Investigating the Effects of Alltech Crop Science (ACS) Products on Plant Defence against Root-Knot Nematode Infestation

Reviewer 1

Dear authors,

The manuscript “Investigating Effects of Alltech Crop Science (ACS) Products on Plant Defence Mechanisms following Root‐Knot Nematode Infestation” has been improved with the revisions of the reviewers.  Some minor revisions are proposed to improve the quality of the manuscript below.

Line 34. Elsen {4} . Those brackets are in journal format?

Corrected to square brackets

Line 38. Would you change Meloidogyne to M. ?

Changed

Lines 51 to 54. Need a reference

Added

Line 66. I would write “among others” because not only Rhizobacterias are responsible for that activation

Edited accordingly

Lines 94. Solanum lycopersicum should be in italics

Changed

Line 94 and 109 Delete variety

Deleted

Line 121. Change days post-treatment by dpt

Changed

Line 96. What is a SETU?

Its South East Technological University (SETU)

Line 118, I understand, in the solid treatment, you remove the plant, mix it with the product and then put the plant back into the pot?

Detailed product treatments should be explained

Detailed explanation about product treatments have been written within this paragraph.

Lone 120-122. What changes between 3dpt and 5dpt and all the other dpt? If it is the same, rewrite the sentence. Proposal  “Plants were uprooted after each dpi to proceed with RNA extraction and then RT‐PCR” and then lines 120 – 126  I would remove it.

Edited accordingly

Line 129. I would remove (100mg per sample) and explain better the complete sentence. Proposal: “Leaf and root tissue, separately, was ground to a fine powder using liquid nitrogen with a sterile porcelain pestle and mortar, and 100mg per sample was used for RNA extraction immediately or kept at -80ºC until it's used.”

Edited

Line 434 and Table 1. Molinari 21, studied JERF3. Is it the same as you study? If it is the same I would change all JERF to JERF3.

Have been changed

Line 456. Change soyabean cyst nematodes to soybean cyst nematodes

changed

Ine477. Change possibly to possible

changed

Table 1. Line 219. I propose to include a column with the references and not in the footnote

A column with references have been included and footnote deleted

Figure 1. needs to be improved.

Figure 2. needs to be improved.

Figure 3 needs to be improved. Foot notes look blurry

Both Figure 1 and 2 have been improved and have been uploaded as a separate word file named “Figure 1, 2 and 3”

Figure 3 has been improved and the clarity of foot notes has been improved.

All Figure 1, 2 and 3 have been improved and have been uploaded as a separate word file named “Figure 1, 2 and 3”

Reviewer 3

The authors need to revise the title of the paper in a more meaningful way. the title is long and has some unnecessary information, suggestion: “Investigating the Effects of Alltech Crop Science Products on Plant Defense Against Root-Knot Nematodes.”

Title has been changed as per the suggestion

The abstract is written in a way lacks logic. It should highlight the salient findings more critically;

Abstract has been edited

In the materials and methods of the manuscript, in the section where the RT-PCR procedures are described and in the statistical analyses, the authors report that they performed a relative expression using the Livak 2001 method; However, it was not clear in the text who was the calibrating sample used in the relationship parameters. TO DESCRIBE!!!

The cycle threshold (C_T values) that were generated by the Roche machine were recorded individually. ΔC_T values were calculated by deducting the individual C_T values from that of the C_T values obtained for the respective Actin gene per each sample [ΔC_T(a target sample)−ΔC_T(Actin)]. Using a reference gene (Actin) as a standard, the ultimate outcome of this method is represented as the mean relative fold change in target gene expression in a target sample compared to untreated control, which acted as a calibrating sample.

The above description has been added in section 2.3 under materials and methods.

Authors should discuss the results integrally. The discussion is based on individual results. I suggest that integrating the results will give more value to the work. I suggest that you discuss by integrating all your results. You can use correlation tests (PCA or Pearson Correlation).

Post-inoculation with M. javanica and treatment with ACS5075, in the root tissues, a positive correlation (r=0.4 and 0.2) has been noted while correlating expression of PR-1 and ACO genes, PR-5 and JERF 3 genes, respectively. This means with an increase in expression of one gene there is a positive increase of fixed proportion in the expression of other genes.

In the leaf tissues, a strong positive correlation (r=0.8, 0.7, 0.6) was also noted while correlating ACO and JERF 3, JERF 3 and PR-1, ACO and PR-1 genes, respectively.

All of the above has been added under the discussion section.

The conclusion is totally confusing. Re-write the conclusion! It needs to be much improved.

Conclusion part has been re-written

Reviewer 2

Introduction

Line 53 Please add a reference concerning the other approaches used to control PPN

Reference has been added

Material and Methods

2.4

How many plants did you use per treatment? Please indicate the number

All the treatments were conducted in triplicates. It has been mentioned in section 2.4.

Results

Lines 237-242 Expression of gene PR1 was referred to the untreated control. I understand that the value of UC is 1, but no reference is inserted in the text or in the legends of the Figures 1 and 2. I recommend adding a small sentence i.e. "compared to untreated control (set as a unit) at all times" in the legends of figures 1 and 2 or a dotted line at value 1 level, in all graphs, which indicates the expression of  the untreated control.

The y-axis in figure 1 and 2 indicates the mean fold change compared to UC. It’s a ratio. If the ratio was equal to 1, it means the expression was not different from that of UC. If the ratio was more or less than 1, it indicates that the expression was different from that of UC.

All of this explanation has been included under ‘statistical analysis’ section.

Lines 248-249 You are comparing expression in roots between 15dpt and 3dpt. According to the graph 1b the fold change is more than 4-fold higher because the value of 3dpt is set at 0.5. Please recalculate.

The values have been recalculated and corrected accordingly

Line 251 Also in this case the value is not correct. Please check and recalculate

The values have been recalculated and corrected accordingly

Figure 1 Please uniform all graphs, some have grid others no.

All the grids have been removed to make them look uniform.

Line 282 You can state significantly lower if there are asterisks.

(*) Symbol indicates mean relative fold change which is significantly different from that of the UC.

This sentence has been indicated in the legends of figure 1, 2 and 3.

Line 311 It seems to me that there are mistakes in the calculation of folds of expression in treated tissues respect to IU. Please check and recalculate.

The fold of expression have been re-calculated and corrected accordingly within the text

Figure 5 shows that protein spots 1, 2 and 3 were present in samples extracted from Nemguard, ACS3048 treated plants and untreated control. For corroborating your statement “However, these protein spots were faintly observed even in the untreated control plants” I suggest to add a graph showing the quantitation of the expressed bands

Figure 5 shows that protein spots 1,2and 3 were present in all the as indicated with the yellow circles.

We have not performed any gel analysis or densitometry, therefore, quantitation of the expressed bands is not possible at this stage. This will be kept in mind for all our future studies.

I have edited the sentence in the manuscript as “However, these protein spots were also observed even in the untreated control plants”.

Minor points:

Introduction

Line 38 Because you already cited the genus Meloidogyne (i.e. Meloidogyne arenaria) you can shorten M. incognita, M. javanica and so on.

edited

Material and Methods

2.1

Line 94 Solanum lycopersicum shoud be in Italic

edited

Line 94 Delete “variety”

deleted

Line 95 Better “roots that were infected with M. javanica”

changed

2.2

Line 109 Delete “variety”

deleted

Lines 117-119 I suggest to rewrite the sentence as “…..4-week old tomato plants were treated individually with 3% (v/v) ACS5075 or 3g ACS3048 only once and were harvested after 3, 5, 9 and 15 days”

rewritten

Lines 119-123 I suggest to move this part “Six replications….including the control” immediately after line 126 “…RKN study[17]”

edited accordingly

Line 126 Add a space between RKN and study

added

Line 127 I suggest to delete “After the completion of each treatment…from the soil” because it is a repetition

deleted

Lines 131-132 Delete “All the treatments were conducted with six replications, therefore”

deleted

Line 132 Twelve (now capital letter)

changed

2.5

Line 215 S. lycopersicum

changed

Results

Line 250 Better to write “trend” instead of “pattern”

Replaced ‘pattern’ with ‘trend’

Line 286 This sentence is incomplete

edited

Figure 3 “housekeeping” not “house keeping”

changed

Discussion

Lines 446-448 Delete this sentence. It has already written.

deleted

Line 456 soybean

changed

Line 473 P. syringae

corrected

Lines 480 and 491 Delete Table 2

Deleted

Reviewer 1

Dear authors,

The manuscript “Investigating Effects of Alltech Crop Science (ACS) Products on Plant Defence Mechanisms following Root‐Knot Nematode Infestation” has been improved with the revisions of the reviewers.  Some minor revisions are proposed to improve the quality of the manuscript below.

Line 34. Elsen {4} . Those brackets are in journal format?

Corrected to square brackets

Line 38. Would you change Meloidogyne to M. ?

Changed

Lines 51 to 54. Need a reference

Added

Line 66. I would write “among others” because not only Rhizobacterias are responsible for that activation

Edited accordingly

Lines 94. Solanum lycopersicum should be in italics

Changed

Line 94 and 109 Delete variety

Deleted

Line 121. Change days post-treatment by dpt

Changed

Line 96. What is a SETU?

Its South East Technological University (SETU)

Line 118, I understand, in the solid treatment, you remove the plant, mix it with the product and then put the plant back into the pot?

Detailed product treatments should be explained

Detailed explanation about product treatments have been written within this paragraph.

Lone 120-122. What changes between 3dpt and 5dpt and all the other dpt? If it is the same, rewrite the sentence. Proposal  “Plants were uprooted after each dpi to proceed with RNA extraction and then RT‐PCR” and then lines 120 – 126  I would remove it.

Edited accordingly

Line 129. I would remove (100mg per sample) and explain better the complete sentence. Proposal: “Leaf and root tissue, separately, was ground to a fine powder using liquid nitrogen with a sterile porcelain pestle and mortar, and 100mg per sample was used for RNA extraction immediately or kept at -80ºC until it's used.”

Edited

Line 434 and Table 1. Molinari 21, studied JERF3. Is it the same as you study? If it is the same I would change all JERF to JERF3.

Have been changed

Line 456. Change soyabean cyst nematodes to soybean cyst nematodes

changed

Ine477. Change possibly to possible

changed

Table 1. Line 219. I propose to include a column with the references and not in the footnote

A column with references have been included and footnote deleted

Figure 1. needs to be improved.

Figure 2. needs to be improved.

Figure 3 needs to be improved. Foot notes look blurry

Both Figure 1 and 2 have been improved and have been uploaded as a separate word file named “Figure 1, 2 and 3”

Figure 3 has been improved and the clarity of foot notes has been improved.

All Figure 1, 2 and 3 have been improved and have been uploaded as a separate word file named “Figure 1, 2 and 3”

Reviewer 3

The authors need to revise the title of the paper in a more meaningful way. the title is long and has some unnecessary information, suggestion: “Investigating the Effects of Alltech Crop Science Products on Plant Defense Against Root-Knot Nematodes.”

Title has been changed as per the suggestion

The abstract is written in a way lacks logic. It should highlight the salient findings more critically;

Abstract has been edited

In the materials and methods of the manuscript, in the section where the RT-PCR procedures are described and in the statistical analyses, the authors report that they performed a relative expression using the Livak 2001 method; However, it was not clear in the text who was the calibrating sample used in the relationship parameters. TO DESCRIBE!!!

The cycle threshold (C_T values) that were generated by the Roche machine were recorded individually. ΔC_T values were calculated by deducting the individual C_T values from that of the C_T values obtained for the respective Actin gene per each sample [ΔC_T(a target sample)−ΔC_T(Actin)]. Using a reference gene (Actin) as a standard, the ultimate outcome of this method is represented as the mean relative fold change in target gene expression in a target sample compared to untreated control, which acted as a calibrating sample.

The above description has been added in section 2.3 under materials and methods.

Authors should discuss the results integrally. The discussion is based on individual results. I suggest that integrating the results will give more value to the work. I suggest that you discuss by integrating all your results. You can use correlation tests (PCA or Pearson Correlation).

Post-inoculation with M. javanica and treatment with ACS5075, in the root tissues, a positive correlation (r=0.4 and 0.2) has been noted while correlating expression of PR-1 and ACO genes, PR-5 and JERF 3 genes, respectively. This means with an increase in expression of one gene there is a positive increase of fixed proportion in the expression of other genes.

In the leaf tissues, a strong positive correlation (r=0.8, 0.7, 0.6) was also noted while correlating ACO and JERF 3, JERF 3 and PR-1, ACO and PR-1 genes, respectively.

All of the above has been added under the discussion section.

The conclusion is totally confusing. Re-write the conclusion! It needs to be much improved.

Conclusion part has been re-written

Reviewer 2

Introduction

Line 53 Please add a reference concerning the other approaches used to control PPN

Reference has been added

Material and Methods

2.4

How many plants did you use per treatment? Please indicate the number

All the treatments were conducted in triplicates. It has been mentioned in section 2.4.

Results

Lines 237-242 Expression of gene PR1 was referred to the untreated control. I understand that the value of UC is 1, but no reference is inserted in the text or in the legends of the Figures 1 and 2. I recommend adding a small sentence i.e. "compared to untreated control (set as a unit) at all times" in the legends of figures 1 and 2 or a dotted line at value 1 level, in all graphs, which indicates the expression of  the untreated control.

The y-axis in figure 1 and 2 indicates the mean fold change compared to UC. It’s a ratio. If the ratio was equal to 1, it means the expression was not different from that of UC. If the ratio was more or less than 1, it indicates that the expression was different from that of UC.

All of this explanation has been included under ‘statistical analysis’ section.

Lines 248-249 You are comparing expression in roots between 15dpt and 3dpt. According to the graph 1b the fold change is more than 4-fold higher because the value of 3dpt is set at 0.5. Please recalculate.

The values have been recalculated and corrected accordingly

Line 251 Also in this case the value is not correct. Please check and recalculate

The values have been recalculated and corrected accordingly

Figure 1 Please uniform all graphs, some have grid others no.

All the grids have been removed to make them look uniform.

Line 282 You can state significantly lower if there are asterisks.

(*) Symbol indicates mean relative fold change which is significantly different from that of the UC.

This sentence has been indicated in the legends of figure 1, 2 and 3.

Line 311 It seems to me that there are mistakes in the calculation of folds of expression in treated tissues respect to IU. Please check and recalculate.

The fold of expression have been re-calculated and corrected accordingly within the text

Figure 5 shows that protein spots 1, 2 and 3 were present in samples extracted from Nemguard, ACS3048 treated plants and untreated control. For corroborating your statement “However, these protein spots were faintly observed even in the untreated control plants” I suggest to add a graph showing the quantitation of the expressed bands

Figure 5 shows that protein spots 1,2and 3 were present in all the as indicated with the yellow circles.

We have not performed any gel analysis or densitometry, therefore, quantitation of the expressed bands is not possible at this stage. This will be kept in mind for all our future studies.

I have edited the sentence in the manuscript as “However, these protein spots were also observed even in the untreated control plants”.

Minor points:

Introduction

Line 38 Because you already cited the genus Meloidogyne (i.e. Meloidogyne arenaria) you can shorten M. incognita, M. javanica and so on.

edited

Material and Methods

2.1

Line 94 Solanum lycopersicum shoud be in Italic

edited

Line 94 Delete “variety”

deleted

Line 95 Better “roots that were infected with M. javanica”

changed

2.2

Line 109 Delete “variety”

deleted

Lines 117-119 I suggest to rewrite the sentence as “…..4-week old tomato plants were treated individually with 3% (v/v) ACS5075 or 3g ACS3048 only once and were harvested after 3, 5, 9 and 15 days”

rewritten

Lines 119-123 I suggest to move this part “Six replications….including the control” immediately after line 126 “…RKN study[17]”

edited accordingly

Line 126 Add a space between RKN and study

added

Line 127 I suggest to delete “After the completion of each treatment…from the soil” because it is a repetition

deleted

Lines 131-132 Delete “All the treatments were conducted with six replications, therefore”

deleted

Line 132 Twelve (now capital letter)

changed

2.5

Line 215 S. lycopersicum

changed

Results

Line 250 Better to write “trend” instead of “pattern”

Replaced ‘pattern’ with ‘trend’

Line 286 This sentence is incomplete

edited

Figure 3 “housekeeping” not “house keeping”

changed

Discussion

Lines 446-448 Delete this sentence. It has already written.

deleted

Line 456 soybean

changed

Line 473 P. syringae

corrected

Lines 480 and 491 Delete Table 2

Deleted

Reviewer 1

Dear authors,

The manuscript “Investigating Effects of Alltech Crop Science (ACS) Products on Plant Defence Mechanisms following Root‐Knot Nematode Infestation” has been improved with the revisions of the reviewers.  Some minor revisions are proposed to improve the quality of the manuscript below.

Line 34. Elsen {4} . Those brackets are in journal format?

Corrected to square brackets

Line 38. Would you change Meloidogyne to M. ?

Changed

Lines 51 to 54. Need a reference

Added

Line 66. I would write “among others” because not only Rhizobacterias are responsible for that activation

Edited accordingly

Lines 94. Solanum lycopersicum should be in italics

Changed

Line 94 and 109 Delete variety

Deleted

Line 121. Change days post-treatment by dpt

Changed

Line 96. What is a SETU?

Its South East Technological University (SETU)

Line 118, I understand, in the solid treatment, you remove the plant, mix it with the product and then put the plant back into the pot?

Detailed product treatments should be explained

Detailed explanation about product treatments have been written within this paragraph.

Lone 120-122. What changes between 3dpt and 5dpt and all the other dpt? If it is the same, rewrite the sentence. Proposal  “Plants were uprooted after each dpi to proceed with RNA extraction and then RT‐PCR” and then lines 120 – 126  I would remove it.

Edited accordingly

Line 129. I would remove (100mg per sample) and explain better the complete sentence. Proposal: “Leaf and root tissue, separately, was ground to a fine powder using liquid nitrogen with a sterile porcelain pestle and mortar, and 100mg per sample was used for RNA extraction immediately or kept at -80ºC until it's used.”

Edited

Line 434 and Table 1. Molinari 21, studied JERF3. Is it the same as you study? If it is the same I would change all JERF to JERF3.

Have been changed

Line 456. Change soyabean cyst nematodes to soybean cyst nematodes

changed

Ine477. Change possibly to possible

changed

Table 1. Line 219. I propose to include a column with the references and not in the footnote

A column with references have been included and footnote deleted

Figure 1. needs to be improved.

Figure 2. needs to be improved.

Figure 3 needs to be improved. Foot notes look blurry

Both Figure 1 and 2 have been improved and have been uploaded as a separate word file named “Figure 1, 2 and 3”

Figure 3 has been improved and the clarity of foot notes has been improved.

All Figure 1, 2 and 3 have been improved and have been uploaded as a separate word file named “Figure 1, 2 and 3”

Reviewer 3

The authors need to revise the title of the paper in a more meaningful way. the title is long and has some unnecessary information, suggestion: “Investigating the Effects of Alltech Crop Science Products on Plant Defense Against Root-Knot Nematodes.”

Title has been changed as per the suggestion

The abstract is written in a way lacks logic. It should highlight the salient findings more critically;

Abstract has been edited

In the materials and methods of the manuscript, in the section where the RT-PCR procedures are described and in the statistical analyses, the authors report that they performed a relative expression using the Livak 2001 method; However, it was not clear in the text who was the calibrating sample used in the relationship parameters. TO DESCRIBE!!!

The cycle threshold (C_T values) that were generated by the Roche machine were recorded individually. ΔC_T values were calculated by deducting the individual C_T values from that of the C_T values obtained for the respective Actin gene per each sample [ΔC_T(a target sample)−ΔC_T(Actin)]. Using a reference gene (Actin) as a standard, the ultimate outcome of this method is represented as the mean relative fold change in target gene expression in a target sample compared to untreated control, which acted as a calibrating sample.

The above description has been added in section 2.3 under materials and methods.

Authors should discuss the results integrally. The discussion is based on individual results. I suggest that integrating the results will give more value to the work. I suggest that you discuss by integrating all your results. You can use correlation tests (PCA or Pearson Correlation).

Post-inoculation with M. javanica and treatment with ACS5075, in the root tissues, a positive correlation (r=0.4 and 0.2) has been noted while correlating expression of PR-1 and ACO genes, PR-5 and JERF 3 genes, respectively. This means with an increase in expression of one gene there is a positive increase of fixed proportion in the expression of other genes.

In the leaf tissues, a strong positive correlation (r=0.8, 0.7, 0.6) was also noted while correlating ACO and JERF 3, JERF 3 and PR-1, ACO and PR-1 genes, respectively.

All of the above has been added under the discussion section.

The conclusion is totally confusing. Re-write the conclusion! It needs to be much improved.

Conclusion part has been re-written

Reviewer 2

Introduction

Line 53 Please add a reference concerning the other approaches used to control PPN

Reference has been added

Material and Methods

2.4

How many plants did you use per treatment? Please indicate the number

All the treatments were conducted in triplicates. It has been mentioned in section 2.4.

Results

Lines 237-242 Expression of gene PR1 was referred to the untreated control. I understand that the value of UC is 1, but no reference is inserted in the text or in the legends of the Figures 1 and 2. I recommend adding a small sentence i.e. "compared to untreated control (set as a unit) at all times" in the legends of figures 1 and 2 or a dotted line at value 1 level, in all graphs, which indicates the expression of  the untreated control.

The y-axis in figure 1 and 2 indicates the mean fold change compared to UC. It’s a ratio. If the ratio was equal to 1, it means the expression was not different from that of UC. If the ratio was more or less than 1, it indicates that the expression was different from that of UC.

All of this explanation has been included under ‘statistical analysis’ section.

Lines 248-249 You are comparing expression in roots between 15dpt and 3dpt. According to the graph 1b the fold change is more than 4-fold higher because the value of 3dpt is set at 0.5. Please recalculate.

The values have been recalculated and corrected accordingly

Line 251 Also in this case the value is not correct. Please check and recalculate

The values have been recalculated and corrected accordingly

Figure 1 Please uniform all graphs, some have grid others no.

All the grids have been removed to make them look uniform.

Line 282 You can state significantly lower if there are asterisks.

(*) Symbol indicates mean relative fold change which is significantly different from that of the UC.

This sentence has been indicated in the legends of figure 1, 2 and 3.

Line 311 It seems to me that there are mistakes in the calculation of folds of expression in treated tissues respect to IU. Please check and recalculate.

The fold of expression have been re-calculated and corrected accordingly within the text

Figure 5 shows that protein spots 1, 2 and 3 were present in samples extracted from Nemguard, ACS3048 treated plants and untreated control. For corroborating your statement “However, these protein spots were faintly observed even in the untreated control plants” I suggest to add a graph showing the quantitation of the expressed bands

Figure 5 shows that protein spots 1,2and 3 were present in all the as indicated with the yellow circles.

We have not performed any gel analysis or densitometry, therefore, quantitation of the expressed bands is not possible at this stage. This will be kept in mind for all our future studies.

I have edited the sentence in the manuscript as “However, these protein spots were also observed even in the untreated control plants”.

Minor points:

Introduction

Line 38 Because you already cited the genus Meloidogyne (i.e. Meloidogyne arenaria) you can shorten M. incognita, M. javanica and so on.

edited

Material and Methods

2.1

Line 94 Solanum lycopersicum shoud be in Italic

edited

Line 94 Delete “variety”

deleted

Line 95 Better “roots that were infected with M. javanica”

changed

2.2

Line 109 Delete “variety”

deleted

Lines 117-119 I suggest to rewrite the sentence as “…..4-week old tomato plants were treated individually with 3% (v/v) ACS5075 or 3g ACS3048 only once and were harvested after 3, 5, 9 and 15 days”

rewritten

Lines 119-123 I suggest to move this part “Six replications….including the control” immediately after line 126 “…RKN study[17]”

edited accordingly

Line 126 Add a space between RKN and study

added

Line 127 I suggest to delete “After the completion of each treatment…from the soil” because it is a repetition

deleted

Lines 131-132 Delete “All the treatments were conducted with six replications, therefore”

deleted

Line 132 Twelve (now capital letter)

changed

2.5

Line 215 S. lycopersicum

changed

Results

Line 250 Better to write “trend” instead of “pattern”

Replaced ‘pattern’ with ‘trend’

Line 286 This sentence is incomplete

edited

Figure 3 “housekeeping” not “house keeping”

changed

Discussion

Lines 446-448 Delete this sentence. It has already written.

deleted

Line 456 soybean

changed

Line 473 P. syringae

corrected

Lines 480 and 491 Delete Table 2

Deleted