The Impact of Forest Fungi on Promoting Growth and Development of Brassica napus L.

Round 1

Reviewer 1 Report

The manuscript “The influence of forest fungi on promoting growth and development of Brassica napus L.” is an interesting work corresponding to the aim and scope of the journal Agronomy (Basel). The study provides primary analysis on ectomycorrhizal and saprophytic fungi typical of forest soils: Collybia tuberosa, Clitocybe rivulousa, Laccaria laccata, Hebeloma mesophaeum, and Cyathus olla, and their influence on the growth and development of Brassica napus L.

Comments:

Abstract:

The abstract is too long. According to the instructions, it should not be longer than 200 words.

 Introduction:

Since the experiment was performed on fungi from the Basidiomycete group, more research results on these fungi should be presented in the introduction. Some of the references do not match the statements they support. On the other hand, some statements are not supported by a reference.

Material and methods:

Why did you sterilize with ethyl alcohol and sodium hypochlorite? Why both?

Why did you choose different strains? Please, explain this. Please be more specific and add information about the location of sampling (coordinates) and the type of the soil. Why was the inoculation done 28 or 56 days after the experiment? Can you explain the method for determining chlorophyll content?

Discussion:

Line 331- In this work, I am not sure that fungi H. mesopheum was used. Please, check it.

Line 378-“ The effect of C. tuberosa on rapeseed plants may be due not only to changes   377

in the number of microorganisms but also to changes in their species composition" Can you present any evidence for this assumption?

Lines 372-381- It is the replication of tables in Results, not needed. 

Line 382- Why not C. tuberose? The results of applying C. tuberose are very similar to the one you got for C. olla.

Line 385- Write the meaning of the PGPF abbreviation.

Line 387- This paper (number 45) does not corroborate this statement. Please, check.

Lines 408-410- Not needed.

Conclusion:

Revision of the Conclusions is required – wider information in shorter volume. Currently, most of the Conclusion contains very few results and many assumptions, predictions, and plans for the future. The biggest part of the Conclusion should provide results.

Lines-466-469- This is unnecessary.

Lines 471-479- This is unnecessary.

References:

-1,4,12,21,23,27,32,34,37,41,54,63,66,67-the references are incorrectly entered.

Comments for author File: Comments.docx

Author Response

Reviewer 1

Thank you for your comments and remarks based on which we have improved the manuscript.

Comments:

1. Abstract:

The abstract is too long. According to the instructions, it should not be longer than 200 words.

Response 1. The abstract was shortened.

2. Introduction:

Since the experiment was performed on fungi from the Basidiomycete group, more research results on these fungi should be presented in the introduction. Some of the references do not match the statements they support. On the other hand, some statements are not supported by a reference.

Response 2.

We have made changes to the text, corrected the citations and supplemented the information on the Basidiomycete mushrooms. Details are provided in the manuscript.

3. Material and methods: Why did you sterilize with ethyl alcohol and sodium hypochlorite? Why both?

Response 3.

This is the standard method of sterilizing plant material, especially in in vitro studies and is commonly used for different species. e.g.

 Gerszberg et al., 2015 In Vitro Cell Dev Biol Plant. 51(1): 80–87

Kamińska et al., 2021 Plant Cell, Tissue and Organ Culture 144: 295–312

Szymczyk et al., 2021 Plant Cell, Tissue and Organ Culture 147:491–502

In earlier studies, it was used to sterilize oilseed rape

4. Why did you choose different strains? Please, explain this. Please be more specific and add information about the location of sampling (coordinates) and the type of the soil.

Response 4.

In our research, we used fungi that we knew to interact with other plants. We used different strains to select the one with the greatest potential to support the growth and development of oilseed rape. We also had partial information on the metabolic activity team of some of the fungi studied. Our earlier studies in which we tested the effect of mycorrhizal fungi on the growth of oilseed rape did not bring any spectacular results. Therefore, we have made an attempt to find fungi positively influencing the growth of this plant among the Basidiomycete group. In the research, we used fungi from anthropogenically degraded soils, contaminated with heavy metals. Detailed data is described in the publication: Hrynkiewicz K, Baum C, and Leinweber P. 2010. Density, metabolic activity, and identity of cultivable rhizosphere bacteria on Salix viminalis in disturbed arable and land-fill soils. Journal of Plant Nutrition and Soil Science 173: 747–756.

5. Why was the inoculation done 28 or 56 days after the experiment? Can you explain the method for determining chlorophyll content?

Response 5.

The first measurement of chlorophyll content was made in the early stage of growth when the plants had at least four leaves (for winter oilseed rape it requires about 28 days of growth). The second measurement was performed when the rape had 8-12 leaves (2 months of growth) developed, which allowed for reliable results for all analyzed plants.  

Discussion:

6. Line 331- In this work, I am not sure that fungi mesopheum was used. Please, check it.

Response 6. Yes, you are right I have changed this information.

7. Line 378-“ The effect of tuberosa on rapeseed plants may be due not only to changes 377 in the number of microorganisms but also to changes in their species composition" Can you present any evidence for this assumption?

Response 7.

We found information on the impact of the presence of fungi on the number of other microorganisms.

Tornberg K., Bååth E., Olsson S. 2003. Fungal growth and effects of different wood decomposing fungi on the indigenous bacterial community of polluted and unpolluted soils. Biology and Fertility of Soils 37(3):190-197.

Olsson PA, Wallander H (1998) Interactions between ectomycorrhizal fungi and the bacterial community in soils amended with various primary minerals. FEMS Microbiol Ecol 27:195–205

Olsson PA, Chalot M, Bååth E, Finlay RD, Söderström B (1996b) Ectomycorrhizal mycelia reduce bacterial activity in sandy soil. FEMS Microbiol Ecol 21:77–86

8. Lines 372-381- It is the replication of tables in Results, not needed.

Response 8. I have deleted this paragraph.

 

9. Line 382- Why not tuberose? The results of applying C. tuberose are very similar to the one you got for C. olla.

Response 9.

The fungus C. ola had a positive effect on oilseed rape. The number of lateral roots and the number of leaves as well as the dry and fresh rape biomass were higher than that of C. tuberose. The fungus C. tuberose did not increase the number of microorganisms in the soil where oilseed rape grew, and in the case of C. olla, an increase in the number of fungi and bacteria was found.

10. Line 385- Write the meaning of the PGPF abbreviation.

Response 10. I have written abbreviation meaning.

11. Line 387- This paper (number 45) does not corroborate this statement. Please, check.

Response 11. Yes, I have now corrected the misquote; it should be:

Hossain, M.M.; Sultana, F.; Islam, S. Plant growth-promoting fungi (PGPF): Phytostimulation and induced systemic resistance; 2017; Vol. 2; ISBN 9789811065934.

12. Lines 408-410- Not needed.

Response 12. I have deleted this paragraph

Conclusion:

13. Revision of the Conclusions is required – wider information in shorter volume. Currently, most of the Conclusion contains very few results and many assumptions, predictions, and plans for the future. The biggest part of the Conclusion should provide results.

Response 13.

We changed the summary as suggested by the Reviewer

14. Lines-466-469- This is unnecessary.
15. Lines 471-479- This is unnecessary.

Response 14 and 15. I have deleted this paragraph

References:

-1,4,12,21,23,27,32,34,37,41,54,63,66,67-the references are incorrectly entered.

The response: I have now corrected the references

 

Author Response File: Author Response.doc

Reviewer 2 Report

The Authors selected some fungi retrieved from forest soil  and tested both in vitro and on pot-grown seedlings their potential effectiveness as plant biostimulants .  To support and explain  their results they evaluated the differential expression of BnMT1 and BnMT3 genes in root-inoculated and non-inoculated rapeseed planlets. and determined quantitatively the total amount of fungi and bacteria in the soil of inoculated  and non-inoculated pots.

The study is designed and developed according to a strictly logical idea. However, in my opinion the experimental design is not adequate to verify the hypothesis and results do not support the assumptions. As a consequence the discussion is not consequential with the results, even though it denotes an updated knowledge of the literature.  

In detail, major concerns are as it follows:

• The Authors have to explain more in detail the criterion they used in choosing the inoculant fungal species;
• M&M : in general the inoculation methods must be described more in detail, in experiments with potted seedlings did you inoculate the control with sterile agar?
•  The results of in vitro tests are dull: there is no proof of a significant stimulant effect of fungi on the biometric characteristics of the rapeseed seedlings; despite this the Authors tested the fungi on pot-grown seedlings;
• All the inoculated fungi produced IAA so apparently the ability to produce this hormone is not related with the supposed biostimulant effect;
• The amount of inoculum was very low (only two agar pieces); you did not verify the rhizosphere competence (the ability to colonize the roots) of inoculated fungi; usually to inoculate the soil in controlled experiments  researchers add the inoculum uniformly to the soil 
• you regarded the total amount of fungi and bacteria in the soil at the end of the experiments (inoculation of potted seedlings) as a proof of the effect of fungi on the rhizosphere microbioma; no qualitative determination was performed; in the era of metagenomic this method appears inadequate and misleading;
• the gene expression was a little bit difficult to explain as the effects of fungi on the biometric characteristics and the physiology of inoculated seedling was erratic: C. olla had no effect on chlorofill content and length of roots but increased the number of leaves, L. laccata improved only  the fresh and dry weight of seedlings, the other fungi had  detrimental effects; none of these results is correlated with the in vitro effect and the production of IAA; I am wondering which is the ratonale of this experiments? Despite this the Authors refer to the literature (actually they demonstrate to be updated on the state of the art) to try to support contradictory results; as a result it was very hard for me as a reader to understand the advantages in using these fungi; 
• Considering all the abovementioned results the conclusions appear very speculative as the application of these fungi as biostimulants in the field is still far from being practically possible . 

An additional critical point of the article is the graphical presentation of the results. The English test needs some minor revisions, expecially as far as the section Results is concerned (the sentences must be consequenzial). 

For other minor text editings and comments see notes in the body of the text (attached file).

Overall my opinion is reject with encouragement to submit after additional experiments and substantial re-editing.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

Thank you for your comments and remarks based on which we have improved the manuscript.

The Authors selected some fungi retrieved from forest soil and tested both in vitro and on pot-grown seedlings their potential effectiveness as plant biostimulants.  To support and explain their results they evaluated the differential expression of BnMT1 and BnMT3 genes in root-inoculated and non-inoculated rapeseed plants. and determined quantitatively the total amount of fungi and bacteria in the soil of inoculated and non-inoculated pots.

1. The study is designed and developed according to a strictly logical idea. However, in my opinion, the experimental design is not adequate to verify the hypothesis and the results do not support the assumptions. As a consequence, the discussion is not consequential with the results, even though it denotes an updated knowledge of the literature.

Response 1.

In our opinion, we have obtained preliminary information on the influence of selected fungi on the growth of oilseed rape in vitro and in a pot experiment. In addition, we found that the presence of some of the analyzed fungi positively affects not only the oilseed rape but also the microorganisms in the rhizosphere. These data form the basis for further research.

In detail, major concerns are as follows:

2. The Authors have to explain more in detail the criterion they used in choosing the inoculant fungal species;

Response 2. An explanation of the choice of material is included in the manuscript.

 

3. M&M : in general the inoculation methods must be described more in detail, in experiments with potted seedlings did you inoculate the control with sterile agar?

Response 3.

We have completed the information on inoculation. Actually, in the control variant, a sterile agar medium of the same size was used for inoculation. The amount of inoculum was very small. In the preliminary in vitro tests, each seedling was inoculated, but due to the very strong growth of the mycelium, the inoculum was limited to 1 agar piece per 2 seedlings (details in the text).

 

 

4. The results of in vitro tests are dull: there is no proof of a significant stimulant effect of fungi on the biometric characteristics of the rapeseed seedlings; despite this, the Authors tested the fungi on pot-grown seedlings;

Response 4.

I understand that for the Reviewer the results of such simple tests may be boring, but the positive effect of fungi on the growth and development of rape seedlings is noticeable, which justifies the continuation of the research.

 

5. All the inoculated fungi produced IAA so apparently the ability to produce this hormone is not related to the supposed biostimulant effect;

Response 5.

In our opinion, the studies suggest that IAA production is not the only factor that has a growth-promoting effect. Of course, we will examine the ability of the fungi studied to produce other hormones or siderophores.

6. The amount of inoculum was very low (only two agar pieces); you did not verify the rhizosphere competence (the ability to colonize the roots) of inoculated fungi; usually to inoculate the soil in controlled experiments researchers add the inoculum uniformly to the soil

Response 6.

The results described in the manuscript are the result of preliminary investigations that led to the selection of fungi that stimulate the growth of oilseed rape. The results are not spectacular but were reliably carried out and provide preliminary observations, the results of which were included in the publication on "Colonization of rapeseed roots by Trichoderma viride", which also shows the involvement of hydrophobins in this process Znajewska et al. 2021 manuscript in progress). So, we see the sense of preliminary investigations.

As for the inoculum used, it was small in comparison with the inoculum we used for experiments under the conditions of field experiments conducted with the above-mentioned fungi for Miscantahus x giganteus. We optimized the content of the inoculum, the larger amount resulted in excessive growth of the fungi in the growth chamber, which had a negative effect on the rape.

In further studies, we want to check microscopically and molecular biologically whether the roots of the rape are colonized by selected fungi.

7. you regarded the total amount of fungi and bacteria in the soil at the end of the experiments (inoculation of potted seedlings) as proof of the effect of fungi on the rhizosphere microbioma; no qualitative determination was performed; in the era of metagenomic this method appears inadequate and misleading;

Response 7.

As mentioned earlier, we have done preliminary research that we will continue in the field, and here we plan to do a qualitative analysis of the microorganisms (under real conditions). However, quantification is not a mistake, it just goes less in-depth.

 

8. the gene expression was a little bit difficult to explain as the effects of fungi on the biometric characteristics and the physiology of inoculated seedling was erratic: C. olla had no effect on chlorophyll content and length of roots but increased the number of leaves, L. laccata improved only the fresh and dry weight of seedlings, the other fungi had detrimental effects; none of these results is correlated with the in vitro effect and the production of IAA; I am wondering which is the ratonale of this experiments? Despite this the Authors refer to the literature (actually they demonstrate to be updated on the state of the art) to try to support contradictory results; as a result, it was very hard for me as a reader to understand the advantages of using these fungi;

Response 8.

These preliminary research results indeed require further analysis to provide clearer evidence of the beneficial effects of L. laccata and C.olla on oilseed rape growth and development.

 

9. Considering all the above mentioned results the conclusions appear very speculative as the application of these fungi as biostimulants in the field is still far from being practically possible

Response 9

We hope that our next results will confirm the possibility of using L. laccata in particular as a component of a biopreparation to support and stimulate the growth of oilseed rape.

 

 

10. An additional critical point of the article is the graphical presentation of the results.

Response 10.

The graphical representation of the results has been changed.

 

11. The English test needs some minor revisions, expecially as far as the section Results is concerned (the sentences must be consequenzial).

Response 11.

 Linguistic proofreading is done.

 

 

 

Author Response File: Author Response.doc

Reviewer 3 Report

Dear Authors,

I have an opportunity to review manuscript entiled:” The influence of forest fungi on promoting growth and development of Brassica napus L.” submitted to Agronomy MDPI journal.

Authors analysed the effect of selected forest soil fungi on the growth and development of Brassica napus L. seedlings.

For the most important and valuable data I consider expression of the metallothioneins B. napus (BnMT1-BnMT3) varied depending on the fungal species. The presence of C. olla significantly in-creased BnMT2 expression in oilseed rape. It was found that BnMT1 expression increased and BnMT3 transcripts decreased in plants growing in the presence of L. laccata.

The introduction and aim of the study are efficient background to analysing results; Moreover, material and methods are accurately described; Please be aware of using actin gene as a reference - in plant-microbe interaction research it is not the most ‘stable choice’, despite using in many research-paper.

I suggest to improved quality of Figure 4;

Very interesting as well as very well written is discussion chapter

In conclusion we can know the authors plan, therefore I suggest to add/widen future prospects coming from Authors analyses to  make the result ‘more visible’ to the wider readers group.

Sincerely

Author Response

Reviewer 3

Thank you for your comments and remarks based on which we have improved the manuscript.

I have an opportunity to review the manuscript entitled:” The influence of forest fungi on promoting growth and development of Brassica napus L.” submitted to Agronomy MDPI journal.

The authors analysed the effect of selected forest soil fungi on the growth and development of Brassica napus L. seedlings.

For the most important and valuable data, I consider the expression of the metallothioneins B. napus (BnMT1-BnMT3) varied depending on the fungal species. The presence of C. olla significantly increased BnMT2 expression in oilseed rape. It was found that BnMT1 expression increased and BnMT3 transcripts decreased in plants growing in the presence of L. laccata.

1. The introduction and aim of the study are the efficient background to analysing results; Moreover, material and methods are accurately described; Please be aware of using actin gene as a reference - in plant-microbe interaction research it is not the most ‘stable choice’, despite using in many research-paper.

Response 1.

Thank you for this important suggestion. In future research, we will use a different reference gene, e.g. 5S rRNA, and we intend to carry out research using the real-time PCR technique.

2. I suggest to improved quality of Figure 4;

Response 2.

We have corrected Figure 4 as suggested by the Reviewer.

Very interesting as well as very well written is discussion chapter

In conclusion, we can know the author's plan, therefore I suggest to add/widen future prospects coming from the Authors analyses to make the result ‘more visible’ to the wider readers group.

Response:

We have corrected the summary as suggested by the Reviewer.

“We intend to continue research to characterize microorganisms in detail. The research will focus on checking the protective role of fungi, especially L. laccata and C. ola against rape pathogens. The long-term goal is to develop a biopreparation that would support the growth of oilseed rape under conditions of dehydration stress and pathogen attack.”

More changes are included in the manuscript.

Author Response File: Author Response.doc

Round 2

Reviewer 2 Report

I have to admit that after the first round of revision the quality of the article has been consistently improved. The Authors addressed properly all the observations of the reviewers. In particular I appreciated the new version of the Introduction and Discussion of the results so I changed my mind.  I remain firm in my opinion that the total amount of microrganisms in the soil is not informative, however as the Authors argued properly on this I respect their opinion. Overall my final opinion is acceptance. I attach the text with very minor corrections (evidenced). 

Comments for author File: Comments.pdf