Phytochemical Profile and Activity against Fusarium Species of Tamarix gallica Bark Aqueous Ammonia Extract

Round 1

Reviewer 1 Report

The research (Phytochemical Profile and Activity against Fusarium Species of a Tamarix gallica Bark Aqueous Ammonia Extract) presented the characterization of the phytochemicals contained in the T. gallica bark aqueous ammonia extract by gas chromatography-mass spectrometry (GC−MS) as well as the evaluation of its antifungal activity for the control of Fusarium spp. Although the manuscript idea is good, some points need handling within the manuscript.

1.       Why did the authors choose an aqueous ammonia solution for extraction?

2.      What is the antifungal mechanism of the extract?

3.     Phytochemicals compounds in the extract of T. gallica must categorize according to their type as phenols, flavonoids, etc and the percentage of each compound must include according to the category.

4.      T. gallica must be in italics, check the whole manuscript.

5.      All the fungal names must be in italics, check the whole manuscript

6.  Antifungal activity plate photos must add to the manuscript.

7.      Antifungal effect experiment is missing compared with a standard antifungal agent.

8.      Discussion part should supplement with new references; also the whole manuscript needs recent references.

Author Response

The research (Phytochemical Profile and Activity against Fusarium Species of a Tamarix gallica Bark Aqueous Ammonia Extract) presented the characterization of the phytochemicals contained in the T. gallica bark aqueous ammonia extract by gas chromatography-mass spectrometry (GC−MS) as well as the evaluation of its antifungal activity for the control of Fusarium spp. Although the manuscript idea is good, some points need handling within the manuscript.

Q1. Why did the authors choose an aqueous ammonia solution for extraction?

Response: Because, for bark, it provides metabolite recovery percentages substantially higher than other extraction media (such as, for example, hydroalcoholic medium), as evidenced in other works involving bark extracts [DOIs: 10.3390/ijms231911882; 10.3390/plants11243415; 10.3390/horticulturae8080672]. In this regard, it should be noted that aqueous ammonia media is widely used in lignocellulosic materials pretreatment, because -as noted in DOI: 10.1016/j.bej.2021.108106]- “it could remove acetyl groups from xylan polymers, selectively decompose and remove lignin from substrates, decrease cellulose crystallinity and enhance porosity with the low release of sugar degradation compounds. Furthermore, aqueous ammonia pretreatment is inexpensive, safe to handle, non-polluting, non-corrosive and recyclable due to its high volatility”. A new paragraph has been included at the beginning of subsection 2.3 to discuss this matter.

Q2. What is the antifungal mechanism of the extract?

Response: It is not possible to assign to the mixture of phytochemicals that constitutes the extract any mechanism, which -in any case- should rather be referred to the most representative phytochemicals (or to the synergy between some of them) and, subsidiarily, to metabolite types/categories.  

Although phenolic, flavonoids and organic acids are not considered antifungals per se, a very recent study on the antifungal properties of the extract of a species of the same genus (Tamarix aphylla) against plant pathogenic fungi [10.3390/microorganisms11010127], published after the submission of our manuscript, has suggested mechanisms of action for their antimicrobial effect. It would be due to their ability to induce hyper acidification via proton donation at the plasma membrane interface of the microorganism and intracellular cytosolic acidification. That may disrupt the H⁺-ATPase required for ATP synthesis [https://doi.org/10.3390/antiox9020165]. Initially, the hydrophobic phenolic compounds are likely to bind on the plasma membrane, cell wall, and lipopolysaccharide–water interface of the cell without penetration [10.3390/antiox10020188]. These phenolic phytochemicals might stack on the plasma membrane, which affects the membrane fluidity and destabilization and results in partial disruption. That allows some phenolics, such as hydroxyl benzoic acid, chlorogenic acid, gallic acid, and lactate, to enter the cytosol. On the other hand, the flavonoid antifungal activity is probably due to their ability to complex with extracellular and soluble proteins and cell walls, as described for lipophilic flavonoids that may disrupt microbial membranes [10.3389/fmicb.2019.00911]. Moreover, the mechanisms of phenol toxicity against microorganisms include enzyme inhibition by the oxidized compounds, possibly through reaction with sulfhydryl groups or through nonspecific interactions with the proteins. A summary of these mechanisms has now been included in subsection 4.1.

Q3. Phytochemicals compounds in the extract of T. gallica must categorize according to their type as phenols, flavonoids, etc and the percentage of each compound must include according to the category.

Response: To the best of the authors' knowledge, the categorization of extract components is of interest when it is not possible to identify individual phytochemicals. When individual assignments are available (as is the case when GC-MS assignments with high Qual values are obtained), the categorization may be regarded as a piece of idle information. Nevertheless, based on identified compounds and their percentages, both categorization and percentualization are feasible: dihydro-3-methylene-2,5-furandione and other alkaloids (10%); 2,6-dimethoxy-phenol, 4-hydroxy-3,5-dimethoxy-benzoic acid, sinapinaldehyde, 1-(2,4,6-trihydroxyphenyl)-2-pentanone, 1-(4-hydroxy-3,5-dimethoxyphenyl)-ethanone, and other phenolic compounds (50%);   syringaldehyde and other flavonoids (10%); trans-squalene and other triterpenes (12%); and hexadecanoic acid methyl ester and related esters (5%).

The following sentence has been added in subsection 3.2, immediately above Table 2 to address the issue raised by the Reviewer: “[…] If aforementioned compounds are grouped into categories, the extract of T. gallica bark consists of phenolic compounds (50%), triterpenes (12%), flavonoids (10%), alkaloids (10%), and fatty acid methyl esters (5%).”

Also, in the discussion, in subsection 4.1, we have mentioned that: “The high phenolics content is in good agreement with that reported in flowers by Boulabaa et al. [DOI 10.1007/s10616-013-9564-4] (135.3 mg GAE/g DW), and would explain the high antioxidant activity observed by Nisar et al. [10.1177/15353702221139208] and by Lefahal et al. [DOI: 10.2174/1386207323666201204141541].”

Q4. T. gallica must be in italics, check the whole manuscript.

Response: We assume that the Reviewer is referring to the title of subsection 4.2.1. Please kindly note that the manuscript was modified by the Editorial Office prior to sending it to peer review (in order to remove all Endnote references, the automatic numbering of figures and tables, and cross-references that we had used in our original submission). The italics were removed at this stage (we have compared both versions). The problem has been corrected and we have double-checked that every binomial name is italicized.

Q5. All the fungal names must be in italics, check the whole manuscript

Response: Please see the response to Q4 above. We have carefully checked the manuscript to find if any binomial names were in plain text instead of italicized, and corrected them accordingly.

Q6. Antifungal activity plate photos must add to the manuscript.

Response: Four new figures (Figures S1, S2, S3, and S4) have been included, as suggested by the Reviewer. The supplementary material section at the end of the manuscript has been updated accordingly.

Q7. Antifungal effect experiment is missing compared with a standard antifungal agent.

Response: Such comparison with standard fungicides is already presented in Table 4. Please kindly note that both mancozeb (a dithiocarbamate non-systemic agricultural fungicide with a market size in 2022 estimated at 795.4 million US$) and fosetyl-Al (an organophosphorus compound derived from ethylphosphite) are widely used fungicides to control various diseases on food and feed crops. Should the Reviewer refer to other fungicides routinely used to treat human and animal diseases, the requested comparison would make little sense, given that World Health Organization (WHO) Global Action Plan on Antimicrobial Resistance requires judicious pesticide use, indicating that conventional antibiotics should not be used for crop protection purposes due to heightened global concern about antimicrobial resistance.

Q8. Discussion part should supplement with new references; also the whole manuscript needs recent references.

Response: Nine new references have been added to the discussion, as requested by the Reviewer:

• Eng. J. 2021, 174, 108106, doi:10.1016/j.bej.2021.108106.
• Frontiers in Microbiology 2019, 10, 911, doi:10.3389/fmicb.2019.00911.
• Antioxidants 2021, 10, 188, doi:10.3390/antiox10020188.
• Antioxidants 2020, 9, 165, doi:10.3390/antiox9020165.
• Microorganisms 2023, 11, 127, doi:10.3390/microorganisms11010127.
• Combinatorial Chemistry & High Throughput Screening 2021, 24, 1671-1678, doi:10.2174/1386207323666201204141541.
• Experimental Biology and Medicine 2022, Online first, doi:10.1177/15353702221139208.
• Plants 2022, 11, 3415, doi:10.3390/plants11243415.
• An introduction to nonparametric statistics, First edition. ed.; CRC Press: Boca Raton, 2020; pp. 224.

Thus, the current version of the ms. includes 32 references published in the past 5 years (an article in 2023, 8 articles in 2022, 8 articles in 2021, 6 articles in 2020, 5 articles in 2019, and 4 articles in 2018).

Reviewer 2 Report

Good morning

The paper presented above, titled "Phytochemical Profile and Activity against Fusarium Species of a Tamarix gallica Bark Aqueous Ammonia Extract" is very interesting and only requires minor amendments to fit into the Publication spectra of the Journal Agronomy. In my honest opinion, these are the minor amendments necessary.

1. Title should remove the letter a before Tamarix.

2. Figure 1 is not self-explanatory as it shows letters a and b but not identifying which is which. 

3. Table 1 and 2 needs to be improved by adding information on the SIMILARITY INDEX of the identified compounds, relative to the program used in identifying the compounds.

4. Table 5. It is best to write the concentration of the plant extracts in mg/ml, not the units used. 

5. The references used are mostly old and can be replaced with the most recent references to upgrade the paper. Example, references 1, 4, 5, 8, 10, 11, 17, 19, 25, 30, 31, 37, 41, 44, 46, 48, 49, 52 and 59 may be replaced with most recent papers published in other Journals.

Author Response

The paper presented above, titled "Phytochemical Profile and Activity against Fusarium Species of a Tamarix gallica Bark Aqueous Ammonia Extract" is very interesting and only requires minor amendments to fit into the Publication spectra of the Journal Agronomy. In my honest opinion, these are the minor amendments necessary.

Q1. Title should remove the letter a before Tamarix.

Response: Corrected.

Q2. Figure 1 is not self-explanatory as it shows letters a and b but not identifying which is which.

Response: Corrected. The figure caption now reads: “(a) Tamarisk of the Paseo de San Pedro, in Llanes (Asturias, northern Spain), (b) trunk of a T. gallica specimen, (c) detail of T. gallica bark.”

Q3. Table 1 and 2 needs to be improved by adding information on the SIMILARITY INDEX of the identified compounds, relative to the program used in identifying the compounds.

Response: To address the Reviewer’s request, we have included the NIST library search reports provided by the external laboratory to which the GC-MS analyses were outsourced. The probability of the identification (i.e., quality of resemblance) has been included in Table 2 (‘Qual’ column). Concerning Table 1, the FTIR band assignments were manually made based on the literature (to the best of the authors’ knowledge, a reliable automatic band assignment software is not available for FTIR spectra), so no similarity indexes can be calculated.

Q4. Table 5. It is best to write the concentration of the plant extracts in mg/ml, not the units used.

Response: Corrected. We used μg/mL to allow direct comparison with our results, but we agree that the readability is improved if mg/mL units are used, as suggested by the Reviewer. Please kindly note that it has been moved to supporting information as per the request of another Reviewer.

Q5. The references used are mostly old and can be replaced with the most recent references to upgrade the paper. Example, references 1, 4, 5, 8, 10, 11, 17, 19, 25, 30, 31, 37, 41, 44, 46, 48, 49, 52 and 59 may be replaced with most recent papers published in other Journals.

Response: Four references ([1], [4], [8], and [11]) have been deleted, but the authors have not found suitable references to replace the rest of the above-cited. Given that -in our view- they are relevant, we have decided not to delete them. In this regard, it is worth noting that, for instance, there is no timeliness requirement in APA Style guidelines, which require that one should “cite the work of those individuals whose ideas, theories, or research have directly influenced your work. The works you cite provide key background information, support or dispute your thesis, or offer critical definitions and data”.

Nonetheless, to address the Reviewer’s suggestion, new (more recent) references have been added to address the issues raised during the peer-review process:

• Eng. J. 2021, 174, 108106, doi:10.1016/j.bej.2021.108106.
• Frontiers in Microbiology 2019, 10, 911, doi:10.3389/fmicb.2019.00911.
• Antioxidants 2021, 10, 188, doi:10.3390/antiox10020188.
• Antioxidants 2020, 9, 165, doi:10.3390/antiox9020165.
• Microorganisms 2023, 11, 127, doi:10.3390/microorganisms11010127.
• Combinatorial Chemistry & High Throughput Screening 2021, 24, 1671-1678, doi:10.2174/1386207323666201204141541.
• Experimental Biology and Medicine 2022, Online first, doi:10.1177/15353702221139208.
• Plants 2022, 11, 3415, doi:10.3390/plants11243415.

The revised version of the ms. includes 32 references published in the past 5 years (an article in 2023, 8 articles in 2022, 8 articles in 2021, 6 articles in 2020, 5 articles in 2019, and 4 articles in 2018), roughly half of the total references cited.

Reviewer 3 Report

The work is very well described that contributes to generating alternatives for the natural control of the Fusarium complex, one of the 10 most pathogenic fungi worldwide.

Some recomnendations:

Abstract:

Line 19: How many Fusarium species?

Line 30 Fusarium spp.

 Introduction

L 62: change chemicals: compounds

Materials and Methods

L226:  Add reference

Results: L236, What was the percentage of similarity in the identification compared with the team's database?

L242- 243, Modifiec:… Chemical structure of….(Figure 2).

Discussion

L325:  I recommend integrating the discussion, starting with the principal and general contribution and after the most relevant compounds, without subtitles.

Table 5. Recommend deleting table 5 and describing the information in the text.

Author Response

The work is very well described that contributes to generating alternatives for the natural control of the Fusarium complex, one of the 10 most pathogenic fungi worldwide.

Some recommendations:

Abstract:

Q1. Line 19: How many Fusarium species?

Response: We have specified that the activity was investigated against four species.

Q2. Line 30 Fusarium spp.

Response: ‘spp’ has been replaced with ‘spp.’

Introduction

Q3. L 62: change chemicals: compounds

Response: ‘Chemicals’ has been replaced with ‘compounds’, as suggested by the Reviewer.

Materials and Methods

Q4. L226:  Add reference

Response: A reference to J. E. Kolassa’s “An introduction to nonparametric statistics” (2020) has been included.

Q5. Results: L236, What was the percentage of similarity in the identification compared with the team's database?

Response: The probability of the identification (i.e., quality of resemblance) has been included in Table 2 (‘Qual’ column).

Q6. L242- 243, Modifiec:… Chemical structure of….(Figure 2).

Response: ‘Chemical structures of’ has been deleted from the caption of Figure 2.

Discussion

Q7. L325:  I recommend integrating the discussion, starting with the principal and general contribution and after the most relevant compounds, without subtitles.

Response: Given that the discussion is lengthy and covers different aspects that are not directly linked to each other, we would rather keep the subsections’ headings. In our view, the use of these subheadings helps to categorize our interpretations into themes.

Q8. Table 5. Recommend deleting table 5 and describing the information in the text.

Response: Table 5 has been moved to the supporting information file (Table S1) and the information has been summarized in the main document.

Round 2

Reviewer 1 Report

The authors gave sufficient answers and did most of the corrections but the manuscript still needs English editing.