Elicitor Activity of Curdlan and Its Potential Application in Protection of Hass Avocado Plants against Phytophthora cinnamomi Rands

Round 1

Reviewer 1 Report

Authors present a greenhouse study in which curdlan is assessed as potential tool to deal with root rot disease in avocado caused by P. cinnamomi. I think introduction could be extended with more information of the defence-related enzymes analysed. MM&MM, results and discussion sections are very well presented. Throughout the manuscript, readers may find some parts difficult to read because an inappropriate use of English.

Other comments are detailed below:

Lines 39-40 (L39-40): the sentence needs to be redrafted.

L41-44: Please, extend a bit more this part for readers who are not familiar with this crop. Why is Hass the most common cultivar? Due to desirable traits in fruit? Is it easier to cultivate? On the other hand, why the pathogen specially affects to this variety?, is it due to its higher extension or it is due to the highly susceptibility levels with respect other varieties?

L45-48: Please, redraft the sentence.

L55: The format of reference entries should be homogenised. Numbers or surnames? Please, follow the author guidelines in this respect.

L59: I suggest to move the last part of the sentence to the beginning of the next paragraph

L72-73: I suggest to modify the sentence into: These elicitors include glycoproteins, carbohydrates (like β-1,3-glucan), chitin, fatty acids, proteins, glycosphingolipids, peptides[8-10]

L76: Please, homogenise the acronym you will use throughout the manuscript (CRDO or Curd?)

L108: Why did you use this 5.316mg/ml dosage? In the work you referenced above, the dosage used in potato was 250 µg/mL. Did you perform any preliminary assay to choose the most appropriate dosage?

L113: You used 2 plants and three replicates. This sentence is confusing here. You should move forward. It becomes clear once you explain the sampling times and sample preparation. Beyond this, I believe some information is still missing. Where did the experiment took place? Green house, or under growth chamber conditions? What about temperature and humidity? Please, give more details

L243: Please, use the treatments tags in the table: Curd+Phy / Phy

L249, L269, L275…: This info should appear in introduction section, instead of MM&MM section

L339-341: Please, remove this.

L362: Why do you think activity of APX and GR was not possible to be measured? Any explanation? Any suspicion?

L430-435: Very long sentence. Difficult to read. Please, redraft

L467-475: Do you know other studies evaluating curdland, or other beta-glucans, under field conditions?  

Author Response

Dear Reviewer 

In the following paragraphs, we added the responses to your questions. We appreciate your contribution to the improvement of the manuscript.

Reviewer 1:

Authors present a greenhouse study in which curdlan is assessed as potential tool to deal with root rot disease in avocado caused by P. cinnamomi. I think introduction could be extended with more information of the defence-related enzymes analyzed. MM&MM, results and discussion sections are very well presented. Throughout the manuscript, readers may find some parts difficult to read because an inappropriate use of English.

Specific comments

Reviewer 1:

Reviewer 1: Lines 39-40 (L39-40): the sentence needs to be redrafted.

Authors: Accepted suggestion.

Reviewer 1: L41-44: Please, extend a bit more this part for readers who are not familiar with this crop. Why is Hass the most common cultivar? Due to desirable traits in fruit? Is it easier to cultivate? On the other hand, why the pathogen specially affects to this variety?, is it due to its higher extension or it is due to the highly susceptibility levels with respect other varieties?

Authors: It has been suggested that organoleptic characteristics and a better post harvest conservation of fruit of Hass avocado has influenced in its increased production worldwide. About pathogen, modifications were made into the text.

Reviewer 1: L45-48: Please, redraft the sentence.

 Authors: Accepted suggestion.

Reviewer 1: L55: The format of reference entries should be homogenized. Numbers or surnames? Please, follow the author guidelines in this respect.

Authors: Accepted suggestion.

Reviewer 1: L59: I suggest to move the last part of the sentence to the beginning of the next paragraph

Authors: Accepted suggestion.

Reviewer 1: L72-73: I suggest to modify the sentence into: These elicitors include glycoproteins, carbohydrates (like ß-1,3-glucan), chitin, fatty acids, proteins, glycosphingolipids, peptides[8-10]

Authors: Accepted suggestion.

Reviewer 1: L76: Please, homogenize the acronym you will use throughout the

manuscript (CRDO or Curd?)

Authors: Accepted suggestion. Curd will be used along  the document

Reviewer 1: L108: Why did you use this 5.316mg/ml dosage? In the work you referenced above, the dosage used in potato was 250 μg/mL. Did you perform any preliminary assay to choose the most appropriate dosage?

Authors: The amount of curland applied per plant, not per leaf, was 13.29 mg (5mL, 2658 mg/L) and not 5.316 mg/mL. A concentration 1000X higher than that reported by Li et al [11] was chosen to improve the possibility of response in avocado trees. The reference was considered to determine the application method (coated foliar surface), but these authors do not specify how much elicitor was applied per plant. Preliminary data (unpublished) show us that the concentration used in our research has no inhibitory effect on the growth of P. cinnamomi. Adittionally, in the present investigation, a trial was previously carried out with a smaller number of plants to establish whether it was better to apply the elicitor 5 days earlier or 1 day earlier, as evaluated in the study carried out by Li et al. (2014), showing us results with the evaluated concentration 24 h before pathogen inoculation.

Reviewer 1: L249, L269, L275…: This info should appear in introduction section, instead of MM&MM section

Authors: The recommendation don’t was realized because the mentioned lines don’t correspond to MM&MM section. Instead, they are in the results section.

Reviewer 1: L339-341: Please, remove this.

Authors: Accepted suggestion.

Reviewer 1: L362: Why do you think activity of APX and GR was not possible to be measured? Any explanation? Any suspicion?

Authors: Modifications were made into the text. In this case we could suppose that avocado defense response under the treatments conditions, is not associated to an increase of this enzymatic pathways, at least under the detection umbral of the applied technique

Reviewer 1: L430-435: Very long sentence. Difficult to read. Please, redraft

Authors: Accepted suggestion.

Reviewer 1: L467-475: Do you know other studies evaluating curdland, or other beta-glucans, under field conditions?

 Authors: The studies carried out in order to elucidate the specific effect of b-glucans on plant defense and the improvement of the tolerance of crops of commercial interest have been carried out mainly in greenhouses, under controlled conditions.

Reviewer 2 Report

Overall comments

The use of elicitors in plant protection is interesting. However, it is difficult to see from these short-term experiments whether curdlan is effective or not. This appears to be an initial pilot experiment. The plants were only monitored up to 9 days after the experimental infection. The trends in the enzymes assayed are difficult to assess and not consistent. 

The Discussion section is excessively long for the data presented. Can the authors either shorten it, concentrating on effects in avocado, or include additional experiments that demonstrate effects more clearly and over longer time-courses?

Specific comments

It might be worth stating that although oomycetes are superficially similar to fungi, they are now considered to be phylogenetically distinct from them and placed in the Stramenopiles. Physiological and structural differences are reasons why many fungicides are not very effective against them. 

Is there a source for the statement that 25% of the production costs are from fungicide inputs? (line 50)

The manuscript is readable but many of the sentences are run together with commas. Perhaps the authors, or a colleague, could look at this when finalising for publication.

Fig 2a, vertical axis should be labelled 0.1 , 0.2 , 0.3 like Fig 2b.

Figs 3a and b should be in the same order as 2a and 2b for consistency. Also vertical axis in Fig 3a should be labelled 1 , 0.8  and so on.

The comment at lines 339 - 341 seems to have been acted upon. 

Line 348 Is there a source for the statement that curdlan can be used in organic agriculture?

Author Response

Dear Reviewer, In the following paragraphs, we added the responses to your questions. We appreciate your contribution to the improvement of the manuscript.

Reviewer 2:

The use of elicitors in plant protection is interesting. However, it is difficult to see from these short-term experiments whether curdlan is effective or not. This appears to be an initial pilot experiment. The plants were only monitored up to 9 days after the experimental infection. The trends in the enzymes assayed are difficult to assess and not consistent.

The Discussion section is excessively long for the data presented. Can the authors either shorten it, concentrating on effects in avocado, or include additional experiments that demonstrate effects more clearly and over longer time-courses?

Authors: About the commentary “The plants were only monitored up to 9 days”, the phenotype and enzyme data do not overlap, because the lesion measurement did not develop at the same rate, it was a slower process. At 3 hai, there was still no lesion increase. At 24 hai there was some lesion noticeable, but not measurable for comparison.  Also, the lesion increase stopped after 9 days, so by 312 hai (13 days) there was no lesion data.

Specific comments

Reviewer 2: It might be worth stating that although oomycetes are superficially similar to fungi, they are now considered to be phylogenetically distinct from them and placed in the Stramenopiles. Physiological and structural differences are reasons why many fungicides are not very effective against them.

Authors: Accepted suggestion.

Reviewer 2: Is there a source for the statement that 25% of the production costs are from fungicide inputs? (line 50)

Authors: This is supported by the reference in the paragraph, corresponding to Romero-Correa et al. (2014).

Reviewer 2: The manuscript is readable but many of the sentences are run together with commas.

Authors: Accepted suggestion. Modifications were made into the text.

Reviewer 2: Perhaps the authors, or a colleague, could look at this when finalizing for publication.

Authors: Accepted suggestion.

Reviewer 2: Fig 2a, vertical axis should be labelled 0.1 , 0.2 , 0.3 like Fig 2b.

Authors: Accepted suggestion. Modifications were made into the text.

Reviewer 2: Figs 3a and b should be in the same order as 2a and 2b for consistency. Also vertical axis in Fig 3a should be labelled 1 , 0.8 and so on.

Authors: Accepted suggestion. Modifications were made into the text.

Reviewer 2: The comment at lines 339 - 341 seems to have been acted upon.

Authors: Modifications were made into the text.

Reviewer 2: Line 348 Is there a source for the statement that curdlan can be used in organic agriculture?

Authors: There are not direct references about it. For that reason, the expression was modified into the text.