Co-Inoculation of an Endophytic and Arbuscular Mycorrhizal Fungus Improve Growth and Yield of Helianthus tuberosus L. under Field Condition
Round 1
Reviewer 1 Report
In the manuscript “Co-inoculation of endophytic fungi and arbuscular mycorrhizal fungi improve growth and yield of Helianthus tuberosus L. under field condition” authors described the effect of co-inoculation of endophytic and arbuscular mycorrhizal fungi on yield production and the growth parameters of sunchoke.
Authors have managed to show that growth-promoting effect of the co-inoculation was greater than those of the chemical fertilizer.
The manuscript in current form presents sufficient novelty, but some questions should be specified to confirm main conclusions. The methods are sufficiently documented and the statistical methods are correctly applied.
Minor comments
In the text authors used abbreviation for endophytic fungi such as Ef. It seems better to use capital letters such as EF, because in the presented form Ef looks similar with a short form of the Latin name.
Page 2, line 63. Please use Italics for the Latin name of plants.
Page 5, line 198. You need to separate the 10%HCl.
Page 11, line 394. Authors assumed that co-inoculation of both EF and AMF improved sunchoke production better than a single inoculation of either one of them. However, the most parameters were similar between co-inoculation of both EF and AMF and AMF single inoculation. Probably it is better to re-phrase this sentence.
Author Response
Point 1: In the manuscript “Co-inoculation of endophytic fungi and arbuscular mycorrhizal fungi improve growth and yield of Helianthus tuberosus L. under field condition” authors described the effect of co-inoculation of endophytic and arbuscular mycorrhizal fungi on yield production and the growth parameters of sunchoke.
Authors have managed to show that growth-promoting effect of the co-inoculation was greater than those of the chemical fertilizer.
The manuscript in current form presents sufficient novelty, but some questions should be specified to confirm main conclusions. The methods are sufficiently documented and the statistical methods are correctly applied.
Response 1: We are grateful for your consideration of this manuscript, and are also very much appreciate your suggestions, which have been very helpful in improving this manuscript.
Point 2: In the text authors used abbreviation for endophytic fungi such as Ef. It seems better to use capital letters such as EF, because in the presented form Ef looks similar with a short form of the Latin name.
Response 2: We have changed the “Ef” to “EPF” throughout the revised manuscript.
Point 3: Page 2, line 63. Please use Italics for the Latin name of plants.
Response 3: Thank you for pointing this out. We have changed the “Verbascum lychnitis” to “Verbascum lychnitis”.
Point 4: Page 5, line 198. You need to separate the 10%HCl.
Response 4: In our study, we followed a modified method described by Koske and Gemma (1989) which 1%HCl solution was used for immersed the root.
Point 5: Page 11, line 394. Authors assumed that co-inoculation of both EF and AMF improved sunchoke production better than a single inoculation of either one of them. However, the most parameters were similar between co-inoculation of both EF and AMF and AMF single inoculation. Probably it is better to re-phrase this sentence.
Response 5: We have revised this sentence in the manuscript on page 10 lines 406-408
“Although, a single inoculation of either EPF or AMF had a significant increase in sunchoke yield. However, plants treated with co-inoculation of both fungi could improve the highest sunchoke yields, suggesting efficient plant growth promotion due to the synergistic effect of EPF and AMF.”
Reviewer 2 Report
This work investigated the potential use of co-inoculation of an EPF strain and an AMF strain could improve plant growth parameters than those of the uninoculated plants. Furthermore, the co-inoculation of EPF and AMF is more beneficial to promote sunchoke yields production than a single inoculation of either one of them. This work clearly demonstrated that synergistic effects of EPF and AMF could promote the yield of sunchoke under field conditions. The findings of this research will further expand people's understanding of the role of EPF and AMF in plant-microorganisms interaction, and also provide a good theoretical basis for the application of EPF and AMF instead of chemical fertilizer or reducing the using of chemical fertilizer. In general, the writing of this paper is reasonable and credible. However, there are some problems that the author should consider and revise:
The line 15 “Endophytic fungi (Ef)” should be “Endophytic fungi (EPF)”, please check!
The line 31 “Helianthus tuberosus L. (Jerusalem artichoke or sunchoke) is a plant native to North America, which belongs to the Asteraceae family.” Should be “Helianthus tuberosus L. (Jerusalem artichoke or sunchoke) is a native plant to North America, which belongs to the Asteraceae family.”.
Line35-38: These two sentences need to be supplemented with references.
Line45: Please replace “producing plant growth hormones” with “promoting plant hormone production”.
The line 63 “Ef could help the Verbascum lychnitis seedlings” shouldn be “EPF could help the Verbascum lychnitis seedlings”.
The line 77 “sterilized sorghum seeds” was how to treatment ?
Is the initial root colonization of Ef and AMF the same for each treatment?
Why can't the Fresh mycelia of strain NMS1.5 be inoculated directly into sunchoke but sorghum seeds?
The line 88 “Then transferring it onto sterile soil in sterile petri dishes……” should be “then transferring them onto sterile soil in sterile petri dishes……”.
The line 214“means at P≤0.05.” should be “means at P≤0.05.
For the line 442 and 452, suggesting further deeper to explain why the AMF and EPF have more efficient function under the field condition than the greenhouse condition.
The results is mixed with discussion. Such as Line 235-237 and Line 291-301.
Figure 5.: Please check whether the letters above the bars of total chlorophyll of T1 and T3 are correct.
Author Response
Point 1: This work investigated the potential use of co-inoculation of an EPF strain and an AMF strain could improve plant growth parameters than those of the uninoculated plants. Furthermore, the co-inoculation of EPF and AMF is more beneficial to promote sunchoke yields production than a single inoculation of either one of them. This work clearly demonstrated that synergistic effects of EPF and AMF could promote the yield of sunchoke under field conditions. The findings of this research will further expand people's understanding of the role of EPF and AMF in plant-microorganisms interaction, and also provide a good theoretical basis for the application of EPF and AMF instead of chemical fertilizer or reducing the using of chemical fertilizer. Ingeneral, the writing of this paper is reasonable and credible. However, there are some problems that the author should consider and revise:
Response 1: We appreciate the positive feedback from the reviewer. We would also like to thank the reviewer for thoughtful comments and constructive suggestions, which help improve the quality of this manuscript.
Point 2: The line 15 “Endophytic fungi (Ef)” should be “Endophytic fungi (EPF)”, please check!
Response 2: We have changed the “Ef” to “EPF” throughout the revised manuscript.
Point 3: The line 31 “Helianthus tuberosus L. (Jerusalem artichoke or sunchoke) is a plant native to North America, which belongs to the Asteraceae family.” Should be “Helianthus tuberosus L. (Jerusalem artichoke or sunchoke) is a native plant to North America, which belongs to the Asteraceae family.”.
Response 3: Thank you for pointing this out. We have changed this in the revised manuscript.
Point 4: Line35-38: These two sentences need to be supplemented with references.
Response 4: The references have been added in the revised manuscript as suggested.
Point 5: Line45: Please replace “producing plant growth hormones” with “promoting plant hormone production”.
Response 5: The change has been made as suggested.
Point 6: The line 63 “Ef could help the Verbascum lychnitis seedlings” shouldn be “EPF could help the Verbascum lychnit is seedlings”.
Response 6: The correction has been made as suggested.
Point 7: The line 77 “sterilized sorghum seeds” was how to treatment ?
Is the initial root colonization of Ef and AMF the same for each treatment?
Why can't the Fresh mycelia of strain NMS1.5 be inoculated directly into sunchoke but sorghum seeds?
Response 7: To sterilize sorghum seeds, the seeds were washed and immersed in tap water overnight. Then, they were boiled at 100 ºC, for 10 min prior to sterilization by autoclaving at 121 ºC, 15 psi, for 1 h before use.
Yes, the initial root colonization of Ef and AMF is the same for each treatment as shown in Figure 2.
Fresh mycelia of strain NMS1.5 could not be inoculated directly into sunchoke because pre-grown mycelia in sorghum seeds allow efficient growth and survival when applied into the soils.
Point 8: The line 88 “Then transferring it onto sterile soil in sterile petri dishes……” should be “then transferring them onto sterile soil in sterile petri dishes……”.
Response 8: The correction has been made as suggested.
Point 9: The line 214“means at P≤0.05.” should be “means at P≤0.05.
Response 9: Done.
Point 10: For the line 442 and 452, suggesting further deeper to explain why the AMF and EPF have more efficient function under the field condition than the greenhouse condition.
The results is mixed with discussion. Such as Line 235-237 and Line 291-301.
Response 10: Thank you for this suggestion. We have discussed this point in the revised manuscript on page 11-12 lines 479-486.
“This is probably because agricultural fields were, in general, regularly disturbed by tillage, naturally-altered environmental conditions and fertilization, all of which could help strengthen fungal adaptability and enhance fungal development in the fields more than when they were subjected to non-disturbed environment under the greenhouse [42, 43]. Moreover, under field conditions, the fungi could better expand its hyphal network and rapidly colonize plant roots, allowing them to occupy a large proportion of diverse neighbouring roots. This, thus, affected the colonization, growth ability and survival of other fungi that naturally occur around the roots [44, 45].”
Point 11: Figure 5.: Please check whether the letters above the bars of total chlorophyll of T1 and T3 are correct.
Response 11: We have re-checked the letters. They are all correct.
Reviewer 3 Report
The general set-up of the study is interesting and the results promising to reduce the use of fertilizers in sunchoke production. However, the number of replicates (4) is far from a good study to compare growth and yield between different treatments. Furthermore, despite of being reviewed already once the manuscript still contains a number of mistakes and a highly repetitive writing style which makes it rather difficult to read. I strongly recommend to get a native speaker to polish the manuscript. Below you will find a number of suggestions for correction, but the list is not exhaustive.
Title:
I suggest to be more precise:
Co-inoculation of an endophytic and arbuscular mycorrhizal fungus improves growth and yield....
Line 23 delete note that
Line 50 protection
Line 51-54 Please put it more simple - while AM fungi only colonizae roots some Ef are also capable to colonize the aboveground parts....
Line 73 The endophytic...
Line 74 which enhanced growth
Line 89 transferred
Line 96 Delete then
Line 218 Effects of teh Ef... of AMF in oder to ensure non-antagonistic
Line 220 Delete The results showed that
Line 223 delete the
Line 224 delete an
Line 230 dele an before the two fungi
Line 233 treatments (delete conditions) is presented. Delete the results showed and reframe the sentence
Line 235 This means that the native agricultural soil also contained AMF and Ef. We cannot rule out that these are different species than the once we used for inoculation. However, in terms of AMF and Ef colonization all treatments....
Line 252 Delete the sentence - competition for nutrients is also a type of antagonism
Line 261 replace . This suggested by indicating
Line 264-269 this should be addressed in the discussion not the results
Line 270-279 This paragraph is irritating at this point - please combine some microscopic pictures with figure 1 and delete Figure 2, 3 and 4 and write a concise text before comparing the colonization rates of the different treatements.
Line 298 delete was
Line 306 delete The results showed that
Line 315 this suggests
Line 316 delete an in front of the two fungi
Line 317 replace in which by and
Line 323 presents
Line 327 replace in some cases with for some parameters
Line 328 what is relatively equal quality? rephrase the whole sentence
Line 333 delete the results showed that
Line 331-333 delete sentence
Line 337 instead of This indicated.... I suggest In summary, AMF could facilitate ... and had a stronger positive influence on many root parameters than chemical fertilizer.
Line 522-531 good summary but highly redundant with the conclusions
Line 344 show
Line 350 fungi of
Line 351-354 please delete sentence
Line 367 that yield of sunchoke plants ...
Line 369-373 verb missing
Line 383 delete of them
Line 392 delete competently
Line 394 could instead of would
Line 396 delete then, of Ef and AMF
Line 398-421 please shorten, this is highly repetitive with the results part
Line 431 I cannot follow this argument
Line 433 under greenhouse and field conditions
Line 436 Under field conditions
Line 437 under greenhouse conditions
Line 445 delete during
Line 446 higher increase
Line 450 Remarkably
Line 453 under field than greenhouse conditions
Line 455 of our work
Line 462 Delete The results showed that
Line 463 showed a higher increase in
Line 465 Therefore, it seems that
Line 466 hormones
Line 471, ...and AMF, we found AMF and Ef colonization in all treatments indicating the existence....
Line 486 resultinger in higher Ef than AMF colonization rates in co-inoculated roots
Line 507 those of
Line 513 AMF may play
Line 522-531 delete - highly redundant
Line 533 of the Ef strain... and the AMF strain
Line 548 demonstrate
Line 549 delete Note that (the wording is exactly the same as in the abstract!)
Line 552-554 please correct, the sentence cannot be understood
Author Response
Point 1: The general set-up of the study is interesting and the results promising to reduce the use of fertilizers in sunchoke production. However, the number of replicates (4) is far from a good study to compare growth and yield between different treatments. Furthermore, despite of being reviewed already once the manuscript still contains a number of mistakes and a highly repetitive writing style which makes it rather difficult to read. I strongly recommend to get a native speaker to polish the manuscript. Below you will find a number of suggestions for correction, but the list is not exhaustive.
Response 1: We would like to thank the reviewer for the careful and thorough reading of this manuscript. Your comments provide useful improvements to our manuscript. In this regard, we will get a native speaker to polish this manuscript as suggested.
Point 2: Title: I suggest to be more precise: Co-inoculation of an endophytic and arbuscular mycorrhizal fungus improves growth and yield....
Response 2: Thank you for your recommendation. The title has been changed to “Co-inoculation of an endophytic and arbuscular mycorrhizal fungus improves growth and yield of Helianthus tuberosus L. under field condition” as suggested.
Point 3: Line 23 delete note that
Response 3: The change has been made in the revised manuscript.
Point 4: Line 50 protection
Response 4: The word has been edited as suggested.
Point 5: Line 51-54 Please put it more simple - while AM fungi only colonizae roots some Ef are also capable to colonize the above ground parts....
Response 5: Thank you for pointing this out. The text has been revised as suggested.
Point 6: Line 73 The endophytic...
Point 7: Line 74 which enhanced growth
Point 8: Line 89 transferred
Point 9: Line 96 Delete then
Point 10: Line 218 Effects of teh Ef... of AMF in oder to ensure non-antagonistic
Point 11: Line 220 Delete The results showed that
Point 12: Line 223 delete the
Point 13: Line 224 delete an
Point 14: Line 230 dele an before the two fungi
Response 6-14: The correction has been made as suggested.
Point 15: Line 233 treatments (delete conditions) is presented. Delete the results showed and reframe the sentence
Response 15: The sentence has been edited and reframed as in the revised manuscript on page 6 lines 232-235.
Point 16: Line 235 This means that the native agricultural soil also contained AMF and Ef. We cannot rule out that these are different species than the once we used for inoculation. However, in terms of AMF and Ef colonization all treatments....
Response 16: It is true that the native agricultural soil contained indigenous AMF and Ef. However, we examined the population of AMF species in the study area before carrying out this experiment, and found that none of those indigenous species was the species that we used for inoculation.
Point 17: Line 252 Delete the sentence - competition for nutrients is also a type of antagonism
Response 17: Thank you for your suggestion. The correction has been made in the revised manuscript.
Point 18: Line 261 replace. This suggested by indicating
Response 18: Done.
Point 19: Line 264-269 this should be addressed in the discussion not the results
Response 19: Many thanks. We have moved these sentences to the discussion in the revised manuscript on pages 11-12 lines 479-486.
Point 20: Line 270-279 This paragraph is irritating at this point – please combine some microscopic pictures with figure 1 and delete Figure 2, 3 and 4 and write a concise text before comparing the colonization rates of the different treatements.
Response 20: Thank you for pointing this out. All of the microscopic pictures have been combined into Figure 1. A concise text to describe this Figure has been rewritten before comparing the colonization rates of the different treatments as suggested.
Point 21: Line 298 delete was
Point 22: Line 306 delete The results showed that
Point 23: Line 315 this suggests
Point 24: Line 316 delete an in front of the two fungi
Point 25: Line 317 replace in which by and
Point 26: Line 323 presents
Point 27: Line 327 replace in some cases with for some parameters
Response 21-27: The correction has been made as suggested.
Point 28: Line 328 what is relatively equal quality? rephrase the whole sentence
Response 28: The whole sentence has been rewritten to clarify this point in the revised manuscript on page 8 lines 342-345 as follows: “Moreover, plants inoculated with AMF had plant roots quality (length, surface area, diameter, and volume) similar to those of the plants co-inoculated with EPF and AMF, which was significantly higher than that of plants in the other treatments.”
Point 29: Line 333 delete the results showed that
Response 29: The phrase “the result showed that” has been deleted as suggested.
Point 30: Line 331-333 delete sentence
Response 30: Done.
Point 31: Line 337 instead of This indicated.... I suggest In summary, AMF could facilitate ... and had a stronger positive influence on many root parameters than chemical fertilizer.
Response 31: Thank you for the suggestion. The sentence has been revised as suggested in the revised manuscript on page 8 lines 351-352 as follows:
“In summary, AMF could facilitate P and K uptake in sunchoke and had a stronger positive influence on many root parameters than chemical fertilizer.”
Point 32: Line 522-531 good summary but highly redundant with the conclusions
Response 32: Many thanks. We have deleted this summary to avoid redundancy.
Point 33: Line 344 show
Point 34: Line 350 fungi of
Point 35: Line 351-354 please delete sentence
Point 36: Line 367 that yield of sunchoke plants ...
Point 37: Line 369-373 verb missing
Point 38: Line 383 delete of them
Point 39: Line 392 delete competently
Point 40: Line 394 could instead of would
Point 41: Line 394 could instead of would
Point 42: Line 396 delete then, of Ef and AMF
Response 33-42: All of these points have been changed accordingly.
Point 43: Line 398-421 please shorten, this is highly repetitive with the results part
Response 43: Thank you for your suggestion. We have shortened the paragraph as suggested.
Point 44: Line 431 I cannot follow this argument
Response 44: To clarify this point, we have rewritten this sentence in the revised manuscript as follows: “This might be because AMF can interact with plants more effectively in the environment where it originated from.”
Point 45: Line 433 under greenhouse and field conditions
Point 46: Line 436 Under field conditions
Point 47: Line 437 under greenhouse conditions
Point 48: Line 445 delete during
Point 49: Line 446 higher increase
Point 50: Line 450 Remarkably
Point 51: Line 453 under field than greenhouse conditions
Point 52: Line 455 of our work
Point 53: Line 462 Delete The results showed that
Point 56: Line 463 showed a higher increase in
Point 57: Line 465 Therefore, it seems that
Point 58: Line 466 hormones
Point 59: Line 471, ...and AMF, we found AMF and Ef colonization in all treatments indicating the existence....
Point 60: Line 486 resultinger in higher Ef than AMF colonization rates in co-inoculated roots
Point 61: Line 507 those of
Point 62: Line 513 AMF may play
Point 63: Line 522-531 delete - highly redundant
Point 64: Line 533 of the Ef strain... and the AMF strain
Point 65: Line 548 demonstrate
Response 45-65: Many thanks. The correction has been made as suggested.
Point 66: Line 549 delete Note that (the wording is exactly the same as in the abstract!)
Response 66: The word “Note that” has been deleted as suggested.
Point 67: Line 552-554 please correct, the sentence cannot be understood
Response 67: To clarify this point, we have rewritten this sentence in the manuscript on page 13 lines 554-556 as follows:
“It is, therefore, the development of a bio-fertilizer containing both EPF and AMF for use in sunchoke fields is worth further study.”
Round 2
Reviewer 2 Report
The authors has revised and done the reply to the comments, I think it can be published in the current.
Reviewer 3 Report
I appreciate that the authors did many corrections and made the figures more concise. However, the manuscript is still full of small English mistakes. In my last review I listed a large number of mistakes but I also stressed that the list is not exhaustive.
A point that has not been addressed yet are the references. Capital and small letters are not consistently used for the Journals and scientific names are not written in italics.
Polishing manuscripts is not the job of the reviewers but the duty of the authors in particular after a second round of revisions, and this manuscript definitely needs corrections before being published.
