Chemical Characterization of Trichoderma spp. Extracts with Antifungal Activity against Cocoa Pathogens

Reviewer 1comments

Response to reviewer comments

Report:

The manuscript entitled “Comparative inhibition analysis of cacao pathogens by extracts obtained

from Trichoderma species” deals with the characterization of chemical compounds in the extract of

four Trichoderma isolates, which exerted growth inhibition toward cocoa pathogens in vitro.

Overall, I think that the manuscript could be reconsidered for publication after major revisions.

Firstly, an accurate revision of the English style and grammar is strongly recommended. I will not

indicate the suggestions for the English language because there are too many errors. I am not an

English mother tongue, but I am expert enough to judge that there is a need for a deep revision of

the language.

The authors appreciate the reviewer's recommendations to improve the manuscript.

Then, I would suggest modifying the title, focusing more on the chemical characterization of the

extracts rather than on the growth inhibition against the pathogens, which was tested in vitro. For instance:

Chemical characterization of Trichoderma sp. extracts with antifungal activity towards cocoa

pathogens

The suggestion was considered, and changes were done in the manuscript.

Abstract

The first two lines resume too poorly the introduction. Please put the study in the right scientific

context and state the objective of the study (see also below)

The suggestion was considered, and changes were done in the manuscript.

Keywords

Eliminate endophyte, add Moniliophthora

The suggestion was considered, and changes were done in the manuscript.

Introduction

1)The first paragraph dedicated to the pathogens is poor. The authors need to enrich it with the help

of a pathologist, describing briefly how the considered pathogens infect cocoa plants, which organs

are the targets, which developmental stage etc. in order to understand how Trichoderma could

counteract the infection by the direct antifungal activity of the metabolites identified in the extract.

The suggestion was considered, and changes were done in the manuscript.

Introduction

2)The ability of Trichoderma species to induce systemic resistance in the host plants is considered

one of the main mechanisms through which this fungus exerts its beneficial activity, beyond direct

antifungal activity or mycoparasitism. Thus, please, dedicate a paragraph to “induced systemic

resistance” in the Introduction section, with adequate references.

The suggestion was considered, and changes were done in the manuscript.

Introduction

3) The objective of the study is not well-focused, because chemical characterization is the core of

this study. Thus the authors need to rephrase it. “this study addresses the chemical characterization

of the extract of four Ecuadorian Trichoderma strains which proved to inhibit the in vitro growth of cocoa pathogens etc…”

The suggestion was considered, and changes were done in the manuscript.

Figure 3 is not easily readable. I suggest making 2 figures, one for M. perniciosa and another for M.

roreri, also because the comparison between the pathogens is not correct from a statistical point of

view.

The suggestion was considered, and changes were done in the manuscript.

Figure 4 and Table 2. For more complete information, the retention time of the compounds should be given in the table, and the compound name related to the major peaks should be reported in the figure. This will facilitate the readers in recognizing the peak/compound

The Figure 1 (before Figure 4) was edited, and number of main chromatographic peaks were added. The retention index is normalized retention time in order to minimize the effects of experimental GC conditions on the magnitude of retention index.

Zellner, B. D. A., Bicchi, C., Dugo, P., Rubiolo, P., Dugo, G., & Mondello, L. (2008). Linear retention indices in gas chromatographic analysis: a review. Flavour and Fragrance Journal, 23(5), 297-314.

Line 126 Substitute “of Trichoderma spp.” with “in the ethyl acetate extracts of the different

Trichoderma spp. isolates”

The suggestion was considered, and changes were done in the manuscript.

Line 170 The sentence is interrupted. Clearly, some text is missing and cannot be evaluated at the

moment. It regards the possible use of the findings of the study, thus it is a very important piece of

text that is lacking. Please check and add the missing text when resubmitting, to permit its evaluation

The interrupted sentence was deleted. The missing text was part of the conclusions. That’s why the interrupted sentence was at the end of the “GCMS metabolite profile” section.

Line 190 Substitute “unpublished data” with an adequate reference or provide the identification

method details. In addition, and more importantly, identify the species for T. sp. C3A isolate, as

done for the other 3 isolates. Nowadays molecular biology methods allow identifying Trichoderma

at the species level.

It is important to indicate that this work was not focus on the molecular identification of Trichoderma strains. However, additional detail about the method identification were added (Line 797-802 and Table 3).

All the Trichoderma isolates were processed in the same way, but the C3A isolate was only identified up to genus. This could be explained because the ITS region used for sequencing is universal for fungi. They may not be so specific and for this reason the sequence of this isolate did not achieve a high identity some specific species. Nevertheless, further work is underway to sequence other genes and obtain more information to arrive at a better identity of it.

Line 201-203 define how long was the incubation period, the size of Petri dish, and which medium

(PDA?)

This information is in “Fungal strains” section. Line 803-809

Trichoderma strains were incubated for 7 days in order to give the culture enough time to form robust mycelia, which are essential for the successful production of enzymes and metabolites [59].

Liu, J.; Liu, G. Analysis of Secondary Metabolites from Plant Endophytic Fungi. In Plant Pathogenic Fungi and Oomycetes. Methods in Molecular Biology; Ma, W., Wolpert, T., Eds.; Humana Press: New York, NY, 2018; Vol. 1848, pp. 25–38.

Line 231-275 give the references for all the methods used

The reference used is in line 997.

Chemical screening tests of ethyl acetate extracts were performed as reported elsewhere [65]

Tiwari, P.; Kumar, B.; Kaur, M.; Kaur, G.; Kaur, H. Phytochemical Screening and Extraction: A Review. Internationale Pharmaceutica Sciencia 2011, 1, 98–106.

Line 293 Percentage data must be transformed before ANOVA. Did you? Otherwise, you need to

repeat ANOVA on transformed data and perform the Tukey test on these new data. Then you can

report percentage data in the figure, but the letters must refer to transformed data.

The suggestion was considered, and changes were done in the manuscript.

Percentage data was transformed, and Anova analysis was repeated. The new significative differences could be observed in Figure 4.

Conclusions could be written more accurately

Line 300 “bioactivity could be attributed to detected compounds” please detail which ones

The suggestion was considered, and changes were done in the manuscript.

Line 301 “strain C10 could be use as raw material” please say it better. A strain cannot be

considered a raw material

The suggestion was considered, and changes were done in the manuscript.

Line 300-302 This is true if direct antagonism is a mechanism that can counteract cocoa infection

by these pathogens. This is why I asked the authors to describe the infection in the Introduction.

Anyway, please add a sentence regarding the necessity to perform further studies to highlight also mycoparasitism ability, possible induced resistance mechanisms, as well as experiments in vivo.

The suggestion was considered, and changes were done in the manuscript.

Line 302-303 delete “and contribute relevant information about endophytic fungi from Ecuador”

The suggestion was considered, and changes were done in the manuscript.

Line 88 and 298 overgrew, overgrowth? Perhaps, did the authors mean “stimulated, stimulation”?

The word correction was considered, and changes were done in the manuscript.

Reviewer 2 comments

Response to reviewer comments

Report: grammar correction, suggestion and questions were placed in a pdf document of the manuscript. Questions were placed below to answer to the reviewer.

The authors appreciate the reviewer's grammar corrections and recommendations to improve the manuscript.

abundant? higher concentrations?

“The most metabolites” was changed by “the largest number of”

How do you justify?

Fungal endophytes produce several compounds such as plant growth hormones (auxins, citokinis and gibberellins), which can help also promote the growth of fungi [56].

Villavicencio, M.; Schuller, L.; Espinosa, F.; Noceda, C.; Sosa, D.; Pérez-Martínez, S. Foliar Endophytic Fungi of Theobroma Cacao Stimulate More than Inhibit Moniliophthora Spp. Growth and Behave More as an Endophytes than Pathogens. agriRxiv 2020, 2020, doi:10.31220/AGRIRXIV.2020.00019.

to what?

The interrupted sentence was deleted. The missing text was part of the conclusions. That’s why the interrupted sentence was at the end of the “GCMS metabolite profile” section.

checked?

10 mg/mL was change to 10 mg/mL

Did the incubation period 7 days? Isn't this a short time?

Trichoderma strains were incubated for 7 days in order to give the culture enough time to form robust mycelia, which are essential for the successful production of enzymes and metabolites [59].

Liu, J.; Liu, G. Analysis of Secondary Metabolites from Plant Endophytic Fungi. In Plant Pathogenic Fungi and Oomycetes. Methods in Molecular Biology; Ma, W., Wolpert, T., Eds.; Humana Press: New York, NY, 2018; Vol. 1848, pp. 25–38.

the method used by Liu and Liu is different from the method used here. It should be revised

The Liu and Liu used by method was used as reference. The sentence was edited as indicated below:

The extraction of secondary metabolites from Trichoderma strains was followed by Liu and Liu method with some modifications [59].

How did you disrupted the mycelia for extract the SM?

Mycelia disruption was not necessary since this work focused on extracellular metabolites. Thus, ultrasonic bath of ethyl acetate containing SFM was not used before reduced rotary evaporation.

Why recovery times extended for 4 days? what is the role of ethyl acetate?

During process it was noted that on the fourth day all extractables in ethyl acetate were obtained after exhaustive extraction. The extractive process was followed until the color fades as recommended protocols for recovery of secondary metabolites [60].

Kjer, J.; Debbab, A.; Aly, A.H.; Proksch, P. Methods for Isolation of Marine-Derived Endophytic Fungi and Their Bioactive Secondary Products. Nature Protocols 2010 5:3 2010, 5, 479–490, doi:10.1038/nprot.2009.233.

? obscure?

The text “indicate that strain C10 could be use as raw material for the development of a potential control agent against witches’ broom, and contribute relevant information about endophytic fungi from Ecuador” was deleted.

Reviewer 3 comments

Response to reviewer comments

Report:

This manuscript deals with antifungal activity of ethyl acetate extracts of Trichoderma species. The authors evaluated the activity, and further analyzed chemical components in the extracts. The examination was carried out carefully, and the results were described in detail. I think the results in this study is important on this field, however it is hard to see which parts of this paper are new findings. So, I think this manuscript should be reconstructed as below before publication.

The authors appreciate the reviewer's recommendations to improve the manuscript.

# In Results and Discussion, the author should describe “Chemical screening”, followed by “GC-MS metabolite profile” and “Antifungal activity”

The suggestion was considered, and changes were done in the manuscript.

# Please discuss the results to highlight the usefulness of C10.

The suggestion was considered, and changes were done in the manuscript.

# In Table 2, retention index is necessary? Can Table 2 be simplified?

Yes, it is necessary.

The retention index is normalized retention time in order to minimize the effects of experimental GC conditions on the magnitude of retention index.

Zellner, B. D. A., Bicchi, C., Dugo, P., Rubiolo, P., Dugo, G., & Mondello, L. (2008). Linear retention indices in gas chromatographic analysis: a review. Flavour and Fragrance Journal, 23(5), 297-314.

# The name of fungi should be expressed as only abbreviation (such as C10) through the manuscript except first part.

The suggestion was considered, and changes were done in the manuscript.

# Overall, “Results and discussion” should be condensed.

The “Results and Discussion” section was improved.