Mortierella elongata Increases Plant Biomass among Non-Leguminous Crop Species

Round 1

Reviewer 1 Report

Review of “Evaluating the impacts of Mortierella elongate isolates on plant growth”

The authors have conducted an experiment by isolating three fungal strains from forests and inoculating them into many crop species to note their effects on plant performance.  While the concept of experimentally determining the effects of fungal strains on performance is compelling with potential important implications, I have a few issues that should likely be addressed.  First, the authors give no background or understanding about the actual fungal strains being used, as well as why they isolated these strains from forests to inoculate into crop species in the greenhouse.  Do these strains commonly associate with these crops?  Why these three isolates? Etc.

Moreover, I don’t really understand the importance (and rationale) for isolating strains from the field (in forest soils, no less) and then inoculating them into agricultural crop species which have likely not been exposed to these strains under field conditions.  What important conclusions can we draw from this? 

Lastly, it looks like the authors ran a one-way ANOVA for every species without accounted for multiple tests run (for multiple species).  If this is the case, a post-hoc p-value correction must be employed (e.g., False Discovery Rate or Bonferroni correction).  Otherwise, there can be no conclusions drawn about differences among species.  The authors don’t specifically test species-differences (they briefly mention this in the Results and Discussion), but they should. 

Overall, I would recommend beefing up a few sections, including the Introduction, Discussion, and parts of the Methods.  The current draft is pretty skeletal.

Minor and housekeeping comments are below

Abstract

Line 16: I would say “of a narrow range of host species”

Giving magnitudes of the effects rather than just directions (e.g., PMI 624 and PMI 93 on average increased/decreased plant growth by XX%.”

Introduction

Line 31: Saprotrophs and root endophytes to which species?

Line 46: This is a really long sentence, and the species names and parenthetical references really break the sentence up into disparate pieces.  Consider rewriting.

Line 53: If you are going to give an example of life phases (senescence), list more than one, as well as list some of the environmental conditions.

Materials and Methods

Line 63: Mean to be “PMI 77, PMI 93, and PMI 624.”

Is there any “natural history” or other additional information about these three isolates?  There is no background about any of these, and thusly it doesn’t read as compelling.  Why these three isolates? 

In addition, I recognize my ecological bias, but I don’t really understand the importance (and rationale) for isolating strains from the field (in forest soils, no less) and then inoculating them into agricultural crop species which have likely not been exposed to these strains under field conditions.  What important conclusions can we draw from this? 

Line 67: I would suggest being more detailed about inoculation techniques rather than citing another study here.

Line 68: Use scientific names for these species

Line 71: This surface sterilization technique is not too common (particularly without any ethanol washes).  Please cite additional studies for this method.

Did you confirm that this surface sterilization method worked?  (e.g., Kaewkla & Franco 2013, Griffin et al. 2019). 

Line 74: Were the pots assembled in a random blocked design?

Line 79: I see a potential big problem here: It looks like the authors ran a one-way ANOVA for every species without accounted for multiple tests run (for multiple species).  If this is the case, a post-hoc p-value correction must be employed (e.g., False Discovery Rate or Bonferroni correction).

Results and Discussion

I highly recommend that the authors separate these two sections and dig a little deeper.

The Agronomy journal author instructions clearly state that these two sections should be separate.

Line 83: Giving the magnitude will greatly help to make the results accessible and more compelling to the reader.  E.g., Mortierella-inoculated watermelon increased plant height by XX% compared to un-inoculated controls.  In addition, this will also break-up and address another concern that I had: the reader is almost bombarded with all results one right after the other in a way that is not easily digestible.  I would suggest dividing the results either A. by plant performance metrics (e.g., height, leaf area, and biomass), or B. by species using subheadings.  This degree of organization will be better for the reader and emphasize the important take-home messages of the data.

In addition, the authors leave a lot to be desired in the Discussion (which is why they should write an independent section).  There are many potentially important topics to include, such as host-specificity, the endophyte-pathogen continuum, potential sources of endophytes (vertically vs horizontally transmitted), etc. etc. etc.  There are quite a few (and good) recent reviews on endophytes to draw inspiration from (e.g., Christian et al. 2015, Eberl et al. 2019, Harrison & Griffin 2020). 

All plant species mentioned should use scientific names.

Line 105: Please list number of replicates.

Author Response

Reviewer1:
Review of “Evaluating the impacts of Mortierella elongata isolates on plant growth”

1) The authors have conducted an experiment by isolating three fungal strains from forests and inoculating them into many crop species to note their effects on plant performance. While the concept of experimentally determining the effects of fungal strains on performance is compelling with potential important implications, I have a few issues that should likely be addressed. First, the authors give no background or understanding about the actual fungal strains being used, as well as why they isolated these strains from forests to inoculate into crop species in the greenhouse. Do these strains commonly associate with these crops? Why these three isolates? Etc.
Response: The clarification was included in the text (“Recent studies have shown that M. elongata is common in agricultural and forest ecosystems, indicating that M. elongata may play an important role in these ecosystems [1,26]. For example, M. elongata accounted for over 15% of total sequences in organic agricultural soils [1]. Given that Mortierella is dominant in soil and rhizosphere niches, and does not show pathogenic tendencies, some Mortierella species may have the potential to provide beneficial activities across diverse ecosystem types. However, no study yet to characterize the host range of any Mortierella isolate.”).

2) Moreover, I don’t really understand the importance (and rationale) for isolating strains from the field (in forest soils, no less) and then inoculating them into agricultural crop species which have likely not been exposed to these strains under field conditions. What important conclusions can we draw from this?
Response: The rationale is addressed in the text (“Based on the broad incidence of M. elongata across geographic ranges and soil types, we hypothesize that most M. elongata isolates can be considered as host-generalists, that are likely to improve the growth of a range of plant species including trees and crops. Further, we expect that plant growth promoting ability will vary between isolates. In this study, we performed paired plant-fungal bioassays to determine the ability of M. elongata isolates in growth promotion across different plant species. We selected M. elongata isolates from different geographic and ecological habitats. For example, M. elongata PMI 93 was isolated from Populus deltoides in NC, USA and may promote Populus growth by manipulating plant defense [2]. M. elongata PMI 77 was isolated from soil in NC and has the ability to provide C and N sources for host plants [7], and it can also interact with bacteria to improve each other’s growth and plant health [13,27]. M. elongata PMI 624 was isolated from P. trichocarpa in CA.”).

3) Lastly, it looks like the authors ran a one-way ANOVA for every species without accounted for multiple tests run (for multiple species). If this is the case, a post-hoc p-value correction must be employed (e.g., False Discovery Rate or Bonferroni correction). Otherwise, there can be no conclusions drawn about differences among species. The authors don’t specifically test species-differences (they briefly mention this in the Results and Discussion), but they should.
Response: We used a one-way analysis (ANOVA) with a Tukey test for post-hoc comparisons to analyze data variances at P < 0.05. The clarification is included in Line 119-121.

4) Overall, I would recommend beefing up a few sections, including the Introduction, Discussion, and parts of the Methods. The current draft is pretty skeletal.
Response: Thanks for your comments. We have substantially added to the introduction and discussion to provide more background and context, and have expanded the methods.

Minor and housekeeping comments are below

Abstract

5) Line 16: I would say “of a narrow range of host species”
Response: The change was made according to the reviewer’s suggestion (Line 19)

6) Giving magnitudes of the effects rather than just directions (e.g., PMI 624 and PMI 93 on average increased/decreased plant growth by XX%.”
Response: We have modified these statements to include the specific magnitudes as suggested (“M. elongata isolates PMI 624 and PMI 93 significantly increased the plant height, leaf area, or plant dry weight of watermelons (Citrullus lanatus), corn (Zea mays), tomato (Solanum lycopersicum), and squash (Cucurbita) on average by about 33.9% compared to PMI 77 did by 14.1%. No significant impacts were observed for any isolate on the growth of okra (Abelmoschus esculentus) or soybean (Glycine max). To the contrary, Glycine max exhibited a significant decrease of 30.6% in height after the inoculation of M. elongata PMI 77.”)

Introduction

7) Line 31: Saprotrophs and root endophytes to which species?
Response: We have clarified it (“Mortierella elongata, a member of the early-diverging Mortierellomycota, is a cosmopolitan fungal species that is often isolated from soils or roots.”).

8) Line 46: This is a really long sentence, and the species names and parenthetical references really break the sentence up into disparate pieces. Consider rewriting.
Response: The sentence was restructured accordingly (“Mortierella may function as a saprotroph that drives soil C cycling [8–10], P dissolution and immobilization [11,12], lipid metabolism [13], and chitin degradation [13,14].” )

9) Line 53: If you are going to give an example of life phases (senescence), list more than one, as well as list some of the environmental conditions.
Response: Good point. We have deleted the example (senescence) to make them consistent (Line 67).

Materials and Methods

10) Line 63: Mean to be “PMI 77, PMI 93, and PMI 624.”
Response: We have corrected. Thanks.

11) Is there any “natural history” or other additional information about these three isolates? There is no background about any of these, and thusly it doesn’t read as compelling. Why these three isolates?
Response: We have supplemented couples of sentences to state the relevance of these three isolates in plant growth in the introduction (“Based on the broad incidence of M. elongata across geographic ranges and soil types, we hypothesize that most M. elongata isolates can be considered as host-generalists, that are likely to improve the growth of a range of plant species including trees and crops. Further, we expect that plant growth promoting ability will vary between isolates. In this study, we performed paired plant-fungal bioassays to determine the ability of M. elongata isolates in growth promotion across different plant species. We selected M. elongata isolates from different geographic and ecological habitats. For example, M. elongata PMI 93 was isolated from Populus deltoides in NC, USA and may promote Populus growth by manipulating plant defense [2]. M. elongata PMI 77 was isolated from soil in NC and has the ability to provide C and N sources for host plants [7], and it can also interact with bacteria to improve each other’s growth and plant health [13,27]. M. elongata PMI 624 was isolated from P. trichocarpa in CA.”).

12) In addition, I recognize my ecological bias, but I don’t really understand the importance (and rationale) for isolating strains from the field (in forest soils, no less) and then inoculating them into agricultural crop species which have likely not been exposed to these strains under field conditions. What important conclusions can we draw from this?

Response: The rationale has been included in the main text (Line 69-85).

13) Line 67: I would suggest being more detailed about inoculation techniques rather than citing another study here.
Response: We have provided more details about inoculation techniques (Line 97-100).

14) Line 68: Use scientific names for these species
Response: We have added the scientific names for these species (Line 104-105).

15) Line 71: This surface sterilization technique is not too common (particularly without any ethanol washes). Please cite additional studies for this method.
Response: We have cited additional two studies (Line 108).

16) Did you confirm that this surface sterilization method worked? (e.g., Kaewkla & Franco 2013, Griffin et al. 2019).
Response: We confirmed this surface sterilization method worked before conducting this bioassay.

17) Line 74: Were the pots assembled in a random blocked design?
Response: Yes, they were. We have supplemented this information (Line 113).

18) Line 79: I see a potential big problem here: It looks like the authors ran a one-way ANOVA for every species without accounted for multiple tests run (for multiple species). If this is the case, a post-hoc p-value correction must be employed (e.g., False Discovery Rate or Bonferroni correction).
Response: We used a one-way analysis of variance (ANOVA) with a Tukey test for post-hoc comparisons to analyze data variances at P < 0.05. The method was included in Line 120-121.

Results and Discussion

19) I highly recommend that the authors separate these two sections and dig a little deeper. The Agronomy journal author instructions clearly state that these two sections should be separate.
Response: In this revision, we have separated this section into results and discussion.

20) Line 83: Giving the magnitude will greatly help to make the results accessible and more compelling to the reader. E.g., Mortierella-inoculated watermelon increased plant height by XX% compared to un-inoculated controls. In addition, this will also break-up and address another concern that I had: the reader is almost bombarded with all results one right after the other in a way that is not easily digestible. I would suggest dividing the results either A. by plant performance metrics (e.g., height, leaf area, and biomass), or B. by species using subheadings. This degree of organization will be better for the reader and emphasize the important take-home messages of the data.
Response: We have rewritten the results and divided the results by plant performance metrics using subheadings.

21) In addition, the authors leave a lot to be desired in the Discussion (which is why they should write an independent section). There are many potentially important topics to include, such as host-specificity, the endophyte-pathogen continuum, potential sources of endophytes (vertically vs horizontally transmitted), etc. etc. etc. There are quite a few (and good) recent reviews on endophytes to draw inspiration from (e.g., Christian et al. 2015, Eberl et al. 2019, Harrison & Griffin 2020).
Response: Thanks for your suggestions. We have added the topics of host-specificity and the endophyte-pathogen continuum into our discussion.

22) All plant species mentioned should use scientific names.
Response: In this revision, scientific names are provided for all plant species.

23) Line 105: Please list number of replicates.
Response: Done.

Author Response File: Author Response.docx

Reviewer 2 Report

This contribution is original and analyses an interesting agronomical topic, providing evidence that the Mucoromycota fungus Mortierella elongata can be either a beneficial generalist increasing the growth of some crops or a fungal specialist; depending on the inoculated strains. This topic has not been studied so far, despite its potential agronomic importance; and then my recommendation is to publish it, after doing some minor corrections:

1. The title is very broad in scope, it should be changed to a more specific one, e. gr. Mortierella elongata strains can act either as a beneficial generalist or fungal specialist on crop growth.
2. It would be interesting in the introduction to include some information of the relevance of Mortierella in bioremediation processes due to its environmental relevance, e.gr. Horel & Schiewer (2020); Cui et al. (2017); Li et al. (2017).

• In the material and methods section it seems that the statistical part has to be carefully checked. It is mentioned that it was used one-way analysis of variance (ANOVA) which is used to determine whether there are any statistically significant differences between the means of three or more independent (unrelated) groups. However, in the Figure 1 there are letters showing differences between treatments. Did the authors use a mean comparison test as Tukey? Please clarify this, the statistical analysis as currently shown, lack of information of specific the statistical method used.

1. Please clarify which were the measure of dispersion used in the data analysis presented in the Figure 1, was standard deviation? Standard error of the mean?
2. In the legend of figure 1 it has to be clearly explained why there are some blank spaces.
3. The authors concluded that “… our study showed that PMI 624 and PMI 93 have more promoting effects on plant growth than PMI 77…”. This statement is inaccurate, actually what really happened is that the authors found that the tested strains have specific promoting effects plant growth depending on the inoculated crops. Then, this has to be changed.

• Despite the fact that plant growth of crops is important, the most important factor from the agronomic point of view is the crop yield. Then in the conclusions it has be include the notion that in future studies it will be compulsory to evaluate the effect of inoculation with Mortierella strains in crop yield.

Please check carefully the English spelling and grammar thoroughly the text, there are some minor details which have to be checked by the authors, e.gr.

In the line 78 where it says “… study of (Orgaz et al. 2005)…” it should say  “… study of Orgaz et al. (2005)…”

In the fourth column in the body of the Figure 1 where it says “… suqash…” it should say “… squash…”

Author Response

Reviewer2

This contribution is original and analyses an interesting agronomical topic, providing evidence that the Mucoromycota fungus Mortierella elongata can be either a beneficial generalist increasing the growth of some crops or a fungal specialist; depending on the inoculated strains. This topic has not been studied so far, despite its potential agronomic importance; and then my recommendation is to publish it, after doing some minor corrections:

• The title is very broad in scope, it should be changed to a more specific one, e. gr. Mortierella elongata strains can act either as a beneficial generalist or fungal specialist on crop growth.

Response: Thanks for your kind suggestion. We have modified the title into “Mortierella elongata promotes plant biomass in diverse non-leguminous crops”

It would be interesting in the introduction to include some information of the relevance of Mortierella in bioremediation processes due to its environmental relevance, e.gr. Horel & Schiewer (2020); Cui et al. (2017); Li et al. (2017).

Response: Yes, agree. We have added this interesting point in the introduction (Line 58-59).

In the material and methods section it seems that the statistical part has to be carefully checked. It is mentioned that it was used one-way analysis of variance (ANOVA) which is used to determine whether there are any statistically significant differences between the means of three or more independent (unrelated) groups. However, in the Figure 1 there are letters showing differences between treatments. Did the authors use a mean comparison test as Tukey? Please clarify this, the statistical analysis as currently shown, lack of information of specific the statistical method used.

Response: We are sorry not to clarify the statistical analysis. We used a one-way analysis (ANOVA) with a Tukey test for post-hoc comparisons to analyze data variances at P < 0.05 (Line 120-121).

Please clarify which were the measure of dispersion used in the data analysis presented in the Figure 1, was standard deviation? Standard error of the mean?

Response: We are sorry to miss it. We supplemented “Vertical bars represent the standard errors of three replicates.” in the figure legend.

In the legend of figure 1 it has to be clearly explained why there are some blank spaces.

Response: We collected data of plant height of all examined plant species and collected the data of leaf area and plant dry weight from most of the plant species. We have re-formatted the figure 1 to avoid the confusion.

The authors concluded that “… our study showed that PMI 624 and PMI 93 have more promoting effects on plant growth than PMI 77…”. This statement is inaccurate, actually what really happened is that the authors found that the tested strains have specific promoting effects plant growth depending on the inoculated crops. Then, this has to be changed.

Response: I have modified this statement accordingly (Line 197-198).

Despite the fact that plant growth of crops is important, the most important factor from the agronomic point of view is the crop yield. Then in the conclusions it has be include the notion that in future studies it will be compulsory to evaluate the effect of inoculation with Mortierella strains in crop yield.

Response: Good point. We have added this statement at the end of conclusion (Line 211-212).

Please check carefully the English spelling and grammar thoroughly the text, there are some minor details which have to be checked by the authors, e.gr.

In the line 78 where it says “… study of (Orgaz et al. 2005)…” it should say  “… study of Orgaz et al. (2005)…”

Response: We have corrected it (Line 117).

In the fourth column in the body of the Figure 1 where it says “… suqash…” it should say “… squash…”

Response: Thanks for pointing it out. We have corrected it.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Please see attached comments.

Comments for author File: Comments.docx

Author Response

Please see the attachment.

Author Response File: Author Response.docx