition zone, Fig. F is a partial enlargement of Fig. E, and the arrow shows the invaginations.”

the arrow shows the invaginations: which one?

 

Response 36: Thank you for the detailed review, and we regret for missing an arrow. We have added the missing arrows to the diagram. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 37: Line 465 “(b)Type of mycelial dispersal.”

is it unique?

 

Response 37: Thank you for the detailed review. In the experiment we found that the mycelium expanded radially, and all the mycelium we observed spread in this pattern. Radioactive diffusion is a more common form of mycelial diffusion and is not unique[3, 4].

 

Point 38: Line 467 “(g) Transverse surface of decay zone, mycelium distributed in xylem rays and axial parenchyma, with the decayed interxylary phloem at the arrow.”

 

Response 38: Thank you for the detailed review, and we regret for missing an arrow. We have added the missing arrows to the diagram. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 39: Line 468 “(h) Fungal hypha in vessel of radial surface which in decay zone.”

 

Response 39: Thank you for the detailed review, and we regret for the inconvenience caused by inadequate description in the manuscript. The figure shows the fungal hyphae in the vessel of the decayed zone, which can be observed clustered in and around the vessel in the radial surface.

 

Point 40: Line 469 “(i) Fungal hypha in xylem rays of radial surface which in transition zone.”

give arrow to indicate hypha

 

Response 40: Thank you for the detailed review, and we regret for missing an arrow. We have added the missing arrows to the diagram. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 41: Line 470 “(j, k) Transverse surface of agarwood zone, inclusions and mycelium in xylem rays, with mycelium at arrows.”

which one?

 

Response 41: Thank you for the detailed review, and we regret for missing an arrow. We have added the missing arrows to the diagram. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 42: Line 472 “(o) Branching hyphae infect adjacent cells through the grains.”

 

Response 42: Thank you for the detailed review, and we regret for the inconvenience caused by our wording. We have changed the grains to the pit in the manuscript.The relevant contents are provided below as a screen dump for your quick reference.

 

Point 43: Line 475 “The degree of fungal enrichment varied widely among zones and there was a decreasing trend from the inside to the outside.”

what enrichment means?

 

Response 43: Thank you for the detailed review, and we regret for the inconvenience caused by inadequate description in the manuscript. Enrichment here means the accumulation of fungus hypha. We have changed enrichment to accumulation in the manuscript.The relevant contents are provided below as a screen dump for your quick reference.

 

Point 44: Line 476 “This phenomenon may be attribute to two factors.”

 

Response 44: Thank you for the detailed review, and we regret for the inconvenience caused by our wording. We have changed attribute to attributed in the manuscript.The relevant contents are provided below as a screen dump for your quick reference.

 

Point 45: Line 445 “Nodulation time had a significant effect (p<0.01) on the number of nuclei, starch grains and sesquiterpene color area.”

Point 46: Line 459 “The median soluble sugar coloring area of 35.3350 was relatively high in the interxylary phloem, followed by 30.9150 in the axial parenchyma, and 16.5450 in the wood rays.”

Point 47: Line 473 “The 18 months of inoculation of A. sinensis had a faster cell death rate after fungal induction.”

Point 48: Line 475 “The correlation among the number of starch grains, area of soluble sugars, and sesquiterpenes with the time of inoculation was not significant. The LSD test for the correlation between the number of starch grains, area of soluble sugars and area of sesquiterpenes and time of inoculation showed that there was a highly significant difference between 12 and 18 months of inoculation in the three dependent variables.”

Point 49: Line 482 “Moreover, the rate of sugar conversion and production of characteristic products was faster in 18 months of inoculation than in 12 months of inoculation.”

Response 45~49: Thank you for your constructive suggestions, and we have rewritten 3.7. Correlation test between parameters based on your suggestions asLine 482. We hope you will find this revised version satisfactory.

 

Point 50: Line 579 “The white wood layer had not started to bear agarwood.”

bear: produce or accumulate?

 

Response 50: Thank you for the detailed review, and we regret for the inconvenience caused by our wording. We have changed bear to produce in the manuscript.The relevant contents are provided below as a screen dump for your quick reference.

 

Point 51: Line 586 “As more agarwood resin formed, it appeared in the vessels and wood fibers adjacent to the parenchyma cells.”

it: represent what?

 

Response 51: Thank you for the detailed review, and we regret for the inconvenience caused by inadequate description in the manuscript.it means agarwood resin here. We have modified the statement in the manuscript. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 52: Line 594 “The content and types of secondary metabolites such as sesquiterpenes increased gradually with the expansion of the agarwood.”

types of secondary metabolites: metabolite analysis was also conduted?

 

Response 52: Thank you for your constructive suggestions. We investigated the species of secondary metabolites in subsequent experiments, which do not appear in this manuscript. We have therefore removed the relevant descriptions.

 

Point 53: Line 596 “This physiological metabolic of converting stored starch into soluble sugar that can participate in physiological reactions occurs mainly in parenchyma cells, and this result is the same as that of Wang Jun.”

 

Response 53: Thank you for your constructive suggestions. We have rewritten this part on your advice, and we hope you will find this revised version satisfactory. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 54: Line 681 “Compared with the 12 months of inoculation in A. sinensis, the xylem of the 18 months of inoculation was more vigorous.”

 

Response 54: Thank you for the detailed review, and we regret for the inconvenience caused by not explaining clearly in the manuscript. In this experiment, more vigorous expressed by the number of nuclei.

In the early stages of inoculation, the fungus decomposes the xylem of the agarwood to obtain the carbon source needed for its own reproduction, while activating the defense reaction of the A. sinensis and weakening the physiological growth, leading to the production of secondary metabolites such as 2-(2-phenylethyl) chromone and sesquiterpene. The secondary metabolites block the vessels and xylem rays, thus cutting off the nutritional supply of the fungus and acting as a barrier to protect the tree, while on the other hand, the chromone acts as an antifungal agent and protects the A. sinensis from damage. The effective reduction of fungal abundance by secondary metabolites leads to a weaker defense response in A. sinensis[1], while the active physiological growth of the trees leads to a longer inoculation time and a decrease in the content of secondary metabolites[2]. In the experiment it was expressed as an increase in the content of nuclei.

 

We would like to take this opportunity to thank you for all your time involved and this great opportunity for us to improve the manuscript. We hope you will find this revised version satisfactory.

 

Sincerely,

 

The Authors

 

references

 

1. Mohamed R. Agarwood: Science Behind the Fragrance. 2016,
2. Sheng M, Yunlin F, Yingjian L, et al. The formation and quality evaluation of agarwood induced by the fungi in Aquilaria sinensis. Industrial Crops &amp; Products 2021, 173,
3. Kang Z. Ultrastructure of Plant Pathogenic Fungi, China Science & Technology Press: Beijing, 1995;
4. Zhang M. Apreliminary study on the of lsolation and identification of endophytic bacteria in white fragrant and distribution. M.D., Guangzhou University of Chinese Medicine, 2017.

 

Author Response File: Author Response.docx

Reviewer 2 Report

 

Title: Tissue Structure Change of Aquilaria sinensis Xylem during fungus induction

 

Dear Author,

General Comments and questions:

Introduction

Due to the scarcity of naturally occurring agarwood,  artificial techniques have been effective for increasing the production of agarwood and meeting market demand.

 

Your article is very interesting.  The readers need some information about A. sinensis more comprehensively. Please re-write again the state of the art of Aquilaria sinensis. Please update the population of natural A. sinensis and the plantation of A. sinensis in your country.  How many natural agarwood productions per year? How many artificial agarwood productions per year? How about their grade between natural and artificial agarwood?  How many percent they can replace natural agarwood? What kind of end product from artificial agarwood? How sustain the artificial agarwood to make mass production? Is there an agarwood plantation on a large scale? 

Specific Comments and questions:
      Please make one paragraph state of the arts according to my comment and question below:

I am curious about so many publications on agarwood production in your country.  

 

How much influence the artificial agarwood has in replacing the existence of natural agarwood? Has it really been mass-produced or is it still on a research scale? How about the success rate (%) of inoculation by fungi compared with chemical induction?  Is it true that the market has accepted artificial agarwood?

 

Could you explain prospect agarwood production by artificial techniques (chemical or biological induction)? How much agarwood artificial agarwood production is in your country per year? Could artificial agarwood replace natural agarwood?

 

Specific comment: why is your focus on Aquilaria sinensis? Other agarwood species, how update Aquilaria yunnanensis?

 

 

Method:

Comments and questions:

Line 100-112.  What kind of fungal species did you find it?  How many fungal species did you find it?

                           Are they fungal pathogens? Are they endophytic fungi?

                          How to identify fungal species? by molecular identification? by morphology?

                       Did you use single fungal species for this experiment? How did you select to effective fungal species in this experiment?

 

 

Line 113-128 :

1. Could you tell us the histories of A. sinensis in this area? How large the plantation and line spacing? Is it monoculture or mix-plantation? Ecological site plantation: altitude, type of soil, type of forests, a diversity of local shrub species near the trees, climate, etc. When A. sinensis flowering and fruiting in this area? Is there any problem with the pest and disease of Aquilaria sinensis?
2. Please show a map or location? Any treatment of A. sinensis plantation, i.e. intensive fertilizer, pesticides, herbicides, etc.
3. Treatment: 1 control;   2. Fungal induction (one species or more than one species?)

How many trees did you use in this experiment? Could you make the layout of the experiment? How select the trees for fungal induction and control experiments?

4. How many holes did you prepare for one tree? How many dosages (mL) of fungal induction per hole or per tree? Did you find some trees that die cause of your treatment?
5. How to take samples, which part of stem, how many replications?
6. Please make an illustration (a figure), how to collect some slices of the stem of standing tree.
7. Line 120: did you make some holes in the control blank groups. How many holes each did you prepare per one tree for control treatment?
8. Why did you use 0.1% HgCl2? other sterilant like H2O2, NaOCl (Clorox), etc.
9. How to separate resinous wood from white wood? Why did you decide to observe 12 months and 18 months? According to your experience, what time the best time for harvest artificial agarwood for the best grade?

 

 

Results :

Could you provide and show xylem structure treated by drill only (the control blank group)?

Could you provide and show xylem structure treated by chemical induction?

If possible, there are comparison xylem structures among the control blank group, chemical induction, and fungal induction.

Discussion:

-could you discuss and explain the difference between structure changes of Xylem by fungal pathogen and endophytic fungi? did you find difference between them

What kind the difference is the role of endophytic fungi residence or fungal pathogen to develop agarwood formation in the xylem.

-What is the best treatment for agarwood formation for human health, by chemical induction or by fungal induction?

Discussion should provide what kind of fungal group for agarwood formation consistency?  was it fungal pathogen or endophytic fungi? You should state your fungal group induction.

 

Conclusion:

You should state the name of the fungal induction. What the name of fungal induction is very important in your research.  So much research will do the same thing, but the difference is fungal induction utilization.

 

 

 

Author Response

Response to Reviewer 2 Comments

 

 

 

Dear Reviewers,

Thank you very much for your time involved in reviewing the manuscript and your very encouraging comments on the merits. We also appreciate your clear and detailed feedback and hope that the explanation has fully addressed all of your concerns.

In the remainder of this letter, we discussed each of your comments individually along with our corresponding responses. The parts that were modified in the manuscript were also organized below.

We have modified all of your suggestions in the manuscript using the Track Changes. Some of the figures may be misaligned in Track Changes, so a PDF version of the hidden Track Changes was submitted at the same time. The number of Line in the response indicates the number of line in Track Changes.

The ANOVA data is organized in the file The Data of ANOVA. Since there are more new pictures and the picture names are different from the previous manuscript, we have organized all the pictures in folder Figure.

 

Point 1: Due to the scarcity of naturally occurring agarwood,  artificial techniques have been effective for increasing the production of agarwood and meeting market demand.

The readers need some information about A. sinensis more comprehensively. Please re-write again the state of the art of Aquilaria sinensis. Please update the population of natural A. sinensis and the plantation of A. sinensis in your country.  How many natural agarwood productions per year? How many artificial agarwood productions per year? How about their grade between natural and artificial agarwood?  How many percent they can replace natural agarwood? What kind of end product from artificial agarwood? How sustain the artificial agarwood to make mass production? Is there an agarwood plantation on a large scale?

 

Response 1:

Thank you for your interest in Aquilaria sinensis cultivation industry, we have added the yield of agarwood and the prospects for the development of artificial agarwood to the manuscript as Line 44. Due to the limited space of the manuscript, more detailed information is as follows.

The original forest of A.sinensis has been severely damaged, coupled with the weak renewal capacity of natural forests, only Hainan, Guangdong, Guangxi and Yunnan still have a small number of plants, estimated to be only 130,000 (information from the survey conducted by the Guangzhou office of the National Endangered Office), and there are not many artificially planted A.sinensis in China, about more than 133km² (Xu Daping, 2016 First National Summit Forum on the Development of the Sinkiang Industry). The current annual demand for agarwood in China is 500 tons and increasing year by year, and the production of agarwood in 2020 is about 118 tons (China National Bureau of Statistics statistics), about 76.4% rely on imports, with the main import region being Vietnam. Due to the scarcity of naturally occurring agarwood, artificial techniques have been effective for increasing the production of agarwood and meeting market demand.

The differences between artificial agarwood and natural agarwood are mainly in the types and relative contents of sesquiterpenoids and 2- (2-phenylethyl) chromones. Short-term formation of artificial agarwood and natural agarwood formed over the years is still a certain gap, but has been able to meet the national standards, and enough to apply in production. Because natural agarwood is so rare and expensive, almost all of the agarwood currently on the market for medicine, daily use and spices is artificial agarwood. It can be seen that artificial agarwood has been able to replace natural agarwood in medicine, daily necessities and spices.

The current A.sinensis plantations usually use traditional agarwood techniques，such as physical and chemical agarwood methods, which generally require older trees (generally more than ten years), with labor-intensive operation and low yield, and some methods even produce chemical pollution and other problems. Therefore artificial agarwood can not form a large-scale industrialization.

At present, the more advanced artificial agarwood-inducing method is whole-tree agarwood-induction technology.This technology has not been developed for a long time, and there are still a lot of shortcomings including high costs, high mortality rate of trees, too long hanging time for injection (3-5 days), or the quality of agarwood does not meet the standard. At present, there is not yet any kind of whole-tree agarwood-induction technology recognized by the industry and can be widely promoted in the production.

 

Point 2: How much influence the artificial agarwood has in replacing the existence of natural agarwood? Has it really been mass-produced or is it still on a research scale? How about the success rate (%) of inoculation by fungi compared with chemical induction? Is it true that the market has accepted artificial agarwood?

Could you explain prospect agarwood production by artificial techniques (chemical or biological induction)? How much agarwood artificial agarwood production is in your country per year? Could artificial agarwood replace natural agarwood?

Specific comment: why is your focus on Aquilaria sinensis? Other agarwood species, how update Aquilaria yunnanensis?

 

Response 2:

Thank you for your interest in the agarwood industry and it is my pleasure to introduce you to the current status of agarwood in China. Currently, the production of artificial agarwood in China has surpassed that of natural agarwood, but there are still many difficulties in large-scale production. Compared with chemical induction, fungal induction has a higher success rate in producing agarwood, because there is almost no death of A. sinensis due to agarwood formation and the agarwood yield is higher. Currently, most of the agarwood products on the market are derived from artificial agarwood, and the market has been able to accept artificial agarwood in medicine and daily necessities.

 

We have added the comparison of Aquilaria yunnanensis and A.sinensis in cultivation techniques and production applications as Line35. Due to the limited space of the manuscript, more detailed information is available here .

There are two main species of Aquilaria trees native to China, A.yunnanensis and A.sinensis[1]. A.yunnanensis is only found in Xishuangbanna and Lincang regions of Yunnan, and A.yunnanensis has high climatic requirements for its habitat, growing better at an average annual temperature of 19-25°C,with an average July temperature of 23°C or more, and an annual rainfall of 1200-2500 mm, and is rarely grown outside Yunnan[2]. The origin of A.yunnanensis is mostly mountainous and hilly, which makes it difficult to carry out large-scale production. In contrast, A.sinensis has a more widespread origin and is distributed in Guangdong, Hainan, Yunnan, Guangxi, Fujian, and Taiwan provinces[3], and has less requirements for temperature, precipitation, and soil, making it more suitable for large-scale cultivation for production. Since almost all of the Aquilaria trees grown in Guangxi are A. sinensis, our research is focused on A. sinensis now. In the future, we will carry out the effect of fungal inducers on A.yunnanensis.

 

Point 3: Line 113.  

What kind of fungal species did you find it? 

How many fungal species did you find it?

Are they fungal pathogens? Are they endophytic fungi?

How to identify fungal species? by molecular identification? by morphology?

Did you use single fungal species for this experiment? How did you select to effective fungal species in this experiment?

 

Response 3:

Thanks for your great suggestion on improving the completeness of our manuscript. We have added the identification of fungal species to the manuscript as Line 127, as well as the names of the main dominant species.

We identified fungal species by high-throughput sequencing. At the family level, 33 families were obtained, of which 11 families were dominant. At the genus level, 37 genera were detected, and 11 genera were the dominant genera. At the species level, 51 species were obtained, and 13 species were the dominant species. Aspergillus penicillioides was the most dominant species with a relative abundance of 31 %. The remaining dominant species were mainly Gongronella butleri (11.38%), Aspergillus sydowii (8.69%), Cladosporium halotolerans (8.36%), etc[4].

The dominant species of endophytic fungi in A. sinensis are mainly Penicillium, Lasiodiplodia, Trichoderma, Aspergillus, Fusarium[5].

Some of the dominant species in the fungal inducers we used belonged to endophytic fungi, and experiments showed that unprocessed A. sinensis also had the presence of these fungi[5]. A large number of forestry practices have shown that unprocessed A. sinensis cannot produce agarwood, but inoculation with endophytic fungi enables to produce agarwood. That means, the mere presence of the original endophytic fungi in the tree without inoculation does not make A. sinensis start to produce agarwood. In our previous study, we found that only drilling but not injecting the fungal also did not make A. sinensis start to produce agarwood (as shown in Fig.1). So we believe that in this experiment, the synergistic effect of fungi and drilling is the key to produce agarwood.

Fig.1 a: Drilling only samples without injection of fungal inducer, no agarwood formation; b: Drilled and injected with fungal inducer, with agarwood formation.

Regarding the selecting to effective fungal species, we first injected a mixed fungal solution into healthy A. sinensis, and later studied the isolation of various fungi for single fungal species inoculation or multiple fungal species inoculation as a way to screen out suitable fungal species.

 

Point 4: Line 131 :

 

Could you tell us the histories of A. sinensis in this area? How large the plantation and line spacing? Is it monoculture or mix-plantation? Ecological site plantation: altitude, type of soil, type of forests, a diversity of local shrub species near the trees, climate, etc. When A. sinensis flowering and fruiting in this area? Is there any problem with the pest and disease of Aquilaria sinensis?

 

Response 4:

Thank you for the detailed review and we regret for the inconvenience caused by not explaining clearly in the manuscript. In the middle of the 20th century, North Sea city in Guangxi Municipality were abundant resources of wild A. sinensis. Later, the wild resources were increasingly depleted due to deforestation, and the planting of A. sinensis plantations started around 2000 (from Beihai Forestry Journal). The average altitude of Beihai is 10~15 m, and the soil type is red soil and limestone soil, while the climate is maritime monsoon climate, and the forest type is subtropical broad-leaved forest. A. sinensis in Beihai, Guangxi usually blooms from mid-March to mid-April. The planting site of this experiment is Senyuan Precious Tree Planting Base, with a total of about 1,000 A. sinensis plants, all planted on flat ground with a row spacing of about 3 m. The planting pattern is monoculture, and no other plants are planted in the forest.

Since A. sinensis is rarely grown on a large scale, no serious pests or diseases have been found, but aphids and leafroller moths often damage young leaves and tips. It has been shown that after feeding by phytophagous insects, A. sinensis releases significantly more herbivore induced plant volatiles than healthy plants to tend to avoid pests and attract natural enemies[6]. Therefore, A. sinensis is rarely infested with insects.

 

Point 5:Please show a map or location? Any treatment of A. sinensis plantation, i.e. intensive fertilizer, pesticides, herbicides, etc.

 

Response 5: Thank you for the detailed review. The map of Senyuan Precious Tree Planting Base, North Sea city in Guangxi Municipality is shown as Fig.2.

Fertilizer was applied twice a year, from February to March before spring sprouting, to promote tending and sprouting, which can accelerate growth. In September to October, we applied organic fertilizer and buried the weeds, which should be applied in furrows. As the age of the tree increases, the amount of fertilizer should be increased accordingly. Weed twice a year, spread the weeds around the roots of the trees and turn them into the soil year by year to increase the soil organic matter. No herbicides or pesticides were used in the experiment.

Fig.2 a: The map of Senyuan Precious Tree Planting Base, North Sea city in Guangxi Municipality.

 

Point 6: Treatment: 1 control;   2. Fungal induction (one species or more than one species?)

How many trees did you use in this experiment? Could you make the layout of the experiment? How select the trees for fungal induction and control experiments?

 

Response 6:

Thank you for the detailed review and we regret for the inconvenience caused by not explaining clearly in the manuscript. The number of samples and controls have been added to the manuscript, as Line 140.

A total of nine trees were sampled for the experiment, including three samples collected at 12 months, three samples collected at 18 months and three blank controls collected at 18 months.

The experimental samples and blank control samples were planted in the same forest to ensure the same temperature, hydrology, soil and other conditions. These A. sinensis were free of pests and diseases before treatment, and the trees were of the same age. Samples and blank control with almost the same tree height and diameter at breast height were selected for sampling.

 

Point 7: How many holes did you prepare for one tree? How many dosages (mL) of fungal induction per hole or per tree? Did you find some trees that die cause of your treatment?

 

Response 7:

Thank you for the detailed review, and we regret for the inconvenience caused by unclearly express in the manuscript. Schematic diagrams of the fungal elicitor injection method have been added to the manuscript as Line 147.

The fungal inducers was injected by drilling holes in a spiral pattern on the body of A. sinensis with a spacing of 8-12 cm between the left and right holes and a spacing of 60 cm between the upper and lower hole layers. Since the diameter at breast height of the tree was about 15 cm, the angle between the two injection holes was 90°, and since the tree was about 5 m high, each tree needed to drill 8 injection holes, and the input volume of each injection hole was 150 ml.

In the experiment, we did not find any death of A. sinensis due to injection of fungal inducers. The fungal inducers we used were based on nutrients such as glucose as substrate, and the fungi were mostly endophytes of A. sinensis, which were less harmful to the tree compared with chemical inducers.

 

Point 8: How to take samples, which part of stem, how many replications?

Please make an illustration (a figure), how to collect some slices of the stem of standing tree.

 

Response 8:

Thank you for the detailed review, and we regret for the inconvenience caused by unclearly express in the manuscript. The schematic diagram of the sample collection have been added to the manuscript as Line 147.

Each tree was sampled at three locations: ground diameter, diameter at breast height and crown branch point, and three sets of replicates were made for each sampling.

The sampling site photos are as Fig.2.

Fig.2 Photos of slices collection

 

 

Point 9: Line 139: did you make some holes in the control blank groups. How many holes each did you prepare per one tree for control treatment?

 

Response 9:

Thank you for the detailed review. In our preliminary experiments we found that drilling alone did not produce agarwood from A. sinensis, as shown in Fig 1 (6 months after drilling, there was still no agarwood formation), so we concluded that artificially injected fungal inducers were the key to agarwood formation and that drilling had minimal effect on agarwood formation. Therefore, we set up a blank control using A. sinensis without treatment at all instead of drilled it.

 

Point 10: Why did you use 0.1% HgCl₂? other sterilant like H₂O₂, NaOCl (Clorox), etc.

 

Response 10: Thank you for the detailed review. The role of 0.1% HgCl₂ in the experiment is to fully kill the fungus and bacteria on the surface of the sample. In the production practice people found that the effect of 0.1% HgCl₂ for A. sinensis samples due to other sterilant.

 

Point 11:How to separate resinous wood from white wood?

 

Response 11:

Thank you for the detailed review, and we regret for the inconvenience caused by unclearly express in the manuscript. The resinous wood has very distinctive characteristics, with the distinctly different color and luster from the white wood. We distinguished the different woods by their color and subsequently used a scalpel to peel off the sinker layer. The wood of A. sinensis are soft and can be split manually.

 

Point 12: Why did you decide to observe 12 months and 18 months? According to your experience, what time the best time for harvest artificial agarwood for the best grade

 

Response 12:

Thank you for your valuable suggestions. In production practice, it was found that A. sinensis artificially inducted by fungal inducers had the best agarwood formation effect at 12 months~18 months after inoculated. In our previous study, we found that the alcohol soluble extract of the samples inoculated at 12 months was higher and the total relative content of sesquiterpenes and chromones was the same as at 18 months of treatment. It means that, the quality of agarwood was higher at 12 months of inoculated compared to 18 months [5]. In terms of time cost, harvesting A. sinensis at 12 months of inoculation could yield higher economic value and higher production efficiency. Therefore, we believe that harvesting at 12 months of inoculation is better.

 

Point 13: Line 131 :

Could you provide and show xylem structure treated by drill only (the control blank group)?

 

Response 13:

Thank you for yours valuable suggestions and we regret for the inconvenience caused by inadequate description in the manuscript. The xylem structure of the blank control has been added in the manuscript.

 

Point 14:Could you provide and show xylem structure treated by chemical induction?

If possible, there are comparison xylem structures among the control blank group, chemical induction, and fungal induction.

 

Response 14:

Thank you for yours valuable suggestions and we regret for the inconvenience caused by inadequate description in the manuscript. We cited previous research results on chemically induction to compare with the structure of our experiment, and the comparative results of the blank control, chemically induced and fungus induced were added to the manuscript as Line 634.

Thank you very much for your constructive suggestions, and we will extend our induction method in the subsequent experiments by conducting a comparison of fungal induction with physical induction and chemical induction to compare the differences in xylem structure of the samples treated with different induction method.

 

Point 15: Discussion:

 

-could you discuss and explain the difference between structure changes of Xylem by fungal pathogen and endophytic fungi? did you find difference between them.

What kind the difference is the role of endophytic fungi residence or fungal pathogen to develop agarwood formation in the xylem.

 

Response 15: Thank you for your constructive suggestions. Some of dominant species in the fungal inducers we used belonged to endophytic fungi, and experiments showed that unprocessed A. sinensis also had the presence of these fungi[5]. At present we have determined that these fungi act synergistically to produce agarwood. We will follow up with experiments to investigate the role played by specific fungal species in agarwood formation.

 

Point 16: What is the best treatment for agarwood formation for human health, by chemical induction or by fungal induction?

 

Response 16:

Thank you for your interest in the agarwood industry. From the perspective of protecting the health of users, we believe that the fungal induction treatment is safer.

The chemical induction method stimulates A. sinensis to produce agarwood by injecting chemicals such as formic acid, or phytohormones such as indole butyric acid, vinblastine and gibberellin. It is unclear whether the chemicals in this method have residues and are harmful to humans.

The fungus draws nutrients in the xylem to multiply continuously and relies on the tree's own channel organization to spread through the tree, continuously stimulating the tree to produce agarwood. The acidic, moist environment of the xylem is also suitable for fungal survival. The fungus-induced method relies on the interaction between the fungus and the trees, which is closer to the natural agarwood. And the fungi we use are mostly endophytes of A. sinensis, which will not destroy the original microbial community structure in the tree, and there will be no chemical residue after harvesting.

Therefore, we believe that the best treatment for artificial induced agarwood for human health is fungal induction.

 

Point 17: Discussion should provide what kind of fungal group for agarwood formation consistency?  Was it fungal pathogen or endophytic fungi? You should state your fungal group induction.

 

Response 17: Thank you for your constructive suggestions. The fungal inducers we used came from wild A. sinensis, which had a high yield of agarwood. We believe that this fungal combination has a better agarwood production effect, and the current experiments have proved that this hybrid fungal species does have high efficiency. In the following experiments, we will isolate these fungi and conduct inoculation experiments on single fungal species and mixed fungal species, expecting to research better fungal species or combinations for agarwood formation.

Some of dominant species in the fungal inducers belonged to endophytic fungi, and experiments showed that unprocessed A. sinensis also had the presence of these fungi[5]. At present we have determined that these fungi act synergistically to produce agarwood.

 

Point 18: Conclusion:

 

You should state the name of the fungal induction. What the name of fungal induction is very important in your research. So much research will do the same thing, but the difference is fungal induction utilization.

 

Response 18: Thank you for yours valuable suggestions and we regret for the inconvenience caused by inadequate description in the manuscript. The fungal species have been added to the manuscript as Line127.

 

We would like to take this opportunity to thank you for all your time involved and this great opportunity for us to improve the manuscript. We hope you will find this revised version satisfactory.

 

Sincerely,

 

The Authors

 

references

1. Wu Z, Lu A, Tang Y, et al. The Families and Genera of Angiosperms in China--A Comprehensive Analysis. China Science Publishing & Media Ltd 2003,
2. Hou Y, Shi F, Xu Y. Aquilaria yunnanensis and its cultivation technology. Yunnan Forestry 2017,
3. Li L, Shou H, Ma Q. Distribution of Aquilaria sinensis (Thymelaeaceae). Journal of Anhui Agri. Sci． 2012, 40, 9254-9256.
4. Sheng M, Yunlin F, Yingjian L, et al. The formation and quality evaluation of agarwood induced by the fungi in Aquilaria sinensis. Industrial Crops &amp; Products 2021, 173,
5. Zhang M. Apreliminary study on the of lsolation and identification of endophytic bacteria in white fragrant and distribution. M.D., Guangzhou University of Chinese Medicine

, 2017.

6. Qiao H, Lu P, Chen J, et al. Repellent and luring effects of pest-induced volatiles of Aquilaria sinensis on the yellow wild borer and its natural enemies. Abstracts of the Seventh Symposium on Plant Chemosensory Effects in China, The 7th Symposium on Plant Chemosensory Effects in China, Kunming, Yunnan, China, 2015;

 

 

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript entitled “Tissue Structure Changes of Aquilaria sinensis Xylem during Fungus Induction” presents comprehensively the changes that occur into induced fungi trees of the genus Aquilaria sinensis.

Merits

The introduction well describes the methods used for trees of the genus Aquilaria sinensis and leading to a stimulation of production agarwood.

The results present an excellent view of the changes occurring in the tree at different levels. The illustrations are a valuable addition to the paper, especially since all stages of the study were documented photographically.

Critique and suggestions

Methods

l. 119-120 “… respectively, and A. sinensis without fungi inoculation as the control blank groups, were collected after 18 months…”

How many trees were inoculated? This is not clear. Line 121 lists three trees, but it is not entirely clear whether these are inoculated or reference trees. How many trees were in each group (inoculated and control)? 

It is standard to measure all biometric features of trees on a sample plot in order to select representative trees, but in this case, it is not clear how the sample trees were selected. Why the measurements of DBH and height were taken.

Control trees were only mentioned in the methods (l. 120). What is the reason for selecting control trees if results from these trees were not presented?

I can't find the type of fungus used for inoculation. Both the species of the fungus (or group of fungi) itself and the basics of its biology should be described. It is the key factor causing the described changes, while we know nothing about the fungi.

Results

The quality of the photographs in Figure 1 is poor, probably due to light reflections and too much contrast. The other photographs present pleasant images.

The abbreviations on the charts (W1, W1.5 etc.) should be explained. The reader guesses that W is the white zone, but what does 1 and 1.5 mean?

Statistical analysis

Statistical analysis is the weakest point in the whole paper. Statistical tests are described poorly in the methods, basically only the analysis of variance was mentioned (l. 154).

The paper does not include any results of the analysis of variance. Descriptions of the assumptions of the analysis and descriptions of the individual stages are missing. Authors mentions the Kruskal-Wallis test (l. 467, Table 2), which may be a nonparametric counterpart to the parametric analysis of variance (One-Way-Anova). Also mentioned is the Tukey test (l. 472), which is a parametric test and may be a part of the analysis of variance. The analysis itself and its assumptions are not presented.

Table 1 shows the “correlation between xylem structure and xylem zone, parenchyma cells tissue types and time of inoculation”. In my point of view, the table does not show any correlations, just significance probability levels. Correlation is determined by correlation coefficients such as Pearson, Spearman, Kendal, etc. A similar situation occurs in Table 3.

In all mentioned analyses, the significance of differences between groups (the variances of each group) such as nuclei, starch, sugar, rather than correlations, was likely determined.

I suggest you correct nomenclature used, and present the assumptions and progress of the analyses performed.

Only Fig. 12 shows a correlation, but lacks descriptions of the X and Y axes. These can be absolute values (e.g., distance from the core on the X axis) or relative values (e.g., z-scores on the Y axis). It is useful to determine correlation coefficients for such relationships.

In summary, the work presents valuable and interesting content, but needs to be completed in the methodological part and thoroughly improved in the statistical part.

Author Response

Response to Reviewer 3 Comments

 

 

 

Dear Reviewers,

Thank you very much for your time involved in reviewing the manuscript and your very encouraging comments on the merits. We also appreciate your clear and detailed feedback and hope that the explanation has fully addressed all of your concerns.

In the remainder of this letter, we discussed each of your comments individually along with our corresponding responses. The parts that were modified in the manuscript were also organized below. For ease of reading, we used highlight words to mark the changes.

We have modified all of your suggestions in the manuscript using the Track Changes. Some of the figures may be misaligned in Track Changes, so a PDF version of the hidden Track Changes was submitted at the same time. The number of Line in the response indicates the number of line in Track Changes.

The ANOVA data is organized in the file The Data of ANOVA. Since there are more new pictures and the picture names are different from the previous manuscript, we have organized all the pictures in folder Figure.

 

Point 1: Methods

 

1. 138-139 “… respectively, and A. sinensis without fungi inoculation as the control blank groups, were collected after 18 months…”

 

How many trees were inoculated? This is not clear. Line 140 lists three trees, but it is not entirely clear whether these are inoculated or reference trees. How many trees were in each group (inoculated and control)?

 

Response 1:

Thank you for the detailed review, and we regret for the inconvenience caused by incomplete description in the manuscript. Sampling protocol and control group have been added to the manuscript as Line 140.

Our experimental samples were collected from the forest farm used for agarwood production by the company that provided us with the fungal inducers, where a total of about 1000 Aquilaria sinensis were inoculated with the fungus. A total of nine trees were sampled for the experiment, three samples were collected at 12 months, three samples at 18 months and three blank controls collected at 18 months。

 

Point 2: It is standard to measure all biometric features of trees on a sample plot in order to select representative trees, but in this case, it is not clear how the sample trees were selected. Why the measurements of DBH and height were taken.

 

Response 2:

Thank you for the detailed review, and we regret for the inconvenience caused by unclearly express in the manuscript.

The experimental samples and the blank control samples were planted in the same forest farm to ensure the same temperature, hydrology, and soil conditions. These A. sinensis were free of pests and diseases before inoculation, and the trees were of the same age.

The diameter at breast height and tree height were measured to ensure that the tree height and diameter at breast height of the samples and the blank control were close to each other, which could ensure homogeneity of the samples.

 

Point 3: Control trees were only mentioned in the methods (l. 120). What is the reason for selecting control trees if results from these trees were not presented?

 

Response 3:

Thank you for the detailed review, and we regret for the inconvenience caused by incomplete descriptions in the manuscript. The blank control was chosen to observe the structure of the xylem of A. sinensis, which did not produce agarwood, for comparison with the inoculated xylem. The images of the blank control have been supplemented and the related analysis is supplemented.

 

Point 4: I can't find the type of fungus used for inoculation. Both the species of the fungus (or group of fungi) itself and the basics of its biology should be described. It is the key factor causing the described changes, while we know nothing about the fungi

 

Response 4:

Thank you for the detailed review and we regret for the inconvenience caused by the missing details in the manuscript. We have added the identification of fungal species to the manuscript as Line 125, as well as the names of the main dominant species.

We identified fungal species by high-throughput sequencing. At the family level, 33 families were obtained, of which 11 families were dominant. At the genus level, 37 genera were detected, and 11 genera were the dominant genera. At the species level, 51 species were obtained, and 13 species were the dominant species. Aspergillus penicillioides was the most dominant species with a relative abundance of 31 %. The remaining dominant species were mainly Gongronella butleri (11.38%), Aspergillus sydowii (8.69%), Cladosporium halotolerans (8.36%), etc[1].

 

Point 5: Results

 

The quality of the photographs in Figure 1 is poor, probably due to light reflections and too much contrast. The other photographs present pleasant images.

 

Response 5: Thank you for the detailed review, I hope that the modified photographs can satisfy you The photographs in Figure 1 have been reprocessed and labeled for easier viewing.

 

Point 6: The abbreviations on the charts (W1, W1.5 etc.) should be explained. The reader guesses that W is the white zone, but what does 1 and 1.5 mean?

 

Response 6: Thank you for the detailed review, and we regret for the error in the manuscript. The W proxy refers to the white zone, the 1 proxy refers to the inoculation time of 1 year (12 months), and the 1.5 proxy refers to the inoculation time of 1.5 years(18 months). The abbreviations in the graphs have been replaced to ensure consistency with the descriptions in the text and have been explained.

 

Point 7: Statistical analysis

 

Statistical analysis is the weakest point in the whole paper. Statistical tests are described poorly in the methods, basically only the analysis of variance was mentioned (l. 154).

 

The paper does not include any results of the analysis of variance. Descriptions of the assumptions of the analysis and descriptions of the individual stages are missing. Authors mentions the Kruskal-Wallis test (l. 467, Table 2), which may be a nonparametric counterpart to the parametric analysis of variance (One-Way-Anova). Also mentioned is the Tukey test (l. 472), which is a parametric test and may be a part of the analysis of variance. The analysis itself and its assumptions are not presented.

 

Table 1 shows the “correlation between xylem structure and xylem zone, parenchyma cells tissue types and time of inoculation”. In my point of view, the table does not show any correlations, just significance probability levels. Correlation is determined by correlation coefficients such as Pearson, Spearman, Kendal, etc. A similar situation occurs in Table 3.

 

In all mentioned analyses, the significance of differences between groups (the variances of each group) such as nuclei, starch, sugar, rather than correlations, was likely determined.

 

I suggest you correct nomenclature used, and present the assumptions and progress of the analyses performed.

 

Response 7: Thank you for your constructive suggestions, and we have rewritten 3.7. Correlation test between parameters based on your suggestions as Line 482. We hope you will find this revised version satisfactory.

 

Point 8: Only Fig. 12 shows a correlation, but lacks descriptions of the X and Y axes. These can be absolute values (e.g., distance from the core on the X axis) or relative values (e.g., z-scores on the Y axis). It is useful to determine correlation coefficients for such relationships.

 

Response 8: Thank you for the detailed review and we regret for the inconvenience caused by the missing details in the manuscript. The description of the X and Y axes in Figure 15 has been added to the figure, and the related description is added to the discussion as Line 607.

 

Point 9: In summary, the work presents valuable and interesting content, but needs to be completed in the methodological part and thoroughly improved in the statistical part.

 

Response 9: Thank you again for your careful review. The methodological part has been refined, and the statistical part has been thoroughly improved.

 

We would like to take this opportunity to thank you for all your time involved and this great opportunity for us to improve the manuscript. We hope you will find this revised version satisfactory.

 

Sincerely,

 

The Authors

 

references

 

1. Sheng M, Yunlin F, Yingjian L, et al. The formation and quality evaluation of agarwood induced by the fungi in Aquilaria sinensis. Industrial Crops &amp; Products 2021, 173,

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

I accepted your responses, but select your responses and write in the text.

Author Response

Response to Reviewer 2 Comments

 

 

 

Dear Reviewers,

 

Thank you very much for your time involved in reviewing the manuscript and your very encouraging comments on the merits. We also appreciate your clear and detailed feedback and hope that the explanation has fully addressed all of your concerns.

In the remainder of this letter, we discussed each of your comments individually along with our corresponding responses. The parts that were modified in the manuscript were also organized below. For ease of reading, we used highlight words to mark the changes.

We have modified all of your suggestions in the manuscript using the Track Changes. The number of Line in the response indicates the number of line in Track Changes.

 

Point:

I accepted your responses, but select your responses and write in the text.

 

Response:

Thank you for your constructive suggestions, and we have selected and added to the text the contents of our responses based on your suggestions.

Due to the limited space of the manuscript, we summarized the contents of the responses before adding them to the manuscript. The content that were added and their locations are organized as follows.

 

 

Point 1: Due to the scarcity of naturally occurring agarwood,  artificial techniques have been effective for increasing the production of agarwood and meeting market demand.

The readers need some information about A. sinensis more comprehensively. Please re-write again the state of the art of Aquilaria sinensis. Please update the population of natural A. sinensis and the plantation of A. sinensis in your country.  How many natural agarwood productions per year? How many artificial agarwood productions per year? How about their grade between natural and artificial agarwood?  How many percent they can replace natural agarwood? What kind of end product from artificial agarwood? How sustain the artificial agarwood to make mass production? Is there an agarwood plantation on a large scale?

Response 1:

Thank you for your valuable suggestions, and we have added the responses to the manuscript as Line 44 and 74.

1. How many natural agarwood productions per year? How many artificial agarwood productions per year? How about their grade between natural and artificial agarwood? How many percent they can replace natural agarwood? What kind of end product from artificial agarwood?

We have added the responses to the manuscript as Line 44. The relevant contents are provided below as a screen dump for your quick reference.

 

2. How sustain the artificial agarwood to make mass production? Is there an agarwood plantation on a large scale?

We have added the responses to the manuscript as Line 75. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 2: How much influence the artificial agarwood has in replacing the existence of natural agarwood? Has it really been mass-produced or is it still on a research scale? How about the success rate (%) of inoculation by fungi compared with chemical induction? Is it true that the market has accepted artificial agarwood?

Could you explain prospect agarwood production by artificial techniques (chemical or biological induction)? How much agarwood artificial agarwood production is in your country per year? Could artificial agarwood replace natural agarwood?

Specific comment: why is your focus on Aquilaria sinensis? Other agarwood species, how update Aquilaria yunnanensis?

Response 2:

Thank you for your valuable suggestions, and we have added the responses to the manuscript.

1. How much influence the artificial agarwood has in replacing the existence of natural agarwood? Has it really been mass-produced or is it still on a research scale? Could you explain prospect agarwood production by artificial techniques (chemical or biological induction)? How much agarwood artificial agarwood production is in your country per year? Could artificial agarwood replace natural agarwood?

We have added the responses to the manuscript as Line 44. The relevant contents are provided below as a screen dump for your quick reference.

 

2. How about the success rate (%) of inoculation by fungi compared with chemical induction?

We have added the responses to the manuscript as Line 681. The relevant contents are provided below as a screen dump for your quick reference.

 

3. why is your focus on Aquilaria sinensis? Other agarwood species, how update Aquilaria yunnanensis?

We have added the responses to the manuscript as Line 35. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 3: Line 113.  

What kind of fungal species did you find it? 

How many fungal species did you find it?

Are they fungal pathogens? Are they endophytic fungi?

How to identify fungal species? by molecular identification? by morphology?

Did you use single fungal species for this experiment? How did you select to effective fungal species in this experiment?

Response 3:

Thank you for your valuable suggestions, and we have added the responses to the manuscript.

1. What kind of fungal species did you find it?

How many fungal species did you find it?

Are they fungal pathogens? Are they endophytic fungi?

How to identify fungal species? by molecular identification? by morphology?

Did you use single fungal species for this experiment?

We have added the responses to the manuscript as Line 137. The relevant contents are provided below as a screen dump for your quick reference.

 

2. How did you select to effective fungal species in this experiment?

We have added the responses to the manuscript as Line 658. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 4: Line 131 :

 

Could you tell us the histories of A. sinensis in this area? How large the plantation and line spacing? Is it monoculture or mix-plantation? Ecological site plantation: altitude, type of soil, type of forests, a diversity of local shrub species near the trees, climate, etc. When A. sinensis flowering and fruiting in this area? Is there any problem with the pest and disease of Aquilaria sinensis?

Response 4:

Thank you for your valuable suggestions, and we have added the responses to the manuscript.

1. Could you tell us the histories of sinensis in this area? How large the plantation and line spacing? Is it monoculture or mix-plantation? Ecological site plantation: altitude, type of soil, type of forests, a diversity of local shrub species near the trees, climate, etc. When A. sinensis flowering and fruiting in this area? Is there any problem with the pest and disease of Aquilaria sinensis? When A. sinensis flowering and fruiting in this area?

We have added the responses to the manuscript as Line 142. The relevant contents are provided below as a screen dump for your quick reference.

 

2. Is there any problem with the pest and disease of Aquilaria sinensis?

We have added the responses to the manuscript as Line 149. The relevant contents are provided below as a screen dump for your quick reference.

Since A. sinensis is rarely grown on a large scale, no serious pests or diseases have been found, but aphids and leafroller moths often damage young leaves and tips. It has been shown that after feeding by phytophagous insects, A. sinensis releases significantly more herbivore induced plant volatiles than healthy plants to tend to avoid pests and attract natural enemies. Therefore, A. sinensis is rarely infested with insects.

 

Point 5: Any treatment of A. sinensis plantation, i.e. intensive fertilizer, pesticides, herbicides, etc.

Response 5: Thank you for your valuable suggestions, and we have added the responses to the manuscript. We have added the responses to the manuscript as Line 142. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 6: How many trees did you use in this experiment? Could you make the layout of the experiment? How select the trees for fungal induction and control experiments?

Response 6:

Thank you for your valuable suggestions, and we have added the responses to the manuscript. We have added the responses to the manuscript as Line 149 and 157. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 7: How many holes did you prepare for one tree? How many dosages (mL) of fungal induction per hole or per tree? Did you find some trees that die cause of your treatment?

Response 7:

1. How many holes did you prepare for one tree? How many dosages (mL) of fungal induction per hole or per tree?

We have added the responses to the manuscript as Line 150. The relevant contents are provided below as a screen dump for your quick reference.

 

2. Did you find some trees that die cause of your treatment?

We have added the responses to the manuscript as Line 681. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 8: How to take samples, which part of stem, how many replications?

Please make an illustration (a figure), how to collect some slices of the stem of standing tree.

Response 8:

Thank you for your valuable suggestions, and we have added the responses to the manuscript. We have added the responses to the manuscript as Line 158 and Figure 2. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 9: Line 139: did you make some holes in the control blank groups. How many holes each did you prepare per one tree for control treatment?

Response 9:

Thank you for your valuable suggestions, and we have added the responses to the manuscript. We have added the responses to the manuscript as Line 155. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 10:How to separate resinous wood from white wood?

Response 10:

Thank you for your valuable suggestions, and we have added the responses to the manuscript. We have added the responses to the manuscript as Line 204. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 11: Why did you decide to observe 12 months and 18 months? According to your experience, what time the best time for harvest artificial agarwood for the best grade

Response 11:

Thank you for your valuable suggestions, and we have added the responses to the manuscript. We have added the responses to the manuscript as Line 687. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 12: Line 131 :

Could you provide and show xylem structure treated by drill only (the control blank group)?

Response 12:

Thank you for your valuable suggestions, and we have added the xylem structure of the control blank group to the manuscript. We have added the responses to the manuscript as Line 234, 282, 296 and 365. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 13:Could you provide and show xylem structure treated by chemical induction?

If possible, there are comparison xylem structures among the control blank group, chemical induction, and fungal induction.

Response 13:

Thank you for yours valuable suggestions and we regret for the inconvenience caused by inadequate description in the manuscript. We cited previous research results on chemically induction to compare with the structure of our experiment, and the comparative results of the blank control, chemically induced and fungus induced were added to the manuscript as Line 664.

Thank you very much for your constructive suggestions, and we will extend our induction method in the subsequent experiments by conducting a comparison of fungal induction with physical induction and chemical induction to compare the differences in xylem structure of the samples treated with different induction method.

 

Point 14: Discussion:

 

-could you discuss and explain the difference between structure changes of Xylem by fungal pathogen and endophytic fungi? did you find difference between them.

What kind the difference is the role of endophytic fungi residence or fungal pathogen to develop agarwood formation in the xylem.

Response 14: Thank you for your valuable suggestions, and we have added the responses to the manuscript. We have added the responses to the manuscript as Line 649. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 15: What is the best treatment for agarwood formation for human health, by chemical induction or by fungal induction?

Response 15:

Thank you for your valuable suggestions, and we have added the responses to the manuscript. We have added the responses to the manuscript as Line 681. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 16: Discussion should provide what kind of fungal group for agarwood formation consistency?  Was it fungal pathogen or endophytic fungi? You should state your fungal group induction.

Response 16: Thank you for your valuable suggestions, and we have added the responses to the manuscript. We have added the responses to the manuscript as Line 649. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 17: Conclusion:

 

You should state the name of the fungal induction. What the name of fungal induction is very important in your research. So much research will do the same thing, but the difference is fungal induction utilization.

Response 17: Thank you for your valuable suggestions, and we have added the responses to the manuscript. We have added the responses to the manuscript as Line 137. The relevant contents are provided below as a screen dump for your quick reference.

 

We would like to express our sincere thanks for your constructive suggestions for this manuscript. We have benefited greatly from your vast knowledge and careful review. It is a great honor to have your review of the paper, which has given us a deeper understanding of manuscript writing. We have revised the manuscript according to your suggestions and hope that you will be satisfied with the revised manuscript.

 

Sincerely,

 

The Authors

Author Response File: Author Response.docx

Reviewer 3 Report

All comments made have been corrected as suggested earlier.

1) The quality of Figure 1, especially the additional labeling, greatly improved the readability of the paper.

2) Reliably presented ANOVA is not questionable and is sufficiently well described.

3) Figure 14 illustrates statistical differences simply, making the paper more reader friendly than the previously posted tables.

5) The reference samples were described.

4) The other method comments (number of trees, number of months on the charts) have been also corrected.

Suggestions under consideration

Figure 14 (l. 517) should be named as “Differences between…” instead of “Significance between”, because figure shows differences (only some of them are significant, ll. 520-521).

I’m not a fan of using the phrase “extremely significant higher” (ll. 505, 508 etc.). Difference between groups may be significant or non-significant at assumed significance level (here 0.05, lines 182-184). If differences between two groups are significant, we believe that there are two groups. If differences are non-significant, there could be probably a single group. One group is no extremely significant higher than the other one (eg. in line 507), difference is not bigger, difference is the same, but “statistically more significant”. Assumed significance level (0.05, lines 182-184) shows which difference we accept and which we don’t, nothing more, we cannot interpret this measure as the strength between groups.

I suggest using rather “statistically more significant” or simply “significant” without suggestion of strength of significance because it was already cleared in the Table 1.

Author Response

Response to Reviewer 3 Comments

 

 

 

Dear Reviewers,

 

Thank you very much for your time involved in reviewing the manuscript and your very encouraging comments on the merits. We also appreciate your clear and detailed feedback and hope that the explanation has fully addressed all of your concerns.

In the remainder of this letter, we discussed each of your comments individually along with our corresponding responses. The parts that were modified in the manuscript were also organized below. For ease of reading, we used highlight words to mark the changes.

 

Point 1: Methods

 

1. Figure 14 (l. 517) should be named as “Differences between…” instead of “Significance between”, because figure shows differences (only some of them are significant, ll. 520-521).

 

Response 1:

Thank you for your suggestions, we have changed Significance to Differences based on your suggestion as Line 539. The relevant contents are provided below as a screen dump for your quick reference.

 

Point 2: I’m not a fan of using the phrase “extremely significant higher” (ll. 505, 508 etc.). Difference between groups may be significant or non-significant at assumed significance level (here 0.05, lines 182-184). If differences between two groups are significant, we believe that there are two groups. If differences are non-significant, there could be probably a single group. One group is no extremely significant higher than the other one (eg. in line 507), difference is not bigger, difference is the same, but “statistically more significant”. Assumed significance level (0.05, lines 182-184) shows which difference we accept and which we don’t, nothing more, we cannot interpret this measure as the strength between groups.

I suggest using rather “statistically more significant” or simply “significant” without suggestion of strength of significance because it was already cleared in the Table 1.

 

Response 2:

Thank you for your careful review and suggestions on nomenclatures, and we regret for the inconvenience caused by wrong expression in the manuscript. We have changed extremely significant to statistically more significant based on your suggestion and modified other related content as Line 517-531. The relevant contents are provided below as a screen dump for your quick reference.

 

We would like to express our sincere thanks for your constructive suggestions for this manuscript. We have benefited greatly from your vast knowledge and careful review. It is a great honor to have your review of the paper, which has given us a deeper understanding of ANOVA. We have revised the manuscript according to your suggestions and hope that you will be satisfied with the revised manuscript.

 

Sincerely,

 

The Authors

Author Response File: Author Response.docx