Mycorrhizal Root Exudates Induce Changes in the Growth and Fumonisin Gene (FUM1) Expression of Fusarium proliferatum
Round 1
Reviewer 1 Report
Introduction
The authors introduced the manscript starting from the agricultural impact of Fusarium proliferatum and of its capability in mycotoxins production. [36]
Then, in few lines switch to the influence of arbuscular mycorrhizal (AM) fungi on F. proliferatum, an the description of the work introduced root exudates effects.[51]
I suggest to the authors to highlight the importance of AM in agricultural production. Furthermore, they should describe and give literature citations on the impact of root exudates on microorganism with agricoltural importance.
The activity, the importance and the meaning of the expression of FUM1 and HOG1 genes is not clearly argued. I suggest to the authors to highlight the most important information and the literature on it. This is important for the comprehension of the results. Otherwise the author present the results before the background clarification.
(HOG1 genes is not described at all in this section.)
Materials and Methods
The authors must be more specific in the section 2.1: how they perform the inoculation of mycorrhizal fungi (Funneliformis mosseae BEG12 64 (Glomerales: Glomeraceae) and Rhizophagus intraradices BEG53 (Glomerales: Glomeraceae)) propagation? (how many cell (OD600))? Otherwise, give references of the protocol.
Results
Just a suggestion: the authors can prepare figures which sum up the data reported in Table 1 and 2 and which makes easier for the reader the interpretation of the results.[163]
The authors should give more information on root exudates composition differences (chemical point of view) in inoculated and not inoculated plants.
Discussion
[259] The authors affirm that the root exudates of inoculated/not inoculated plants contain mycotoxin: this is a statement that must be demonstrated (if they affirm it). This may request a deeper analysis of root exudates content.
The discussion includes literature that should be cited in the introduction,addressing the reader in the meaning of this work. Furthermore, this section should evidence the innovation of the manuscript.
Minor typo
[172] add measures units on the numeric data
[192] typo “after 5 days, the influence”
Author Response
Response to Reviewer 1 Comments
We are greatly indebted to You for your corrections and comments on our manuscript.
I have accepted all of your corrections and your comments, let see my remarks and comments written in red.
Point 1: The authors introduced the manuscript starting from the agricultural impact of Fusarium proliferatum and of its capability in mycotoxins production. [36]
Then, in few lines switch to the influence of arbuscular mycorrhizal (AM) fungi on F. proliferatum, an the description of the work introduced root exudates effects.[51]
I suggest to the authors to highlight the importance of AM in agricultural production. Furthermore, they should describe and give literature citations on the impact of root exudates on microorganism with agricultural importance.
Response 1: The Introduction was improved with literatures to highlight the importance of arbuscular mycorrhizal fungi in agriculture moreover, rewritten following your recommendations. See lines in corrected manuscript 49-57.
Point 2: The activity, the importance and the meaning of the expression of FUM1 and HOG1 genes is not clearly argued. I suggest to the authors to highlight the most important information and the literature on it. This is important for the comprehension of the results. Otherwise the author present the results before the background clarification.
Response 2: The function of FUM1 and HOG1 genes in general were corrected, and improved in the Introduction part for clarifying the background of this work. See lines in corrected manuscript 34-43, 60-63.
Point 3: (HOG1 genes is not described at all in this section.)
Response 3: It was improved, added. See lines in corrected manuscript 60-63.
Point 4: The authors must be more specific in the section 2.1: how they perform the inoculation of mycorrhizal fungi (Funneliformis mosseae BEG12 64 (Glomerales: Glomeraceae) and Rhizophagus intraradices BEG53 (Glomerales: Glomeraceae)) propagation? (how many cell (OD600))? Otherwise, give references of the protocol.
Response 4: Mixture of Funneliformis mosseae BEG12 (Glomerales: Glomeraceae) and Rhizophagus intraradices BEG53 (Glomerales: Glomeraceae)) propagules were used for all inoculation treatments at the same rate (16 g plot-1). The infective propagules were determined by MPN (most probable number) test following the method of Feldmann and Idzack (1994). This part of the manuscript was improved see lines in corrected manuscript 74-77.
Point 5: Just a suggestion: the authors can prepare figures which sum up the data reported in Table 1 and 2 and which makes easier for the reader the interpretation of the results.[163]
Response 5: Corrected accordingly. Table 1. is replaced with Figure 1., demonstrating nicely how influence different treatments the plant biomass. See lines in corrected manuscript 162-166. However, we should like to retain Table 2. as Table 1., because only small differences are between different treatments and table form offers better visibility. See lines in corrected manuscript 173-178.
Point 6: The authors should give more information on root exudates composition differences (chemical point of view) in inoculated and not inoculated plants.
Response 6: The highlight of the root exudates composition is improved in the manuscript see line in corrected manuscript 52-55, 238-244, 246-254.
Point 7: [259] The authors affirm that the root exudates of inoculated/not inoculated plants contain mycotoxin: this is a statement that must be demonstrated (if they affirm it). This may request a deeper analysis of root exudates content.
Response 7: We are total agreed with you, deeper analysis is required to confirm the occurrence of mycotoxin in root exudates. Our results indicate only that root exudates both from –AM and +AM plants contain mycotoxin production regulator and/or stronger mycotoxin production inducer substrates. (See lines in corrected manuscript 276-278) There is no confirmation in that work about mycotoxin in root exudatum. On the bases of our in vitro test could be measured only the change in gene expression responsible for fumonisin production. So indirect way to show the potential of occurrence of mycotoxin. In the future we would like to extend our measuring for mycotoxin as well, using HPLC, it is only the first step to show: we are working on it.
Point 8: The discussion includes literature that should be cited in the introduction,addressing the reader in the meaning of this work. Furthermore, this section should evidence the innovation of the manuscript.
Response 8: The Discussion part was improved and modified. Literatures were cited in the Introduction for clarifying the meaning of the work. See line in corrected manuscript 47-63.
Point 9: [192] typo “after 5 days, the influence” Corrected, see line in corrected manuscript 202
[172] add measures units on the numeric data
Response 9: . As we mentioned in Material and Methods, the gene expression was calculated using the ΔΔCT method (Livak and Schmittgen 2001) with Fusarium proliferatum histone H3 gene as an internal control. To determine the relative change in FUM1 and HOG1 expressions, the comparative CT (ΔΔCT) method was performed and we used the fold change unit for to measure the differential expression level of both genes among each others, at two dates. See lines in corrected manuscript 180-181.
Thank you again for the most useful and helpful critical remarks.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Mayer et al. manuscript present an interesting functional approach to characterize the effect of mycorrhizal colonization under different nutrient conditions and the impact that it has on the production of mycotoxins and the expression of genes involved in this process. The experiments are well designed and provide a basis for conducting more research on this area, like testing the direct impact on colonization by the fungal pathogen in a controlled assay co-inoculating both mycorrhiza and pathogen. Nonetheless, the paper provides data in an area that has conflicting results, and this is a valuable input. However, in general, the manuscript requires some adjustment since some of the sentences are not clear enough, some of the language is very colloquial and some sections of the manuscript could use more work and input. I attached a PDF with different notes, but I will cover some points here:
Introduction
- The introduction is very superficial, and the authors can take the opportunity to elaborate on the selection of the genes evaluated with RT qPCR. For instance, HOG gene is not spelled out and the importance of the gene is explained until the discussion and it is superficial.
- Line 35-36 Fusarium is known by its genetic and phenotypic diversity, which is also reflected in the broad host range of this pathogen.
- Line 48 is very general and should be mentioned before lines 43 - 45, which are more specific examples of the impact of arbuscular mycorrhiza
- In general, I think this section could use more work and more information, it is very light and it doesn't set up the stage for the type of work done for the paper.
Materials and Methods
- Line 68 The composition of the Long Ashton solution should be briefly mentioned even if it is a common solution in other fields.
- Line 81-82 Not sure how the concentration of root exudates was measured? and what specific components of the root exudates were quantified before adjusting the concentration.
Results
- In general, this section is very clear, some sentences need some re-organization and there is some information in the paragraph that is repetitive and it is also presented on the tables.
- Scientific binomials and other technical words should be in italics (e.g. in vitro, Rhizopus).
- Line 180 Spell out the HOG gene and address the reason for selecting this gene (maybe this explanation should be part of the introduction).
Discussion
- Line 218-219 This is a potential overstatement since root extracts were purified and tested on Fusarium pure cultures. None of the experiments looked at the interaction of Fusarium with the arbuscular mycorrhiza. Therefore, suggesting based on in vitro evidence that root exudates modify the whole rhizosphere community is an overreaching statement since the authors don't have appropriate evidence to support this.
- Discussion of the response of FUM1 and HOG to the exposure to root extracts at different time points is not covered well enough, I suggest the author going back through the discussion and develop some points more thoroughly. Especially the added effects of nutrients and the response of the plants to it.
References
- Scientific binomials should be in italics.
Comments for author File: Comments.pdf
Author Response
Response to Reviewer 2 Comments
We are greatly indebted to You for your corrections and comments on our manuscript.
I have accepted all of your corrections and your comments, let see my remarks and comments written in red.
Point 1: The introduction is very superficial, and the authors can take the opportunity to elaborate on the selection of the genes evaluated with RT qPCR. For instance, HOG gene is not spelled out and the importance of the gene is explained until the discussion and it is superficial.
Response 1: The Introduction part was improved with literatures moreover rewritten to clarify the background. The functions of FUM1 and HOG1 genes in Fusarium proliferatum were also added, See lines in corrected manuscript 34-43, 60-63.
Point 2: Line 35-36 Fusarium is known by its genetic and phenotypic diversity, which is also reflected in the broad host range of this pathogen.
Response 2: Corrected accordingly. See lines in corrected manuscript 34-36.
Point 3: Line 48 is very general and should be mentioned before lines 43 - 45, which are more specific examples of the impact of arbuscular mycorrhiza
Response 3: As I mentioned in Response 1., the Introduction was rewritten following your suggests. See lines in corrected manuscript 47-57.
Point 4: In general, I think this section could use more work and more information, it is very light and it doesn't set up the stage for the type of work done for the paper.
Response 4: The Introduction part was improved and rewritten.
See lines in corrected manuscript 30-63.
Point 5: Line 68 The composition of the Long Ashton solution should be briefly mentioned even if it is a common solution in other fields.
Response 5: It was completed, see lines in corrected manuscript 79-81.
Point 6: Line 81-82 Not sure how the concentration of root exudates was measured? and what specific components of the root exudates were quantified before adjusting the concentration.
Response 6: We followed the method of Lioussanne et al. 2009 similar to Pinior et al. 1999 and Vierheilig et al. 2003 for comparison the effects of different exudates originated from 1 g root fresh weights.
Root exudate solutions were adjusted to a ratio of 1 g root fresh weight by adding distilled water when required. On this way the effects of various root exudates remain comparable, however roots from different treatments showed significant differences in weights. (See lines in corrected manuscript 94-95.
Point 7: In general, this section is very clear, some sentences need some re-organization and there is some information in the paragraph that is repetitive and it is also presented on the tables.
Response 7: This section was improved and reorganized. See lines in corrected manuscript 173-175.
Point 8: Scientific binomials and other technical words should be in italics (e.g. in vitro, Rhizopus).
Response 8: Improved in the main text an also in References part. See lines in corrected manuscript 244, 253.
Point 9: Line 180 Spell out the HOG gene and address the reason for selecting this gene (maybe this explanation should be part of the introduction)
Response 9: The importance of HOG1 and the justification for its examination are included in the Introduction. See lines in corrected manuscript 60-63.
Point 10: Line 218-219 This is a potential overstatement since root extracts were purified and tested on Fusarium pure cultures. None of the experiments looked at the interaction of Fusarium with the arbuscular mycorrhiza. Therefore, suggesting based on in vitro evidence that root exudates modify the whole rhizosphere community is an overreaching statement since the authors don't have appropriate evidence to support this.
Response 10: We accept it, the overreaching statement was removed, moreover rewritten this part improving with new references, following your suggest. See lines in corrected manuscript 227-244.
Point 11: Discussion of the response of FUM1 and HOG to the exposure to root extracts at different time points is not covered well enough, I suggest the author going back through the discussion and develop some points more thoroughly. Especially the added effects of nutrients and the response of the plants to it.
Response 11: The Discussion part was rewritten and improved following your suggest and recommendation. See lines in corrected manuscript 227-281.
Point 12: Scientific binomials should be in italics.
Response 12: Corrected, see lines in corrected manuscript 252-252 and in References.
Thank you again for the most useful and helpful critical remarks.
Author Response File: Author Response.pdf
