Bark Extract of Uncaria tomentosa L. for the Control of Strawberry Phytopathogens^†

Round 1

Reviewer 1 Report

The manuscript is very well written and covers alternative bio fungicide as well as for comparison purposes, the results of experiments conducted with four conventional synthetic fungicides. 

I recommend some changes in order to improve the manuscript.

Lines 98 - 100: I suggest restructuring this part in order to avoid too many 'of'.

Lines 219-224: Please exclude references from the results section. References can be moved to the discussion.

Line 291-296: According to which methodology was the degree of severity determined? Is it the same as the severity of the disease explained in the material and methods section? 

In the discussion section, this is the essential part: Hence, it would be comparable to that of Mancozeb, for which full inhibition was attained at concentrations below 150 μg∙mL‐1 thus it should be emphasized more. Lines 355-360 could be explained further to highlight the previous observation. 

In the conclusion section authors state that immersion in a 1000 μg∙mL‐1 conjugate complex solution for 5 minutes resulted in a severity decay of 0.5. I wonder what is the cost rate for obtaining such solutions and would it be economically justified in organic strawberry production?

Author Response

Comments and Suggestions for Authors

The manuscript is very well written and covers alternative bio fungicide as well as for comparison purposes, the results of experiments conducted with four conventional synthetic fungicides.

I recommend some changes in order to improve the manuscript.

Q1.  98 - 100: I suggest restructuring this part in order to avoid too many 'of'.

Response: The sentence has been rewritten and two ‘of’ are no longer necessary. It now reads: “The extract was prepared from a composite sample consisting of the bark of ten Uncaria tomentosa specimens from La Merced, Chanchamayo, Peru. The bark samples, with a golden yellow (or light brown) color, were thoroughly mixed, dried, and reduced to a fine powder.”

Q2. Lines 219-224: Please exclude references from the results section. References can be moved to the discussion.

Response: The indicated paragraph has been deleted from the results section and has been integrated into subsection 4.1 of the discussion (“Octyl isobutyrate, the octyl ester of isobutyric acid (previously identified in Mangifera indica L. [24]), and mitraphylline, a pentacyclic oxindole (found in the leaves of Mitragyna speciosa (Korth.) Havil. [25] and in cat's claw bark along with several isomeric alkaloids [26]), were the two main phytocompounds present in the extract. Regarding the observed antimicrobial activity, […]”).

Q3. Line 291-296: According to which methodology was the degree of severity determined? Is it the same as the severity of the disease explained in the material and methods section?

Response: Yes, it is the same empirical scale explained in subsection 2.6. Nonetheless, to avoid potential confusion, we have added a clarification in the lines of section 3.4 indicated by the Reviewer (“[…] with a degree of severity of only 0.5 according to the empirical scale proposed by Romanazzi et al. [16]”).

Q4. In the discussion section, this is the essential part: Hence, it would be comparable to that of Mancozeb, for which full inhibition was attained at concentrations below 150 μg∙mL‐1 thus it should be emphasized more. Lines 355-360 could be explained further to highlight the previous observation.

Response: We have modified the indicated lines to highlight that Metalaxyl and Maconzeb, according to the MIC values reported in the literature (Table 7), would also be the most effective among the four selected synthetic fungicides, and we have re-written the last sentence to emphasize the fact that their activity would be lower than that of the COS-U. tomentosa conjugate complex (thus reinforcing the statement made in the previous paragraph).

Q5. In the conclusion section authors state that immersion in a 1000 μg∙mL^‐1 conjugate complex solution for 5 minutes resulted in a severity decay of 0.5. I wonder what is the cost rate for obtaining such solutions and would it be economically justified in organic strawberry production?

Response: The price of cat’s claw bark in natural product wholesalers is approximately 25 EUR/kg, out of which 720 g of extract (lyophilized) may be obtained, so that the final price of the extract turns out to be 35 EUR/kg. Concerning chitosan, the wholesale price is approximately 40 EUR/kg, which increases to 55 EUR/kg due to COS preparation costs. Hence, the cost of the reagents to prepare the conjugate complexes (in a 1:1 ratio) would be in the 45-50 EUR/kg range. Although this price is higher than that of synthetic fungicides (e.g., 8 EUR/kg for Mancozeb 75-80% formulates), 1 liter of a 1000 μg∙mL^‐1 conjugate complex solution would cost around 0.05 EUR, which would be affordable for organic strawberry production. We have discussed this in subsection 4.1 (below Table 7).

Reviewer 2 Report

The paper entitled “Bark extract of Uncaria tomentosa L. for the control of strawberry phytopathogens” is an interesting work about the protection of various pathogens of interest. The work is well written, with a relevant introduction and updated literature. The M&M are performed correctly, and the results are consistent with the methodologies. The conclusions are consistent with the results and the speculative aspects are limited. I suggest publication after minor revisions.

L.33. No in vivo pre-harvest tests have been performed, therefore it is appropriate to describe this aspect only as a theoretical potential

L.107. This part needs a more detailed description. How was the absence of infections assessed? What is the symptom picture of plants? How were the symptoms assessed?

L.115-119. A list of fungicides is difficult to understand in the absence of some details relating to the spectrum of action. Why were these products chosen? Against which pathogens (among those being tested) are they active?

L.122. Please indicate the code of the isolates.

L129-134. No literature was reported here. Why was this procedure used? For what purpose was it chosen?

L.186. What about the numerical consistency of groups?

General remarks for M&M: no statistical methods are reported. It would be interesting to know the phytotoxicity of the extract or at least report the need to evaluate it in the conclusions

Author Response

Comments and Suggestions for Authors

The paper entitled “Bark extract of Uncaria tomentosa L. for the control of strawberry phytopathogens” is an interesting work about the protection of various pathogens of interest. The work is well written, with a relevant introduction and updated literature. The M&M are performed correctly, and the results are consistent with the methodologies. The conclusions are consistent with the results and the speculative aspects are limited. I suggest publication after minor revisions.

Q1. L.33. No in vivo pre-harvest tests have been performed, therefore it is appropriate to describe this aspect only as a theoretical potential

Response: We agree with the Reviewer that the statement made about its potential application in preharvest was speculative, so the final sentence of the abstract has been re-written. It now reads: “Because of this effectiveness, higher than that attained with conventional synthetic fungicides, the bark extracts of cat’s claw may hold promise for strawberry crop protection.”

Q2. L.107. This part needs a more detailed description. How was the absence of infections assessed? What is the symptom picture of plants? How were the symptoms assessed?

Response: Please kindly note that the requested information was provided below, in subsection 2.6: the absence of natural infection was ensured by superficial disinfection with NaOCl 3% solution for 2 min; disease severity was determined by visual inspection of the percentage of surface infected (according to Romanazzi’s empirical scale), and absence of infection was verified by SEM inspection, as in [Agronomy 2021, 11(6), 1246; https://doi.org/10.3390/agronomy11061246].

Q3. L.115-119. A list of fungicides is difficult to understand in the absence of some details relating to the spectrum of action. Why were these products chosen? Against which pathogens (among those being tested) are they active?

Response: The fungicides included in the list were selected either due to their weak risk of resistance, their favorable toxicological and/or environmental profile, or their significant pathogen control ability. A clarification has been included at the end of subsection 2.1.

Q4. L.122. Please indicate the code of the isolates.

Response: The codes for two of the isolates have been indicated. In the case of B. cinerea, it was not available, so a reference to the article in which details on its provenance are provided has been included instead. The text now reads: “The fungal isolates of B. cinerea (code not available, but details on its provenance are provided in [17]), P. cactorum (CRD Prosp/59), and V. dahliae (MYC-1134) were supplied […]”

Q5. L129-134. No literature was reported here. Why was this procedure used? For what purpose was it chosen?

Response: A reference to (expired) US patent US2823223A on ‘Chemical products from bark digested in ammonia’ has been included. Please kindly note that the original 1958 procedure has been substantially modified, incorporating Green Chemistry techniques.

Concerning the choice of this procedure, digestion in an aqueous ammonia solution allows the dissolution of the polyphenols and other bioactive compounds of interest contained in the bark of U. tomentosa. The first paragraph in subsection 2.3 has been updated to clarify this point.

Q6. L.186. What about the numerical consistency of groups?

Response: We noted in the following paragraph that we carried out 3 repetitions with 15 fruits/(repetition*treatment), but we have moved this information to the line indicated by the Reviewer to avoid potential confusion. Concerning whether this number of fruits may suffice to obtain representative results, please kindly note that the chosen number of fruits per treatment and repetition is higher than that used by other authors: e.g., in [Agronomy 2021, 11(6), 1246; https://doi.org/10.3390/agronomy11061246] they used 6 fruits for each treatment and replicate. Further, we have now clarified that “[…] ensuring that all fruits were in the 20-30 g/fruit weight range and that they were larger than 25 mm in diameter” to indicate that fruits in each group were homogeneous.

Q7. General remarks for M&M: no statistical methods are reported.

Response: We thank the Reviewer for bringing this point to our attention: indeed, we showed the results of the ANOVA followed by Tukey’s HSD test in Table 5, but we had not included a statistical analysis subsection in Materials & Methods. We have now included the missing subsection (2.7) and a table footer in Table 5 to clarify the meaning of the letters next to the average±s.d. severity values obtained for each treatment.

Q8. It would be interesting to know the phytotoxicity of the extract or at least report the need to evaluate it in the conclusions

Response: The last sentence in subsection 4.2 has been updated to include this point, stating the need to evaluate phytotoxicity in addition to dosage and number of applications. It now reads: “[…] (although field experiments would be needed to confirm this point, to optimize the dosage and number of applications, and to exclude phytotoxicity).”