Defense Enzymes in Mycorrhizal Tomato Plants Exposed to Combined Drought and Heat Stresses

Round 1

Reviewer 1 Report

The article titled "Defense enzymes in mycorrhizal tomato plants exposed to combined drought and heat stresses" investigates the role of AMF fungi on tomato plants tolerance against environmental stresses in a climate change perspective. 
This is a nicely written paper that makes a contribution to the understanding of the ecology of AMF fungi in agricultural systems. The experiment is well designed and allows to properly address the research questions. I have however few  suggestions to improve the result presentation of the manuscript.

Abstract

L21 - What are "Normal" conditions? define normal or change (maybe natural?)

Introduction

L43 "...Climate Change report that,..." change report with reports and delete the comma

L44-46 Please re-phrase.

L52-53 Any reference?

L67 Only genus and species should be written in italics. Change Phylum Glomeromycota

L96 "Washed several times with sterile distilled water" How many times for how long?

L100 "different mycorrhizal fungi inoculum" change with different mycorrhizal fungal inocula

L100-103 Are these fungi deposited and/or cryopreserved? Are they available? Accession numbers?

L110 change inoculums with inocula

L115-120 Why haven't you tried a complex inoculum with all the three fungal strains?

L118-120 check the punctation, at the moment it's not clear 

L121-122 what do you mean with "stress treatments were implemented"?

L124 Change normal with natural?

L133 "four plants of each treatment.." change of with for

L135 "visual inspection method" add a reference

L155 delete "received"

L181 change "per" with "described by"

L205 change "by" with "with"

L207 change "by" with "with"

L233 there is an extra space before non-AMF

L247 "Non-AMF, No AM" Why you change it? keep being consistent through the manuscript. Change the figure legend and use the same name for all the figures.

L285-288 please re-phrase

L327-331 put somewhere a reference to fig 3A

L386 change establishes with establish

L401 change impedes with impede

L405 Rhizophagus irregularis in italics

L408 Spell out the full name of E. angustifolia

L428-431 please re-phrase

L471 Any reference?

Figures

All the figures should be self explanatory.  in all the captions there is no explanation for the Am1, Am2 and Am3. please indicate in the captions of all the figures to which fungus those names are referred to.

Author Response

Response to Reviewer 1

Dear Sir/Madam,

We are very grateful and would like to thank the reviewer for the careful and thorough reading of this manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. The comments are encouraging; therefore, we have studied these carefully and have made corresponding corrections that we hope will meet with your approval.

Overall comment: regarding English language minor/spelling check, introduction, research design, methodology, results and discussion, and conclusion.

Response: Thank you for the observations. The manuscript underwent a thorough language check to correct the minor mistakes, spellings.

The suggested corrections were resolved for every mentioned part.

1. comment: L21 - What are "Normal" conditions? define normal or change (maybe natural?)

Response:  “non-stress” was used to replace “normal” (Line 20)

2. comment: L43 "...Climate Change report that,..." change report with reports and delete the comma

Response:  Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 45

3. comment: L44-46 Please re-phrase.

Response:  Sentence was re-phrased.

Therefore, the negative effect of global warming on horticultural crop growth needs more attention. Line: 46-48

4. comment: L52-53 Any reference?

Response:  Reference was added and the sentence was rephrased. Line: 54-59.

Moreover, drought is a limiting in agriculture yield production, therefore we can assume that the co-occurrence of these two abiotic stresses (drought, heat) can be even more harmful [4,7].It has been reported that the physiological and biochemical processes of plants are affected by combined stresses as well as the photosynthesis, synthesis of protein and energy, moreover, it has a degrading effect on the ionic and osmotic balance that induces oxidative stress in tomato plant [7].

5. comment: L67 Only genus and species should be written in italics. Change Phylum Glomeromycota

Response:  Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 71

6. comment: L96 "Washed several times with sterile distilled water" How many times for how long?

Response: Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 99-101

Solanum lycopersicum (L.) var. MoneyMaker seeds (Sieberz Ltd, Hungary) were surface sterilized by immersion into 2.5% sodium hypochlorite containing 0.02% (v/v) Tween-20 for 30 min, then seeds were washed several times with sterile distilled water for 10 min.

7. comment: L100 "different mycorrhizal fungi inoculum" change with different mycorrhizal fungal inocula

Response: Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 105

8. comment: L104-107 Are these fungi deposited and/or cryopreserved? Are they available? Accession numbers?

Response: These fungi are available in our lab. They are continously maintained and propagated to obtain enough amount as our experiments start.

9. comment: L110 change inoculums with inocula

Response: Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 114, 115

10. comment: L115-120 Why haven't you tried a complex inoculum with all the three fungal strains?

Response: Thank you for your suggestion, this study only focused on the effects of single fungal strains to know which strain could enhance plant tolerance to the combined stresses. Combined AM inoculation requires three combinations of two strains and one combination of three strains to clearly identify contribution of each strain in multiple inoculation. This will lead to double our treatments and workload. Therefore, multiple inoculation of AMF species could be our other experiments.

11. comment: L118-120 check the punctation, at the moment it's not clear

Response: The sentence was revised. Line: 125-130

For all 4 tomato treatments, a total of 96 pots were used, where 3 stress effects were examined:  control with no stress (NoS); simultaneous heat and drought stress (D+H); simultaneous drought and heat shock stress (D+HS).

12. comment: L121-122 what do you mean with "stress treatments were implemented"?

Response: The sentence was revised. Line: 131-132

All plants were grown under the same non-stress conditions for six weeks, after that different stress applications were implemented (Figure S1).

13. comment: L124 Change normal with natural?

Response: We used “non-stress” instead of “normal”. Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 138

14. comment: L133 "four plants of each treatment.." change of with for

Response: Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 149

15. comment: L135 "visual inspection method" add a reference

Response: Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 152

16. comment: L155 delete "received"

Response: Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 171

17. comment: L181 change "per" with "described by"

Response: Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 201-202

18. comment: L205 and L2017 change "by" with "with

 Response: The whole sentence was rewritten. Line: 219-220

19. comment: L233 there is an extra space before non-AMF

Response: Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 285

20. comment: L247 "Non-AMF, No AM" Why you change it? keep being consistent through the manuscript. Change the figure legend and use the same name for all the figures.

Response: Figure legends and whole manuscript were corrected.

21. comment: L285-288 please re-phrase

Response: Sentence was re-phased. Line: 304-311

Defense enzyme activity ( CAT, PPO, POD, and GST) was stimulated and increased both in leaves and roots when they were exposed to the studied combined stresses. However, there was a slight difference between the inoculated and non-inoculated plants because the activity was significantly higher in mycorrhiza treated tomato plants.

22. comment: L327-331 put somewhere a reference to fig 3A

Response: Figure 3 reference was put in the main text. Line: 357

23. comment: L386 change establishes with establish

Response: Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 433

24. comment: L401 change impedes with impede

Response: Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 448

25. comment: L405 Rhizophagus irregularis in italics

Response: Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 452

26. comment: L408 Spell out the full name of E. angustifolia

Response: Correction was made with MS Office Word, in Track changes mode, as it was requested. Line: 455-456

27. comment: L428-431 please re-phrase

Response: Sentence was re-phrased. Line: 479-484

MDA content (is considered an end product of lipid peroxidation) decreased in plants treated with different AM compared to No-AM plants under stress treatments (Figure 1).

28. comment: L471 Any reference?

Response: Reference was added. Line: 524

29. comment: All the figures should be self explanatory. in all the captions there is no explanation for the Am1, Am2 and Am3. please indicate in the captions of all the figures to which fungus those names are referred to.

Response: The legend of all figures was redone so it would be self explanatory.

We would like to express our great appreciation to the reviewer for the comments on our paper and thank for the interest on this topic. We  are  confident  that  the  new  version  of  the  manuscript  will  be  greatly improved. We hope that the reviewer will find our responses to their comments satisfactory, and we are willing to finish the revised version of the manuscript including any further suggestion that the reviewer may have.

If you have any further queries, please do not hesitate to contact us.

Kind regards,

Katalin Posta

Author Response File: Author Response.docx

Reviewer 2 Report

Line 30, I don’t think you need to repeat keywords that are already appeared in the title.

Line 45, more attention is good, more and more sounds very colloquial.

Line 54, better explain “it” (combined stresses) instead of using “it”.

Line 61, very wired when you use “however” here.

Line 106, better use ‘in addition’ rather than ‘on the other hand’.

Line 113, 23/28 °C with 16/8 hours? But in your abstract, you mention 26/20 °C with 16/8 h.

Line 122, what’s your control field capacity?

Line 125-127, for heat stress, are you applying temperature along with 14-day drought? And for heat shock, what does “till the end” mean, are you apply 45°C for 6 hours only at the end of drought, like in day 14 from 18:00-24:00? Then probably it’s better use “at”, please also put more details here.

Table 1, why some of you Df in AMF is 3 and some is 7?

Line 202-204, this sentence is really hard to understand, please revise. Are you comparing the decreased root colonization with no-stress?

Table S2, I think this table is only for D+HS, but in your table title, you put both D+H, and D+HS, please double check.

Line 223-229, I think better put this paragraph after you finish talking about POD, H2O2, etc.

Table S1 and S2, are you using AMF colonization percentage to perform your correlation with other parameters?

You mention a lot the correlations in your results but not too much in your discussion, I’m wondering why you perform the correlation analysis, you only need to do the correlation between AMF and other parameters if this is the most important. Also, I suggest you do a PCA for a better visualization your results.

Another point I’m concerned is about the effects of combined D+H or D+HS, first you didn’t discuss many differences between D+H and D+HS, why heat shock performs little bit differently compares to long-term heat? Only repeating your results without any discussion for this will lead to your well-measured results to nowhere.

Another thing is, you discussed a lot about stress responses (e.g., POD, PPO, MDA, H2O2, etc.), as we know both drought and heat stress will lead to the change of these parameters, but what’s the combined effects caused by D+H? This leads to my other concern, you only did D+H without doing D or H alone, how can you say that the combined effects will be different? You did a good discuss about AMF effect on stress, but how does these combined stresses alter AMF function? Again, without doing D or H stress treatment alone, it’s hard to identify this.

Author Response

Dear Sir/Madam,

We are very grateful and would like to thank the reviewer for the careful and thorough reading of this manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. The comments are encouraging; therefore, we have studied these carefully and have made corresponding corrections that we hope will meet with your approval.

We would like to express our great appreciation to the reviewer for the comments on our paper and thank for the interest on this topic. We  are  confident  that  the  new  version  of  the  manuscript  will  be  greatly improved. We hope that the reviewer will find our responses to their comments satisfactory, and we are willing to finish the revised version of the manuscript including any further suggestion that the reviewer may have.

If you have any further queries, please do not hesitate to contact us.

Kind regards,

Katalin Posta

Let see more details in attaced file.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

I like this responses, very good and declares most of the concerns I have, I would like to suggest accept but just with a minor changes, could you incorporate some of your responses for comment 16 (the last one) into your manuscript introduction to make it more complete (talking some of your previous study and link to your current study). 

Author Response

Dear Sir/Madam,

Thank you for taking the time to re-read our manuscript after the revision. We are delighted that You found our responses good and satisfactory. Moreover, we are pleased to know that we could answer all your questions and declared Your concerns.

Overall comment: English language and style are fine/minor spell check required

Response: Thank you for the observation, the manuscript has been carefully revised to eliminate minor spell checks.

1. comment: I would like to suggest accept but just with a minor changes, could you incorporate some of your responses for comment 16 (the last one) into your manuscript introduction to make it more complete (talking some of your previous study and link to your current study).

Response:  Thank you for the suggestion, some details from comment 16 were incorporated into the Introduction part. Lines: 89-100.

Even in our previous study, we examined the effect of AMF on plant tolerance to individual drought, individual heat shock, and combined drought and heat shock [25]. Our previous results showed that the combined drought and heat shock caused more severe damage to tomato plants than individual stresses, but AMF alleviated the negative impacts of the single stresses and the combined stress. We expanded the experiment with combined drought and heat stress together combined drought and heat shock based on our previous results. However, in this study, we only focus on two combinations of drought and heat, which frequently occurs in field conditions and under climate change. In our viewpoint, research on combinations of these stresses is more realistic because under field cultivation or natural conditions, not single stresses but multiple stresses take place. Therefore, we support the point that combined stresses (combined drought and heat stresses in our study) are unique and more natural stresses.

Hereby, we would like to express our great appreciation for these valuable feedbacks, we believe that all the corrections improved the quality of the manuscript.

If you have any further questions, please do not hesitate to contact us.

Kind regards,

   Katalin Posta

Author Response File: Author Response.docx