The Effect of Microbial Fertilizer on the Growth, Rhizospheric Environment and Medicinal Quality of Fritillaria taipaiensis

Round 1

Reviewer 1 Report

The manuscript is generally well prepared and contains interesting data. However, most of the analyzes in this study were performed spectrophotometrically, so the results are approximate. The use of HPLC would be better for the analysis of specific parameters, so the study would be better suited for publication in Horticulturae. I have some other important comments and some useful improvements that need to be made before a positive recommendation for publication.

In some respects the discussion is very sound and poor. In this study, all the results are the description of how the biochemical index changes, the authors should explore the further reasons for the changes. An important point that needs to be considered is the colonization with AM fungi - if the authors did not evaluate and find arbuscules, typical structures of AM fungi, then it cannot be said that the colonization with AM fungi was successful.

There are several comments/suggestions for the manuscript as below:

Title: I suggest to replace the »quality« with other word or few words, which would better describe the aim of the study.

INTRODUCTION:

Line 35: Please check the spelling of the authority for a binomial name.

Line 47: ...showed lower quality - compared to what?

Line 57: italics Trichoderma

The reference list should be updated. Some of the references used in this study are outdated (e.g. some in the introduction section on AM fungi, before 2000 and others). Please provide new reports.

Line 68: ...promote their growth and development - please provide the appropriate and more recent reference here. Also, there is a missing reference when you refer to "and improve their yield and quality" (line 70). I suggest at least using these two: Saini, I.; Aggarwal, A.; Kaushik, P. Inoculation with mycorrhizal fungi and other microbes to improve the morpho-physiological and floral traits of Gazania rigens (L.) Gaertn. Agriculture 2019, 9, 51 and Vosnjak, M., Likar, M., & Osterc, G. (2021). The Effect of Mycorrhizal Inoculum and Phosphorus Treatment on Growth and Flowering of Ajania (Ajania pacifica (Nakai) Bremer et Humphries) Plant. Horticulturae, 7(7), 178.

Line 68: missing comma after development

MATERIALS AND METHODS:

Line 82: Please give the name of the country and city and the website for "INVAM" and the product purchased. Same for NIFDC.

Line 89: These were probably standards for uracil, guanine, uridine, adenine, thymidine, and adenosine?

Line 95: ...used in this study.

Line 97:... uniform layer of microbial agents was applied...? How was this applied? Please specify.

Line 98: Did you use bulbs or seedlings? Please specify.

Line 105: When was the harvest of bulbs, roots, and soils assesed? Please provide the date the bulbs were planted and the date they were harvested. At what phenological stage were the bulbs harvested? There is missing information.

Line 109: explain what FAA is?

Line 110: please give more information about the method used. The material used (trypan blue, etc.), under what conditions, etc...

Line 111: The authors refer to the method of Trouvelot et al. (1986) for quantifying mycorrhizal structures, but given the importance of this method in the MS, I think it would be interesting to explain the method in detail in the MS and indicate which mycorrhizal parameters were assessed.

Line 113: You only used part of the leaf for biochemical analysis? Why was only the part of the leaf from the base to the upper and middle part used? Please explain.

Line 114: Please briefly provide some additional information about the extraction procedure for pigment analysis (material and method). What pigments were analyzed? Was the analysis performed with HPLC? There is a lot of information missing that is important.

Line 115, 116: Please provide the references for enzymatic analysis (nitrogen blue tetrazole method, guaiacol method and ultraviolet spectrophotometry). Also indicate the method used for the analysis of MDA and soluble sugar content. Were these methods all spectrophotometric?

Line 120: Please provide more information on the counting of AM fungal spores. Did you observe only the spores or other AM structures as well?

What about the arbuscules, which are the main structures of AM fungi? Did you also examine the arbuscules? Please provide data and information. If no, how confident are you that colonization with AM fungi and not another fungus was successful?

Line 137, 138: Can you briefly provide more information on the procedure used to determine total alkaloids (extraction procedure...)? No need for this reference at the end.

Line 142: one-way AMOVA? Please provide the statistical analysis methods used in your study and explain how many factors (with levels) were included in the individual ANOVA model for a particular parameter. Please also provide a copy of the table ANOVA (in supplemental material) showing the significance (with p-values) of the factors or interactions tested for a particular parameter.

RESULTS:

Line 154: here the values are probably reported as mean and standard error? Please make this clear in all figures and tables. It is not clear now.

Line 187: you do not always need to highlight the age of the bulbs. Or is it important?

Table 4 and 5: here the values are probably presented as mean and standard error?

DISCUSSION:

Line 234: ... forming a typical arbuscular mycorrhizal structure. If no assessment of arbuscules has been made, you can not say that. Typical arbuscular mycorrhizal structures are just arbuscules. If no arbuscules are present, then it is questionable whether colonization by AM fungi was successful.

Line 245: ... for the high yield of F. taipaiensis??? Did this study also evaluate the yield of F. taipaiensis???? This would be very interesting information.

Lines 242-253: The discussion of biochemical indices, enzymes, MDA and soluble sugars is solid and poor. The discussion should be extended and the authors should investigate the other reasons for the changes.

REFERENCES: The current reference style does not match the style of the journal Horticulturae, please refer to the submission guidelines for authors. Some references do not contain all the information (Dong et al. etc...).

Author Response

Dear Reviewer:

Thank you for your letter and for the comments concerning our manuscript entitled “The Effect of Microbial Fertilizer on the Quality of Fritillaria taipaiensis P. Y. Li” . Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in the paper. The main corrections in the paper and the responds to the editor and reviewer’s comments are as flowing:

Reviewer #1: The manuscript is generally well prepared and contains interesting data. However, most of the analyzes in this study were performed spectrophotometrically, so the results are approximate. The use of HPLC would be better for the analysis of specific parameters, so the study would be better suited for publication in Horticulturae. I have some other important comments and some useful improvements that need to be made before a positive recommendation for publication.

In some respects the discussion is very sound and poor. In this study, all the results are the description of how the biochemical index changes, the authors should explore the further reasons for the changes. An important point that needs to be considered is the colonization with AM fungi - if the authors did not evaluate and find arbuscules, typical structures of AM fungi, then it cannot be said that the colonization with AM fungi was successful.

Comments:

1. Title-I suggest to replace the »quality« with other word or few words, which would better describe the aim of the study.

Response: It is really true as reviewer suggested that the word “quality” could not fully express the aim of our study, so we have removed this word and changed the title into “The Effect of Microbial Fertilizer on Fritillaria taipaiensis P. Y. Li”.

2.INTRODUCTION-Line 35: Please check the spelling of the authority for a binomial name.

Response: It is really true as reviewer suggested that the standardized Latin name of Chuanbeimu should be Fritillaria cirrhosa D. Don, and we have replaced it with the correct form (1. Introduction, L35).

3.INTRODUCTION-Line 47: ...showed lower quality - compared to what?

Response: We are so sorry for our negligence of not giving out the clear information. The three-year-old F. taipaiensis seedlings showed a lower quality in the content of active constituents compared to one and two-year-old F. taipaiensis seedlings (1. Introduction, L47-48).

4.INTRODUCTION-Line 57: italics Trichoderma

Response: We have corrected it into italic type as “Trichoderma spp” (1. Introduction, L58).

5.INTRODUCTION-The reference list should be updated. Some of the references used in this study are outdated (e.g. some in the introduction section on AM fungi, before 2000 and others). Please provide new reports.

Response: Thank you very much for this valuable suggestion. It is true as the reviewer said some of the references used in this study are outdated. We have replaced them with the new reports.

6. INTRODUCTION-Line 68: ...promote their growth and development - please provide the appropriate and more recent reference here. Also, there is a missing reference when you refer to "and improve their yield and quality" (line 70). I suggest at least using these two: Saini, I.; Aggarwal, A.; Kaushik, P. Inoculation with mycorrhizal fungi and other microbes to improve the morpho-physiological and floral traits of Gazania rigens (L.) Gaertn. Agriculture 2019, 9, 51 and Vosnjak, M., Likar, M., & Osterc, G. (2021). The Effect of Mycorrhizal Inoculum and Phosphorus Treatment on Growth and Flowering of Ajania (Ajania pacifica (Nakai) Bremer et Humphries) Plant. Horticulturae, 7(7), 178.

Response: We have replaced the reference in the sentence of “promote their growth and development...” with the more appropriate and recent literatures (1. Introduction, L68-70, Reference [22-29]). And it is true that as the reviewer suggested that there is a missing reference in “and improve their yield and quality”, we have cited these two literatures there as reference [28, 29] according to the reviewer’s suggestion(1. Introduction, L70, Reference [28, 29]).

7.INTRODUCTION-Line 68: missing comma after development.

Response: Thank you for your attention, we have added a comma there (1. Introduction, L69).

8. MATERIALS AND METHODS-Line 82: Please give the name of the country and city and the website for "INVAM" and the product purchased. Same for NIFDC.

Response: The International Culture Collection of (Vesicular) Arbuscular Mycorrhizal Fungi (INVAM) is Morgantown, USA, and its website is https://invam.wvu.edu/home (2.1. Microbial Inoculum, L93-94). China National Institutes for Food and Drug Control (NIFDC) is in Beijing, China, its website is https://www.nifdc.org.cn/nifdc/ (2.3. Reference Drugs, L104-105). And the batch numbers of the uracil, guanine, uridine, adenine, thymidine, and adenosine reference we bought from NIFDC is 100469-200401, 140631-201205, 110887-200202, 886-200001, 101215-201401 and 110879-200202, respectively (2.3. Reference Drugs, L103-104).

9.MATERIALS AND METHODS-Line 89: These were probably standards for uracil, guanine, uridine, adenine, thymidine, and adenosine?

Response: Yes, they are the standard references for uracil, guanine, uridine, adenine, thymidine, and adenosine in the the determination of nucleoside content. Their Batch numbers were 100469-200401, 140631-201205, 110887-200202, 886-200001, 101215-201401 and 110879-200202, respectively. And they were obtained from the China National Institutes for Food and Drug Control (NIFDC) (Beijing, China, https://www.nifdc.org.cn/nifdc/ ) (2.3. Reference Drugs, L103-105).

10. MATERIALS AND METHODS-Line 95: ...used in this study.

Response: We are very sorry for our incorrect writing, we have re-written this part according to the reviewer’s suggestion that replaced the word “paper” with “study” (2.4. Cultivation Management and Sample Collection, L112).

11. MATERIALS AND METHODS-Line 97:... uniform layer of microbial agents was applied...? How was this applied? Please specify.

Response: We are very sorry for the unclear expression. And we have corrected it into “the F. taipaiensis bulbs were planted on an uniform layer of microbial agents and were covered by 5~6 cm of the surface soil” (2.4. Cultivation Management and Sample Collection, L114-115).

12. MATERIALS AND METHODS-Line 98: Did you use bulbs or seedlings? Please specify.

Response: We used the bulbs for planting, so sorry for the unclear expression, and we have corrected it (2.4. Cultivation Management and Sample Collection, L116).

13. MATERIALS AND METHODS-Line 105: When was the harvest of bulbs, roots, and soils assesed? Please provide the date the bulbs were planted and the date they were harvested. At what phenological stage were the bulbs harvested? There is missing information.

Response: On, August 27, 2017, the F. taipaiensis bulbs were planted. And on May 29, 2018, the bulbs, fibrous roots, leaves, and rhizospheric soils of F. taipaiensis at the beginning of blooming stage were harvested: the leaves were used to determine the activity of protective enzymes and the contents of photosynthetic pigments, malondialdehyde, soluble sugar, and soluble protein immediately after harvest; the bulbs were washed and dried at 45 ℃ for analysis; the fibrous roots were preserved for mycorrhizal colonization rate and infection intensity analysis; rhizospheric soil was collected and preserved for soil microbial quantity and soil enzyme activity determination. (2.4. Cultivation Management and Sample Collection, L114-115, L125-129)

14. MATERIALS AND METHODS-explain what FAA is?

Response: We are very sorry for the unclear expression. And we have added the formulation to this part: 75% (V/V) ethano·90 mL + glacial aceticacid·5 mL + 40% (V/V) formaldehyde·5mL (2.5. Analysis of Mycorrhizal Colonization Rate, L132).

15. MATERIALS AND METHODS-Line 110: please give more information about the method used. The material used (trypan blue, etc.), under what conditions, etc...
Response: We are very sorry for the unclear expression. In this part, fifty root segments with uniform thickness of each basket were randomly selected and stained according to the method of Philips et al. (1970) to calculate the mycorrhizal colonization rate and the mycorrhizal infection intensity: The root segments of F. taipaiensis with uniform thickness about 1 cm long were treated by 10% KOH under 90 ℃ water bathe for 45 min, and then were acidified by 2% HCl for 5 minutes after 3~5 times of distilled water washing; after acidification, the root segments were stained by 0.05% trypan blue 90 ℃ water bathe for 30 min, and then were decolorized in glycerol lactate solution (1:1:1) for 24 h (2.5. Analysis of Mycorrhizal Colonization Rate, L132-137).

16. MATERIALS AND METHODS-Line 111: The authors refer to the method of Trouvelot et al. (1986) for quantifying mycorrhizal structures, but given the importance of this method in the MS, I think it would be interesting to explain the method in detail in the MS and indicate which mycorrhizal parameters were assessed.

   Response: We assessed the colonization rate and the infection intensity in this study according to method of Trouvelot et al. (1986). Colonization rate = (number of infected root segments / total number of examined root segments) × 100%; Infection intensity = (95·n5 +70·n4 + 30· n3 + 5·n2 + n1) / total number of examined root segments × 100%, where n5 means number of root segments with level 5 infection, n4 means number of root segments with level 4 infection, etc. (2.5. Analysis of Mycorrhizal Colonization Rate, L139-142)

17. MATERIALS AND METHODS-Line 113: You only used part of the leaf for biochemical analysis? Why was only the part of the leaf from the base to the upper and middle part used? Please explain.

Response: We only use the healthy mature leaves from the base to the upper and middle parts in our explain, and they have the similar sizes. These materials can more accurately reflect the physiological and biochemical indices of leaves. (2.6. Determination of Physiological and Biochemical Indices of Leaves, L144)

18. MATERIALS AND METHODS-Line 114: Please briefly provide some additional information about the extraction procedure for pigment analysis (material and method). What pigments were analyzed? Was the analysis performed with HPLC? There is a lot of information missing that is important.

   Response: We are very sorry for the unclear expression. And we have added the missing information to this part: the contents of the mail photosynthetic pigments in the leaves such as chlorophyll a, chlorophyll b and carotenoid were determined according to the method in the literature via spectrophotometry; the activities of superoxide dismutase (SOD), peroxidase (POD) were determined by the nitrogen blue tetrazole method and the guaiacol method, respectively via spectrophotometry, and the activity of catalase (CAT) was determined by the ultraviolet absorption method; the content of malondialdehyde (MDA) and soluble sugar was determined using a thiobarbituric acid method and anthrone method, respectively via spectrophotometry; the content of soluble protein was determined by Coomassie brilliant blue method via spectrophotometry. (2.6. Determination of Physiological and Biochemical Indices of Leaves, L145-151)

19. MATERIALS AND METHODS-Line 115, 116: Please provide the references for enzymatic analysis (nitrogen blue tetrazole method, guaiacol method and ultraviolet spectrophotometry). Also indicate the method used for the analysis of MDA and soluble sugar content. Were these methods all spectrophotometric?

Response: All these analysis were according to the method from “Reference [42]:  Zhang, Z.L.; Qu, W.J.; Li, X.F. Plant Physiology Experiment Instruction. 1st ed.; Higher Education Press: Beijing, China, 2009.”, we are so sorry for our negligence of not giving out these information, and we have added them to these parts (2.6. Determination of Physiological and Biochemical Indices of Leaves, L146-151;Reference [42] ).

20. MATERIALS AND METHODS-Line 120: Please provide more information on the counting of AM fungal spores. Did you observe only the spores or other AM structures as well? What about the arbuscules, which are the main structures of AM fungi? Did you also examine the arbuscules? Please provide data and information. If no, how confident are you that colonization with AM fungi and not another fungus was successful?

Response: We are so sorry for the unclear expression, and we have removed this part in the manuscript. We just counted the spores in the preparation stage of the experiment for the  propagation culture, and we did not count the spores after the inoculation in the experiment. For the short survival period of arbuscules, the mycorrhizal colonization rate might be underestimated if we just use the abundance of arbuscules to calculate it. Thus in this study, we examined the either endophytic hyphae or the arbuscules of AM fungi in the root to calculate the mycorrhizal colonization rate and the mycorrhizal infection intensity and we did not examine the arbuscules specifically. As we have only ten days for revision, it would be hard for us to prepare the materials and calculate the data again from the beginning. We are so sorry for our thoughtless consideration, and we will examine the abundance of arbuscules in our future study, thank you very much for your valuable suggestion.

21. MATERIALS AND METHODS-Line 137, 138: Can you briefly provide more information on the procedure used to determine total alkaloids (extraction procedure...)? No need for this reference at the end.

Response: In the determination of total alkaloids, 2 g of dried F. taipaiensis bulb powder (No. 3sieve passed) from each sample was used to extract the total alkaloids. The total alkaloids were extracted by chloroform-methanol (4:1) mixed solution and then were determined by ultraviolet-visible spectrophotometry according to the literature using sipeimine as a reference standard (2.9. The Quality Analysis of Bulb, L170-172)

22. MATERIALS AND METHODS-Line 142: one-way AMOVA? Please provide the statistical analysis methods used in your study and explain how many factors (with levels) were included in the individual ANOVA model for a particular parameter. Please also provide a copy of the table ANOVA (in supplemental material) showing the significance (with p-values) of the factors or interactions tested for a particular parameter.

Response: We are for sorry for mistake typing one-way ANOVA as one-way AMOVA, we have corrected it (2.10. The Data Analysis, L176). The data were analyzed by a single factor (one-way ANOVA) and Tukey for multiple comparisons, respectively, via SPSS 22.0 (SPSS, Inc., Chicago, IL, USA) and Excel 2003 (α=0.05). A p < 0.05 indicated statistical significance. In the determination of ecological characteristics (Fig 1), the values are means ± standard deviation of 3 sampling replicates (50 root segments each replicate) randomly selected from each group. In the determination of protection enzyme activity (Fig 2), contents of MDA, soluble sugar, and soluble protein (Fig 3), photosynthetic pigment content (Table 1), nucleoside content (Table 4) and alkaloid content (Table 5), the values are means ± standard deviation of 5 plants randomly selected from each group. And in the determination of rhizosphere microbial number (Table 2) and soil enzyme activity (Table 3), the values are means ± standard deviation of 5 soil samples randomly selected from each group. We have added this statement as a footer in for the figure and tables. We have prepared a copy of the table ANOVA showing the significance of the factors and interactions tested for the particular parameter as a supplemental material with this revision.

23. RESULTS-Line 154: here the values are probably reported as mean and standard error? Please make this clear in all figures and tables. It is not clear now.

Response: The data were analyzed by a single factor (one-way ANOVA) and Tukey for multiple comparisons, respectively, via SPSS 22.0 (SPSS, Inc., Chicago, IL, USA) and Excel 2003 (α=0.05). A p < 0.05 indicated statistical significance. In the determination of ecological characteristics (Fig 1), the values are means ± standard deviation of 3 sampling replicates (50 root segments each replicate) randomly selected from each group. In the determination of protection enzyme activity (Fig 2), contents of MDA, soluble sugar, and soluble protein (Fig 3), photosynthetic pigment content (Table 1), nucleoside content (Table 4) and alkaloid content (Table 5), the values are means ± standard deviation of 5 plants randomly selected from each group. And in the determination of rhizosphere microbial number (Table 2) and soil enzyme activity (Table 3), the values are means ± standard deviation of 5 soil samples randomly selected from each group. We have added this statement as a footer in for the figure and tables. And we have explained that in the footers of tables and figure after revision.

24. RESULTS-Line 187: you do not always need to highlight the age of the bulbs. Or is it important?

Response: It is true as the reviewer suggested that we do not have to always mention the age of the bulb.

25. RESULTS-Table 4 and 5: here the values are probably presented as mean and standard error?

Response: In Table 4 and Table 5, the values are means ± standard deviation of 5 plants randomly selected from each group.

2.6 DISCUSSION-Line 234: ... forming a typical arbuscular mycorrhizal structure. If no assessment of arbuscules has been made, you can not say that. Typical arbuscular mycorrhizal structures are just arbuscules. If no arbuscules are present, then it is questionable whether colonization by AM fungi was successful.

Response: It is true as the reviewer suggested that typical arbuscular mycorrhizal structures are just arbuscules, for we did not calculate the abundance of arbuscules specifically, we could not say that. So we replaced that sentence as “the inoculation increased the mycorrhizal infection intensity of F. taipaiensis” (4. Discussion, L277-278).

27. DISCUSSION-Line 245: ... for the high yield of F. taipaiensis??? Did this study also evaluate the yield of F. taipaiensis???? This would be very interesting information.

Response: We did not evaluate the yield of F. taipaiensis in this study, that statement was based on the reports before, we are very sorry about the unclear expression, and we have removed that part.

28. DISCUSSION-Lines 242-253: The discussion of biochemical indices, enzymes, MDA and soluble sugars is solid and poor. The discussion should be extended and the authors should investigate the other reasons for the changes.

Response: We have re-written this part in the manuscript, and added the content of the discussion of biochemical indices, enzymes, MDA and soluble sugars (4. Discussion, L289-311).

29. REFERENCES-The current reference style does not match the style of the journal Horticulturae, please refer to the submission guidelines for authors. Some references do not contain all the information (Dong et al. etc...).

Response: We have revised the format of the reference to match the style of the journal Horticulturae and checked the reference again to correct the mistakes.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors performed an interesting study addressing the relevance of plant microbe interactions towards the production of medicinal active compounds. Te experimental design includes one medicinal species, two AMF fungi, one putatively P solubilizing bacteria, and one putatively K solubilizing bacteria species. The pot experiment includes the interaction of the plant with each of the microbial taxa and with the mix of all of them. The parameters analysed cover plant physiology, soil functionality and microbial community. The results are clear and show a benefit of using this type of inoculants to promote the medicinal potential of the plant (Fritillaria taipaiensis).

The introduction is well written and leads the reader towards the main issue of the manuscript. However there are several concerns that need to be clarified before considering the work for publication:

• What is the statistical unit, the plant or the basket? How many baskets per treatment were present in the experiment? When we read n=5, does the n refer to plants of the same basket or to 5 baskets?
• What was the composition of the media where the plants were grown?
• The Authors provide references to the distinct methods used, but at least a mention to the principle of each method used should be present in the paper. Like this we have to go many other works to obtain essential information about the results.
• The taxonomy of Glomeromycota is very complex and changed a lot in the recent years. Please refer to the taxonomic system your are using.
• What was the criteria for choosing these two AMF species?
• The claim that Bacillus mucilaginosus and Bacillus megaterium are P and K solubilizers (respectively) is not enough to associate the main contribution of these bacteria to plant performance (medicinal active compounds) to one or another of these functions. Did the authors test if Bacillus mucilaginosus is a good P solubilizer under the growth conditions used and the same for Bacillus megaterium. Microbes are very complex entities and they have multiple modes of action depending on the conditions. This may be one of the reasons why not such a big difference was observed in plants inoculated with each of the bacteria group. Please clarify.
• Why do NPK soil concentrations decrease after three years of culture (Introduction line 49). I do not understand. How is the crop usually managed?

Author Response

Dear Reviewer:

    Thank you for your letter and for the comments concerning our manuscript entitled “The Effect of Microbial Fertilizer on the Quality of Fritillaria taipaiensis P. Y. Li” . Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in the paper. The main corrections in the paper and the responds to the editor and reviewer’s comments are as flowing:

Reviewer #2: The authors performed an interesting study addressing the relevance of plant microbe interactions towards the production of medicinal active compounds. Te experimental design includes one medicinal species, two AMF fungi, one putatively P solubilizing bacteria, and one putatively K solubilizing bacteria species. The pot experiment includes the interaction of the plant with each of the microbial taxa and with the mix of all of them. The parameters analysed cover plant physiology, soil functionality and microbial community. The results are clear and show a benefit of using this type of inoculants to promote the medicinal potential of the plant (Fritillaria taipaiensis).

The introduction is well written and leads the reader towards the main issue of the manuscript. However there are several concerns that need to be clarified before considering the work for publication.

Comments:

1. What is the statistical unit, the plant or the basket? How many baskets per treatment were present in the experiment? When we read n=5, does the n refer to plants of the same basket or to 5 baskets?

Response: In our study, there were three baskets of F. taipaiensis for each treatment group, a total of 30 F. taipaiensis bulbs were planted in each basket. In the determination of ecological characteristics (Fig 1), the values are means ± standard deviation of 3 sampling replicates (50 root segments each replicate) randomly selected from each group. In the determination of protection enzyme activity (Fig 2), contents of MDA, soluble sugar, and soluble protein (Fig 3), photosynthetic pigment content (Table 1), nucleoside content (Table 4) and alkaloid content (Table 5), the values are means ± standard deviation of 5 plants randomly selected from each group. And in the determination of rhizosphere microbial number (Table 2) and soil enzyme activity (Table 3), the values are means ± standard deviation of 5 soil samples randomly selected from each group. We have added this statement as a footer in for the figure and tables.

2. What was the composition of the media where the plants were grown?

Response: We are very sorry for the unclear expression. And we have added the missing information to this part: The culture medium was the mixture of humus soil and sand with a ratio of 3:1. The plants grown outdoors, each basket was watered according to the real time climatic conditions and was supplemented with 1000 mL Hoagland’s nutrient solution once a week. (2.4. Cultivation Management and Sample Collection, L111, L123-124)

3. The Authors provide references to the distinct methods used, but at least a mention to the principle of each method used should be present in the paper. Like this we have to go many other works to obtain essential information about the results.

Response: We are very sorry for our negligence of not giving out these information. And we added them to these parts after revision (In the section of “Materials and Methods”, marked).

4. The taxonomy of Glomeromycota is very complex and changed a lot in the recent years. Please refer to the taxonomic system your are using.

Response: The two AM fungi species applied in this study were from the Glomeromycetes (according to the taxonomic system published on the Arthur Schüßler: http://www.amf-phylogeny.com/), which was the main group of arbuscular mycorrhizal fungi: Claroideoglomus claroideum from Claroideoglomeraceae family and Racocetra coreoidea from Gigasporaceae family (2.1. Microbial Inoculum, L90-93).

5. What was the criteria for choosing these two AMF species?

Response: The two AM fungi we chosen in the study were the typical species of the main group in Glomeromycetes, both of them were widely used in promoting the growth of plants (2.1. Microbial Inoculum, L90-93; Reference [38]).

6. The claim that Bacillus mucilaginosusand Bacillus megateriumare P and K solubilizers (respectively) is not enough to associate the main contribution of these bacteria to plant performance (medicinal active compounds) to one or another of these functions. Did the authors test if Bacillus mucilaginosus is a good P solubilizer under the growth conditions used and the same for Bacillus megaterium. Microbes are very complex entities and they have multiple modes of action depending on the conditions. This may be one of the reasons why not such a big difference was observed in plants inoculated with each of the bacteria group. Please clarify.

Response: It is absolutely right that Bacillus mucilaginosus and Bacillus megaterium can not just be simply described as P and K solubilizers. For many literatures reported these two species as P and K solubilizers respectively (Reference [30-36]), we chose them as the P and K solubilizing bacteria in our study. We are so sorry for our negligence of not testing whether they are good P and K solubilizers respectively by ourselves, we will be more rigorous in our future research, thank you for you valuable suggestions. And we added the information of these two species and the genus they belong to in the section of Introduction (1. Introduction, L72-80).

7. Why do NPK soil concentrations decrease after three years of culture (Introduction line 49). I do not understand. How is the crop usually managed?

Response: We are so sorry for the unclear expression, and we have added the missing information to that part in the manuscript (1. Introduction, L50). After the long term artificial disturbance during the cultivation, the contents of nutrients in the cultivated soil such as available N, available P, available K, and organic matter may significantly decrease due to the plant absorption and leaching.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Most of the comments on the revision have been answered with success. However, I have some minor improvements and further clarifications to improve the quality.

Specific suggestions are listed below:
- The title, as it is now formed, is not okay, I think it is worse than the previous version. In my opinion it is too broad and does not offer much information. I suggest replacing the word "quality" with another word or a few words that better describe the aim of the study. Or maybe you can use the word "quality" and also other words that will describe the aim of the study.

- The authors should rather use "mycorrhizal colonization" instead of "arbuscular mycorrhizal colonization". If no arbuscules were detected, you cannot be sure that colonization with AM fungi was successful. Just evaluating spores and endophytic hyphae cannot be an indicator of successful colonization with AM fungi. These structures could belong to any other mycorrhizal fungi. I suggest avoiding arbuscular mycorrhizal colonization because based on these results, one cannot be sure that colonization with AM fungi was successful. So please avoid statements such as "As shown in Figure 1, the roots of F. taipaiensis in different microbial inoculum treatment groups were infected with AM fungi... (line 189) (replace with infected with mycorrhizal fungi) and also in the conclusion section "the inoculation of beneficial microorganisms in rhizospheric soil can form typical arbuscular... (can form mycorrhiza)".

Author Response

Please see the attachment。

Author Response File: Author Response.docx