Development of a Novel Emulsion Formulation of Trichoderma asperelloides PSU-P1 Conidia against Stem Canker on Dragon Fruit Caused by Neoscytalidium dimidiatum

Round 1

Reviewer 1 Report

This is a very interesting study with potential practical application. I read the manuscript with great interest. Honestly, after I read this, I started thinking of preparing the emulsion myself. The manuscript is well written and most parts are clear except for some sections mentioned below. I recommend publication of this manuscript after minor changes.

Abstract-lines 16-17: This sentence somehow seems as if it came from nowhere. Is there any other control method other than biological which was implemented as a control option against the disease? Especially because you mentioned “appropriate formulation”. So, was there an inappropriate formulation, too?

Line 18- Again you said “new” formulation…was there any previous formulation that did not work?

Line 20. I think p value is not necessary in the abstract

Lines 35, 36. What does this mean “prolongs T. asperelloides PSU-P1 conidia by up to 6 months”, did you mean that it prolongs the viability of the conidia?

Introduction: lines 59-60 What did you mean by conditions in agricultural fields? Which problem?

line 73- Fresh conidia? Did they ever try to use dry conidia?

line 75-What is active conidia, did you mean viable conidia?

lines 76-77- How did you conclude this? I didn’t get why it is an effective method in agricultural fields. Why are agricultural fields so specific? Please elaborate on this. Why do they differ compared to e.g. forestry fields. Are all the agricultural fields the same for the application of Trichoderma, or are there any differences related to the crops that are planted in those fields, e.g. cornflower compared to fruit trees? Also, you didn’t mention if Trichoderma formulations were applied to the soil or if the plants were sprayed with Trichoderma.

Discussion. Line 247. Twin goals? Please modify this.

Line 248. I would say the first goal is a consequence of the second goal. If you reduce the severity of the disease, you will improve the shelf life of dragon fruit. Also, the goal of improving the shelf life was not mentioned in the introduction.

Line 254. Any emulsion or the same emulsion as yours-with coconut oil, tween 20 and conidia?

The manuscript is well written and the quality of English language is ok.

Author Response

This is a very interesting study with potential practical application. I read the manuscript with great interest. Honestly, after I read this, I started thinking of preparing the emulsion myself. The manuscript is well written and most parts are clear except for some sections mentioned below. I recommend publication of this manuscript after minor changes.

Answer: Thank you for your comments and gave valuable suggestions to improve this manuscript.

 

Abstract-lines 16-17: This sentence somehow seems as if it came from nowhere. Is there any other control method other than biological which was implemented as a control option against the disease? Especially because you mentioned “appropriate formulation”. So, was there an inappropriate formulation, too?

Answer: We have revised as “The development of a new formulation may offer an alternative way to address the problem of stem canker on dragon fruit caused by Neoscytalidium dimidiatum”

 

Line 18- Again you said “new” formulation…was there any previous formulation that did not work?

Answer: We have removed “new” from this sentence.

 

Line 20. I think p value is not necessary in the abstract

Answer: We have removed p value from the abstract.

 

Lines 35, 36. What does this mean “prolongs T. asperelloides PSU-P1 conidia by up to 6 months”, did you mean that it prolongs the viability of the conidia?

Answer: That’s right, we have revised as “this study prolongs viability of T. asperelloides PSU-P1 conidia”

 

Introduction: lines 59-60 What did you mean by conditions in agricultural fields? Which problem?

Answer: It mean several factors in agricultural field such as high temperature, light intensity, wind and moisture may effect on use of biological control agent. Therefore, we have revised as “However, conditions in agricultural fields such as temperature, light, wind and moisture represent a major limitations on the use of biocontrol agents”

 

line 73- Fresh conidia? Did they ever try to use dry conidia?

Answer: We have cultured Trichoderma on PDA and harvested only spore, therefore, we have never tried to use dry conidia.

 

line 75-What is active conidia, did you mean viable conidia?

Answer: Yes, we mean viable conidia. We have revised as “viable conidia”

 

lines 76-77- How did you conclude this? I didn’t get why it is an effective method in agricultural fields. Why are agricultural fields so specific? Please elaborate on this. Why do they differ compared to e.g. forestry fields. Are all the agricultural fields the same for the application of Trichoderma, or are there any differences related to the crops that are planted in those fields, e.g. cornflower compared to fruit trees? Also, you didn’t mention if Trichoderma formulations were applied to the soil or if the plants were sprayed with Trichoderma.

Answer: Based on your suggestion, we have better replaced with specific application of this formulation as “tin controlling plant disease”.

 

Discussion. Line 247. Twin goals? Please modify this.

Answer: We have revised as “The goals”

 

Line 248. I would say the first goal is a consequence of the second goal. If you reduce the severity of the disease, you will improve the shelf life of dragon fruit. Also, the goal of improving the shelf life was not mentioned in the introduction.

Answer: We have revised as “The goals of this study were to develop a formulation and to improve the protection of dragon fruit against stem canker.”

 

Line 254. Any emulsion or the same emulsion as yours-with coconut oil, tween 20 and conidia?

Answer: This citation used different ingredient of emulsion formulation. Therefore, emulsion formulation with coconut oil, tween 20 and conidia in water phase is a new formulation reported in this study

 

Comments on the Quality of English Language

The manuscript is well written and the quality of English language is ok.

Answer: Thank you very much.

Reviewer 2 Report

This results showed the formulation of Trichoderma  up to 6 months, and  effectively inhibits N. dimidiatum in vitro and reduces stem canker in vivo, and provided the biocontrol agent for this disease.  pls see the note below.

1. provide the control of inhibition rate test (the N. dimidiatum were incubated at 28±2C for 7 days). 

2. describe in detail how to measure the inhibition of this disease, Using dripping and spraying for the formulation of Trichoderma , how much each treatment. Enviromental condition?

3. how effective is in a field application?

Author Response

This results showed the formulation of Trichoderma up to 6 months, and  effectively inhibits N. dimidiatum in vitro and reduces stem canker in vivo, and provided the biocontrol agent for this disease.  pls see the note below.

Answer: Thank you for your comments and gave valuable suggestions to improve this manuscript.

1. Provide the control of inhibition rate test (the N. dimidiatum were incubated at 28±2°C for 7 days). 

Answer: Control of inhibition rate was the same as viability test as we mentioned “For the control plate, the agar plug of N. dimidiatum was placed on one side of the PDA plate only.”

2. Describe in detail how to measure the inhibition of this disease, Using dripping and spraying for the formulation of Trichoderma, how much each treatment. Enviromental condition?

Answer: For in vivo test, ability of formulation to reduce disease development was conducted on dragon fruit cladodes which incubated in a moist box. We have added volume for dropping formulation onto dragon fruit cladodes (1 mL). This study, we have tested ability of emulsion formulation in vivo only, the experiment on field observation should be verify in near future.

3. How effective is in a field application?

Answer: As we mentioned above, we have developed the formulation and tested antifungal ability against stem canker as in vivo and tested viability and antifungal ability up to 6 month. However, for field condition, experiments need to be conducted for further study.

Reviewer 3 Report

The manuscript with the title “Development of A Novel Emulsion Formulation of Trichoderma asperelloides PSU-P1 Conidia Against Stem Canker on Dragon Fruit Caused by Neoscytalidium dimidiatum” presents a series of experiments aiming to identify a suitable emulsion for the biocontrol of a common disease in dragon fruit crop. Biological control methods are highly sought. But their optimization is required. The manuscript has great potential in this regard.

Abstract
is excessively long and too detailed in regards with the Material and Method. Please make it brief and insist only on the novel aspects and results.


Introduction
Dragon fruit (the species on which the study was conducted is mentioned only once in the Introduction). Line 63 – please also give at least the up to date accepted scientific name of this plant species (is this Selenicereus costaricensis?) and give us a brief phrase e.g. “this is tropical crop cultivated for ….”
At the end of the introduction, the aim and objectives shall have their own paragraph and be clearly delimited. After reading the paper it becomes clear that one objective was to test mixing time of various formulation and to recommend the best one, another objective was to test inhibition capacity etc. Please delimit clearly the objectives, 2-3 or how many are fit for your experiment.

Material and Method
The statistical test applied seems all right. Although only post-hoc test (Tukey, respectively t-test) results are presented. Data distribution (that help to choose the most appropriate test: parametric or distribution free test) are not mentioned. Nor ANOVA results presented, which usually have to precede the application of post-hoc test (and why two different tests?).


Results

The Material and Method announced 12 experimental variants, but these were only to test mixing time (presented in figure 1), and not inhibition rate. That was a different experiments with its own variants (?). Not clear here.

Table 2 from Results presents a new set of variants that were further tested?  New experimental factors suddenly appear at results section: temperature etc.…

Please note that the exact same experimental variants presented at Material and Method section (M&M) shall have corresponding results at the Results section - same number of variants same names.
If two or more separate sets of experiments were performed please make separate subchapters at M&M section with the respective factors and variants.

Table 2, figure 3, figure 6 – appear to present plus/minus values, but what are they? Standard deviation, standard error or minimum-maximum?

Experimental variants have to be presented at M&M section clearly and explicitly.

RECOMMENDATIONS

The Results shall be presented in the order of the objectives presented at the end of the introduction (objectives are steps proposed for reaching an aim, name them first!).

Material and Method shall have subchapters with the assays conducted. The experimental variants shall be found identically corresponding at Results section.

Results section shall start with ANOVA results and descriptive statistics (average values, minimum, minimum, standard deviation, CV, p…).

Conclusions have to follow the same structure and order of the objectives given at the end of the introduction. Name your objectives first and answer to each of them in Conclusions.

Best regards.

some syntax mistakes

Author Response

The manuscript with the title “Development of A Novel Emulsion Formulation of Trichoderma asperelloides PSU-P1 Conidia Against Stem Canker on Dragon Fruit Caused by Neoscytalidium dimidiatum” presents a series of experiments aiming to identify a suitable emulsion for the biocontrol of a common disease in dragon fruit crop. Biological control methods are highly sought. But their optimization is required. The manuscript has great potential in this regard.

Answer: Thank you for your comments and gave valuable and tremendous suggestions to improve this manuscript.

Abstract
is excessively long and too detailed in regards with the Material and Method. Please make it brief and insist only on the novel aspects and results.

Answer: We have revised as suggestion by removed some materials and methods.


Introduction
Dragon fruit (the species on which the study was conducted is mentioned only once in the Introduction). Line 63 – please also give at least the up to date accepted scientific name of this plant species (is this Selenicereus costaricensis?) and give us a brief phrase e.g. “this is tropical crop cultivated for ….”

Answer: We have added more detail about dragon fruit and dragon fruit variety used in this study.

At the end of the introduction, the aim and objectives shall have their own paragraph and be clearly delimited. After reading the paper it becomes clear that one objective was to test mixing time of various formulation and to recommend the best one, another objective was to test inhibition capacity etc. Please delimit clearly the objectives, 2-3 or how many are fit for your experiment.

Answer: We have separated objective into the last paragraph of introduction and specify objective to fit with experiment.

Material and Method
The statistical test applied seems all right. Although only post-hoc test (Tukey, respectively t-test) results are presented. Data distribution (that help to choose the most appropriate test: parametric or distribution free test) are not mentioned. Nor ANOVA results presented, which usually have to precede the application of post-hoc test (and why two different tests?).

Answer: We have revised as “Mixing time, disease development, viability and antifungal ability were assessed. Data were achieved the parametric criteria of normality and homogeneity of variance necessary for the analysis of variance (ANOVA) and were subjected to one-way analysis of variance (ANOVA). Tukey’s test and T-testing were used to analyze statistically significant differences at a 95% significance level (p < 0.05).

For experiment composed of two treatment we used student t-test and we used f-test analysis and means comparison by Tukey’s test.

Results

The Material and Method announced 12 experimental variants, but these were only to test mixing time (presented in figure 1), and not inhibition rate. That was a different experiments with its own variants (?). Not clear here.

Answer: We have revised in materials and method in section 2.2 “The experiment was composed of two sets in order to get the most appropriate vegetable oil and to get the fastest mixing time. Firstly, to get the most appropriate vegetable oil, viscosity of formulation was tested by varied amounts of vegetable oils in 12 formulations as shown in Table 1 and mixing times were measured. Vegetable oil with the fastest mixing time was selected. Secondly, the selected vegetable oil was varied in volumes and added with additive dextrose in 6 formulations as shown in Table 2 and mixing time was measured to get the fastest mixing time formulation.”

Table 2 from Results presents a new set of variants that were further tested?  New experimental factors suddenly appear at results section: temperature etc.…

Answer: We have revised and rearrangement of materials and method of Table 1 and 2 to corresponding with results of Fig. 1 and Table 3, respectively.

Please note that the exact same experimental variants presented at Material and Method section (M&M) shall have corresponding results at the Results section - same number of variants same names.
If two or more separate sets of experiments were performed please make separate subchapters at M&M section with the respective factors and variants.
Answer: Thank you for this concern, we have revised and rearrangement the set of experiments, especially in materials and methods section 2.2, which showed table 1 and 2 to correspond with Fig. and Table 3, respectively.

Table 2, figure 3, figure 6 – appear to present plus/minus values, but what are they? Standard deviation, standard error or minimum-maximum?

Answer: We have added “Values are expressed as means ± standard deviation (SD).”

Experimental variants have to be presented at M&M section clearly and explicitly.

Answer:

RECOMMENDATIONS

The Results shall be presented in the order of the objectives presented at the end of the introduction (objectives are steps proposed for reaching an aim, name them first!).

Answer: We have revised the results correspond with revised objective of this study.

Material and Method shall have subchapters with the assays conducted. The experimental variants shall be found identically corresponding at Results section.

Answer: We have revised according to this comments.

Results section shall start with ANOVA results and descriptive statistics (average values, minimum, minimum, standard deviation, CV, p…).

Answer: We have added results with data of minimum to maximum and standard deviation (SD) in results especially in section 3.1, 3.3 and 3.4.

Conclusions have to follow the same structure and order of the objectives given at the end of the introduction. Name your objectives first and answer to each of them in Conclusions.

Answer: We have revised the conclusion correspond to revised objective of this study.

Best regards.

Round 2

Reviewer 3 Report

Dear authors,

All my comments were addressed and resolved. Please see below some small details that need adjustment. Also, please revise English style and grammar where necessary. The tense used is not right in some places.


Line 61 – change from KIN to SKIN

Line 88 – objectives (with plural add “s”) were …

Conclusions
Line 345: “formulation composed of coconut oil, DW, and tween 20 in a ratio of 30:60:10 required a great mixing time.” Instead of “great” I think it should be “shortest” mixing time. According to table 3 this was the fastest ones to mix/emulsify.


Caption of: figure 1, figure 3 and figure 6 it is good the authors added “Values are expressed as means ± standard deviation (SD) which indicate by error bars”, but please delete “which indicate by error bars”, in order to avoid any possible confusion with standard error which is a different thing. If you wish to be very precise you could say e.g. “…means ± standard deviation (SD) indicated by whiskers” or whiskers denote standard deviation from mean.


Caption of figure 4, figure 5, I do not think here is necessary to add “Values are expressed as means ± standard deviation (SD) which indicate by error bars” because the standard deviation whiskers are not visible on these graphs. Maybe they would be visible if the scale would start at 2 or 2.5 in figure 4, and at 30% in figure 5, because this would enlarge a bit the graph line and marks.

Note
in vitro and in vivo shall be written with italics because are in latin

Best regards.

English style and grammar

Line 63-64 “… commonly cultivated for consume”, replace with “… for consumption”

Line 64 “According to Thailand locates in tropical…” maybe authors want to say “Thailand is located in tropical and sub-tropical climate suitable to the cultivation of dragon fruit.”

 

Author Response

Reviewer 3

Dear authors,

All my comments were addressed and resolved. Please see below some small details that need adjustment. Also, please revise English style and grammar where necessary. The tense used is not right in some places.

Answer: Thank you for your comments, we have carefully checked in this manuscript according to your comments.


Line 61 – change from KIN to SKIN

Answer: We have revised with yellow highlight.

Line 88 – objectives (with plural add “s”) were …

Answer: We have revised with yellow highlight.

Conclusions
Line 345: “formulation composed of coconut oil, DW, and tween 20 in a ratio of 30:60:10 required a great mixing time.” Instead of “great” I think it should be “shortest” mixing time. According to table 3 this was the fastest ones to mix/emulsify.

Answer: We have revised as “the shortest mixing time.


Caption of: figure 1, figure 3 and figure 6 it is good the authors added “Values are expressed as means ± standard deviation (SD) which indicate by error bars”, but please delete “which indicate by error bars”, in order to avoid any possible confusion with standard error which is a different thing. If you wish to be very precise you could say e.g. “…means ± standard deviation (SD) indicated by whiskers” or whiskers denote standard deviation from mean.

Answer: We have revised according to reviewer comments.


Caption of figure 4, figure 5, I do not think here is necessary to add “Values are expressed as means ± standard deviation (SD) which indicate by error bars” because the standard deviation whiskers are not visible on these graphs. Maybe they would be visible if the scale would start at 2 or 2.5 in figure 4, and at 30% in figure 5, because this would enlarge a bit the graph line and marks.

Answer: We have removed this “Values are expressed as means ± standard deviation (SD) which indicate by error bars” from figure caption of Figs. 4 and 5.

Note
in vitro and in vivo shall be written with italics because are in latin

Answer: We have italicized in vitro and in vivo throughout this manuscript.

Best regards.

 

Comments on the Quality of English Language

English style and grammar

Line 63-64 “… commonly cultivated for consume”, replace with “… for consumption”

Answer: We have revised as “for consumption”

Line 64 “According to Thailand locates in tropical…” maybe authors want to say “Thailand is located in tropical and sub-tropical climate suitable to the cultivation of dragon fruit.”

Answer: We have revised according to reviewer comments.