The Preharvest Application of Essential Oils (Carvacrol, Eugenol, and Thymol) Reduces Fungal Decay in Lemons

Round 1

Reviewer 1 Report

In this manuscript, the authors found that the use of Carvacrol at 100 µl/L could be a potential alternative to the current fungicides used during the postharvest storage of lemon fruits, maintaining their fruit quality. The data are clean and convincing, and the manuscript is relatively well written. However, there are still several minor points that need to be addressed. 

- Explain why all quality analyses were not performed every period? just 0 and 35. day.  Although the study is based on the prevention of deterioration, periodic analysis should have been performed in the storage study.

- The treated substances have quite pungent odors. In particular, the smell of carvacrol, which was recommended as a result of the study, is very pungent. The smell may have affected the taste. Why wasn't the taste test performed?

- Other revisions are marked on the text. 

Reviewer,

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

Thank you very much for all the valuable comments. We really appreciate all the useful comments that you have provided and that will help improving our original manuscript. Please find below a detailed list of all your comments and suggestions and the corresponding answers and modification performed in the revised manuscript. All changes have been included and highlighted in purple ink in the manuscript.  

Comment 1. Explain why all quality analyses were not performed every period? just 0 and 35. day.  Although the study is based on the prevention of deterioration, periodic analysis should have been performed in the storage study.

All quality analyses were performed on every period; however, data was not relevant as the main differences were observed at harvest and after 35 days of storage. Although, if you think it could be better to present them too, we are happy to supply this data and include it as supplementary material, or we can even think about changing the graphs or presenting this data in a table.

A new phrase has been added to results, indicating that even though the analytical parameters were studied weekly for 35 days, data is only presented at harvest and after 35 days of storage:

Line 214- Quality parameters were analysed weekly, although data has only been presented at harvest and after 35 days of storage.

Besides, few changes have also been made to clarify the fact that these analytical parameters were only represented at those days:

Line 215- Weight losses (WL) were studied and presented after 35 days of storage at 10 ^°C in ‘Fino’ (Figure 3A) and ‘Verna’ lemon fruit (Figure 4A).

Line 239- TSS and TA were also studied and represented at harvest and after 35 days of storage (Figure 3D, 4E, Figure 4D, 4E).

Comment 2. The treated substances have quite pungent odors. In particular, the smell of carvacrol, which was recommended as a result of the study, is very pungent. The smell may have affected the taste. Why wasn't the taste test performed?

That’s a very good question, it could have been interesting to do a taste test of the resulted lemons. However, as the solution with EOs was applied 5 days before harvest in the lemon trees and they are volatiles compounds, we were not expecting any residual smell on the lemons. Besides, when they were transported and handed into the lab no smell was noticed at any time, even though we were working at room temperature until they were stored. It also needs to be considered the fact that lemons are not consumed with the peel.

Revision marked on the text. Keywords alphabetical order, abbreviation changes, change the word ‘lemon fruits’ to ‘lemon fruit’, missing reference 1.

All of these changes have been carried out and marked on the manuscript.

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The research studied the effect of carvacrol, eugenol and thymol individual and their combination in preharvest on fungal incidence of lemon fruits, which can provide theoretical guidance and technical support for the storage and preservation disease control of lemon fruits. However, the present results were not insufficient to support the conclusion and too little data in the MS, the author had better supplement some data related to diseases, for example, PAL, C4H, 4CL, etc.

 

1.     Abstract:

(1) There were too many descriptive statements in line 9-13 and they can be included in the introduction. Abstract had better go straight to the point and get to the point.

(2) Line 20, “results have shown that” should be changed to “results showed that”.

2.     Introduction:

(1)   Line 28-29 should be added the references.

(2)   Line 89-90, reference 28 is strawberry, 29 and 30 are tomato, 31-33 are lettuce, 34 is cherry tomato and 35 is strawberry. They can't correspond with the contents in line 89-90.

(3)   The introduction should be added some latest research progress.

3.     Materials and Methods:

(1)   The information about the lemon trees was not detailed. For example, tree age, row spacing, and where the fruit was harvested, etc?

(2)   To what extent of the foliar spraying has not been explained in the MS.

(3)   Is it appropriate to determine the harvest maturity based on the diameter size?

(4)   The concentration of CV, EG and TH was 100, 500 and 1000 µg/mL in line 105-109, why it changed to 100, 500 and 1000 µL/L in line 172-173??  µg/mL and µL/L are the same unit?????

4.     Results:

(1) The concentration of CV was conducted in 100, 500 and 1000 µg/mL and the better effect was 100 µg/mL? If 50 µg/mL showed the same effect as 100 µg/mL, if so, the reagent cost is an issue to consider. The author also mentioned in the last sentences of the conclusions.

(2) Why were Fig 3 and Fig 4 not done with significant differences (p< 0.05) with different lower-case letters like Fig 1 and Fig 2.

5.     Discussion:

(1)   Discussion need to be improved and should be precise. For example, line 268-269, if is this the first study in elucidating the effect of pure EOs applied in preharvest against fungal decay in citrus fruit.

(2)   Line 344 should leave a blank space.

6.     Reference:

The format of the references is too messy.

(1)   Where is reference 1?

(2)   Line 392, pp,

(3)   Line 409, vol,

(4)   Line 423 and 429, no pages,

(5)   Line 434, no doi, etc

All the references should be consistent.

The English is ok and just pay attention to tenses and some sentences.

Author Response

Dear reviewer,

Thank you very much for all the valuable comments. We really appreciate all the useful comments that you have provided and that will help improving our original manuscript. Please find below a detailed list of all your comments and suggestions and the corresponding answers and modification performed in the revised manuscript. All changes have been included and highlighted in orange ink in the manuscript. 

Comment 1. Abstract.

• There were too many descriptive statements in line 9-13 and they can be included in the introduction. Abstract had better go straight to the point and get to the point.

These descriptive statements are already included in the introduction (line 45-50) and the first two phrases have been summarised.

 

Line 9-Lemon fruit postharvest losses are mainly due to the presence of fungal diseases. Current postharvest decay strategies rely on synthetic chemical fungicides; however, consumers are demanding fruit free of any chemical residue. The use of new natural alternatives to fungicides, including essential oils, is emerging due to their potential antimicrobial activity.

 

• Line20 “results have shown that” should be changed to “results showed that”.

 

Line 19-Results showed that carvacrol at the lowest concentration (100 µL/L) provided the lowest fungal incidence with non-negative effect on the lemon fruit quality parameters during storage, while the highest concentrations and the combination of essential oils resulted in the opposite effect.

Comment 2. Introduction:

• Line 28-29 should be added the references.

References have been added

• Line 89-90, reference 28 is strawberry, 29 and 30 are tomato, 31-33 are lettuce, 34 is cherry tomato and 35 is strawberry. They can't correspond with the contents in line 89-90.

All reference numbering has been updated and text presented as follows:

 

Line 96- Few studies have been carried out in preharvest, such as those on tomatoes [31,32], lettuce [33–35] and strawberry [36,37].

 

• The introduction should be added some latest research progress.
  Two latest research works have been added to the introduction.

 

Line 58- Therefore, the biological effect of EOs have been widely documented by different authors, elucidating their capability to disrupt the permeability of mycelial cell walls and inducing plant defense mechanisms [9–13].

 

 

Comment 3. Material and Methods

• The information about the lemon trees was not detailed. For example, tree age, row spacing, and where the fruit was harvested, etc?

 

The information about the lemon trees has been included (tree age and spacing) as follows:

 

Line 106- . Two different lemon cultivars were used for these experiments, 12-year-old ‘Fino 49’ lemon trees grafted on Citrus macrophylla and 15-year-old ‘Verna’ lemon trees grafted on Citrus aurantium planted at 7 x 5 m.

 

The location and date of harvest have already been included:

 

Line 103- The experiments were carried out in two consecutive harvest seasons (2021-22 and 2022-23) in an outdoor commercial plot of lemon trees located in Fortuna (Murcia, Spain) under organic agronomic practices.

 

Line 108- ‘Fino 49’ lemon trees were treated and harvested in January and ‘Verna’ lemon trees were treated and harvested in June.  

 

• To what extent of the foliar spraying has not been explained in the MS.

Treatments were applied to leaves and lemon fruits by sprying.

 

Line 119-  All treatments were applied by spraying 5L per tree in leaves and lemon fruit.

 

• Is it appropriate to determine the harvest maturity based on the diameter size?

 

The harvest maturity was determined based on the diameter size and the characteristic yellow colour, this information has been added:

 

Line 122- ‘Fino’ and ‘Verna’ lemon fruit were harvested at a yellow commercial ripening stage when they presented the optimal diameter (around 55 mm) and they were then transferred to the lab within 2 hour period.

 

• The concentration of CV, EG and TH was 100, 500 and 1000 µg/mL in line 105-109, why it changed to 100, 500 and 1000 µL/L in line 172-173?? µg/mL and µL/L are the same unit?????

 

The unit was µL/L. It has been corrected in the Material and Methods section and reviewed in the whole manuscript.

Comment 3. Results

• The concentration of CV was conducted in 100, 500 and 1000 µg/mL and the better effect was 100 µg/mL? If 50 µg/mL showed the same effect as 100 µg/mL, if so, the reagent cost is an issue to consider. The author also mentioned in the last sentences of the conclusions.

 

A new phrase has been added in the conclusions regarding the future work needed on the optimisation of the concentration and the number of applications:

 

Line 395- Although, future work on the optimization of the EO concentration and the number of applications will be required, to reduce the stress that the fruit is suffering in presence of the preharvest treatment and to effectively control the fungal incidence.

 

• Why were Fig 3 and Fig 4 not done with significant differences (p< 0.05) with different lower-case letters like Fig 1 and Fig 2.

 

We have now included the significant difference letters, based on the statistical analysis, in the figure 3 and 4 and the statistical table, where the interaction of parameters is also studied for each of the quality parameters.

 

Besides, the captions of the figures have been modified as follows:

 

Line 233- Figure 3. Effect of preharvest treatment of Carvacrol at 100 µl/L (CV), Carvacrol and Eugenol at 100 µl/L (CV + EG), Carvacrol and Thymol at 100 and 500 µl/L (CV + TH) and Carvacrol, Eugenol and Thymol at 100, 100 and 500 µl/L (CV + EG + TH) in Weight loss (A), Firmness (B), Hue Angle (C), Total Soluble Solids (D), Titratable acidity (E) and Total Phenolic Content (F) in ‘Fino’ lemon fruit at harvest and 35 days of storage at 10 ^°C. Significant differences (p < 0.05) between treatments are presented with different lower-case letters. No significant differences are presented with a ‘ns’.

 

Line 255- Figure 4. Effect of preharvest treatment of Carvacrol at 100 µl/L (CV), Carvacrol and Eugenol at 100 µl/L (CV + EG), Carvacrol and Thymol at 100 and 500 µl/L (CV + TH) and Carvacrol, Eugenol and Thymol at 100, 100 and 500 µl/L (CV + EG + TH) Weight loss (A), Firmness (B), Hue Angle (C), Total Soluble Solids (D), Titratable acidity (E) and Total Phenolic Content (F) in ‘Verna’ lemon fruit at harvest and 35 days of storage at 10 °C. Significant differences (p < 0.05) between treatments are presented with different lower-case letters. No significant differences are presented with a ‘ns’.

Comment 4. Discussion

• Discussion needs to be improved and should be precise. For example, line 268-269, if is this the first study in elucidating the effect of pure EOs applied in preharvest against fungal decay in citrus fruit.

 

Discussion has been improved:

 

Line 279- This is the first work on the effect of pure EOs applied in preharvest against fungal decay in citrus fruit.

 

Line 294- Previous in vitro studies have already evaluated the antifungal activity of Thymus and Oreganum species EOs at different concentrations (50-4000 µl/L) against P. digitatum, P. italicum and G. citri-aurantii, achieving the best results when these cultures were subjected to the highest concentrations [14,16–19,21,23–25,43]. Besides, the antifungal effect of the studied EOs when they were applied in postharvest fruits has also been widely studied in citrus against Penicillium spp. [21,28], Alternaria alternata [27] and G. citri-aurantii [18,22], among others.

 

Line 311- Therefore, the present study is the first one in evaluating the effect of EOs in preharvest to control fungal decay of lemon fruit.

 

Line 325- The application of a higher content of EOs can result harmful for the lemon fruit, reducing lemon fruit quality and directly affecting the integrity of the cells and, therefore, becoming more susceptible to fungal decay [52]. In the present study the EOs showed a preventive effect, significantly reducing the fungal incidence and increasing the total phenolic content in lemon fruits. Although the mechanism of action of EOs is still not clear, this effect could be due to an increase of the Phenylalanine ammonia-lyase (PAL), enzyme implicated in the phenylpro-panoid pathway leading to the synthesis of phenolic compounds, and therefore their accumulation plays an important role in the plant defence mechanisms[53,54].

 

Line 329- Although this is the first study where EOs are applied in preharvest in citrus fruit to evaluate its antifungal effect during postharvest storage, there are few previous studies where these EOs have been applied in preharvest but in different crops.

 

Line 334- While in the present study, this increase in firmness was only observed in ‘Fino’ lemon fruit treated in preharvest with 100µl/L of carvacrol and not in the lemon fruit treated with a combination of carvacrol and thymol

 

• Line 344 should leave a blank space.

I am sorry but we do not understand what you mean with this comment, it would be great if you can clarify it. 

Comment 5. References. All the references should be consistent. The format of the references is too messy.

References format has been reviewed and missed information added.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Entitled: "Preharvest application of essential oils (carvacrol, eugenol and thymol) reduces fungal decay in lemon fruits". This work analyzed the impact of different concentrations individually and in combination applied in preharvest on the fungal incidence and quality parameters of lemon fruit. I find the topic of the paper is some application value. However, I do not think that the manuscript in its current form is convincing enough. Here is a list of my questions and comments:

1. Keywords

Authors should reconsider the order of keywords. The “quality” doesn’t use italics;

2. Materials and Methods

Some key experimental procedures and parameters are unclear. Please recheck and describe them in detail;

L97-100. Are lemon trees grown indoors or outdoors?

L105. Please explain the reasons for choosing the three concentrations.

L110. What is the composition of the solution?

L112. The description of foliar spraying treatments is unclear. Do the treatments involve all the leaves on the tree? Why do you treat the leaves and not the fruit? As mentioned in L302-314, the antimicrobial effect of EO treatments can be achieved by interacting with cellular membranes and destructing organelle of fungus. How to explain the antifungal effect of RO in this experiment?

L115-122. This is a confusing description. How many fruits were selected for each of the treatments?

3. Results

The significance letter mark of figure 1 and figure 2 is wrong. Figure 3 and figure 4 do not indicate significant differences.

L250. Why do you measure the total phenolic content of the peel instead of the whole fruit?

4.Discussion

 

Authors should focus on the reasons and potential mechanisms for the impact of different Eos treatments on lemon, rather than the difference between the two fruits. 

 Minor editing of English language is required

Author Response

Dear reviewer,

Thank you very much for all the valuable comments. We really appreciate all the useful comments that you have provided and that will help improving our original manuscript. Please find below a detailed list of all your comments and suggestions and the corresponding answers and modification performed in the revised manuscript. All changes have been included and highlighted in green ink in the manuscript. 

Comment 1. Keywords. Authors should reconsider the order of keywords. The “quality” doesn’t use italics.

The order of the keywords has been changed. As this comment has already been suggested from Reviewer one, the changes has been included in purple ink:

Line 24- Keywords: Citrus limon; fungal incidence; postharvest losses; preharvest treatment; quality.

Comment 2. Materials and Methods. Some key experimental procedures and parameters are unclear. Please recheck and describe them in detail.

• L97-100. Are lemon trees grown indoors or outdoors?

Lemon trees were grown outdoor in a commercial plot located in Fortuna, Murcia.

Line 104- The experiments were carried out in two consecutive harvest seasons (2021-22 and 2022-23) in an outdoor commercial plot of lemon trees located in Fortuna (Murcia, Spain) under organic agronomic practices.

• Please explain the reasons for choosing the three concentrations.

The three concentrations selected for the experiment was based on previous studies on oranges and lemons, where concentrations between 400 -1000 µL/L was providing the best results. We included lower concentrations as in preharvest lower concentrations of EOs are normally used.

First, we have specified the concentrations that were used for those specific studies:

• Line 72- In oranges, the use of thymol coating at 2% was tested against digitatum development, reducing the decay incidence [26].
• Line 76- A different study using the dipping method checked the effect of Thymus vulgaris, whose main components are carvacrol and thymol, on “Navel” oranges, resulting in a great reduction on fruit decay at 400 µL/L
• Line 78- In “Valencia” oranges the optimal concentration of Thymus vulgaris applied in postharvest that achieved the highest fungal decay reduction was 1000 µL/L [18]. Besides, the combined application of eugenol, thymol and carvacrol (250 µL/L) in sweet cherries significantly reduced the ripening process and increased the antioxidant content, parameters that improve the defense system of the fruit [29].
• Line 84- Both EOs were applied with wax in the packing lines at different concentrations (10-500 µL/L), resulting in a higher reduction in the infected fruit surface in those lemon fruit treated with 500 µL/L [21,30].

Then, in the material and methods section, three references regarding the selected concentrations have been added:

Line 112- Three blocks of five lemon trees were selected for the experiment for each of the treatments. In the harvest season 2021-22, lemon trees were treated with Carvacrol (CV), Eugenol (EG) and Thymol (TH) at three different concentrations (100, 500 and 1000 µL/L) according to previous work [18,29,40].

• What is the composition of the solution?

The composition of the control solution has been specified in the MM section in line 116, the order of those two phrases has been changed and then the control solution has been specified.

Line 117- All EO treatments were prepared by diluting the pure EOs (Sigma-Aldrich, Madrid, Spain) in tap water with 0.1% of wetting solution. In both seasons control trees were treated with an aqueous solution containing 0.1% of wetting solution.

• The description of foliar spraying treatments is unclear. Do the treatments involve all the leaves on the tree? Why do you treat the leaves and not the fruit? As mentioned in L302-314, the antimicrobial effect of EO treatments can be achieved by interacting with cellular membranes and destructing organelle of fungus. How to explain the antifungal effect of EO in this experiment?

Treatments were applied to leaves and lemon fruits by sprying. This comment has also been suggested by the reviewer 2, and therefore changes are presented in orange ink:

Line 119- All treatments were applied by spraying 5L per tree in leaves and lemon fruit.  

The antifungal effect of the EOs when they are applied at preharvest can be explained with the fact that once EOs get in contact with leaves and lemon fruit, as it has been previously reported, an increase on the phenolic content is observed and therefore an increase in the shikimic acid and phenylpropanoid metabolism pathways, enhancing the defense system of the plant. Therefore, the EOs applied in the fruit and leaves is acting therefore as a fungicide.

A new paragraph has been added in the discussion to clarify this effect:

Line 326- In the present study the EOs showed a preventive effect, significantly reducing the fungal incidence and increasing the total phenolic content in lemon fruits. Although the mechanism of action of EOs is still not clear, this effect could be due to an increase of the Phenylalanine ammonia-lyase (PAL), enzyme implicated in the phenylpropanoid pathway leading to the synthesis of phenolic compounds, and therefore their accumulation plays an important role in the plant defense mechanisms[53,54].

• L115-122. This is a confusing description. How many fruits were selected for each of the treatments?

Line 124- Then, lots of 15 fruits (three replicates of five lemons each) uniform in size, colour and without any physical damage were selected for each of the treatments and sampling days and stored for 35 days at 10 ^⁰C and 85% of relative humidity (RH).

Comment 3. Results. The significance letter mark of figure 1 and figure 2 is wrong. Figure 3 and figure 4 do not indicate significant differences.

Figure 1 and Figure 2 has been changed; significance differences letters have been put in the correct order. And the legend has been modified to clarify the order of the significance letters:

Line 190- Figure 1. Fungal decay incidence (%) in ‘Fino’ (A) and ‘Verna’ (B) lemon fruit from the 2021-22 harvest season control and treated at preharvest with Carvacrol (CV), Eugenol (EG) and Thymol (TH) at three different concentrations (100, 500 and 1000 µl/L) after 7, 14, 21, 28 and 35 days of storage at 10 °C. Significant differences (p < 0.05) between treatments are presented with different lower-case letters, corresponding to the order of the treatments in the legend. 

Line 208- Figure 1. Fungal decay incidence (%) in ‘Fino’ (A) and ‘Verna’ (B) lemon fruit from the 2022-23 harvest season control and treated at preharvest with Carvacrol (CV), Eugenol (EG) and Thymol (TH) at three different concentrations (100, 500 and 1000 µl/L) after 7, 14, 21, 28 and 35 days of storage at 10 °C. Significant differences (p < 0.05) between treatments are presented with different lower-case letters, corresponding to the order of the treatments in the legend. 

For Figure 3 and Figure 4, statistical differences between treatments have been added to the graph. Besides, the significant differences between time and the interaction of both parameters are included in the Table.  As this comment has also been suggested by reviewer 2, the comments are presented in orange ink.

• Why do you measure the total phenolic content of the peel instead of the whole fruit?

The total Phenolic Content (TPC) was measured on the peel, as the main expected changes directly related to the fungal incidence are occurring in the peel. TPC changes that occurs in the peel will directly affect the fungal decay, as the peel is the first barrier that the pathogens get in contact with.

Comment 4. Discussion. Authors should focus on the reasons and potential mechanisms for the impact of different Eos treatments on lemon, rather than the difference between the two fruits.

The discussion has been improved and a few sentences have been included providing more information about the potential mechanism for the impact of different EOs. There was already a sentence about this:

Line 326- In the present study the EOs showed a preventive effect, significantly reducing the fungal incidence and increasing the total phenolic content in lemon fruits. Although the mechanism of action of EOs is still not clear, this effect could be due to an increase of the Phenylalanine ammonia-lyase (PAL), enzyme implicated in the phenylpropanoid pathway leading to the synthesis of phenolic compounds, and therefore their accumulation plays an important role in the plant defense mechanisms[53,54].

There was already a sentence where the potential mechanism involved when essential oils are applied in fruit:

Line 383- . This increase has been associated with the fact that applying EOs to the surface of the fruits and leaves on the tree may induce a stressful environment, resulting in an increase of the shikimic acid and phenylpropanoid metabolism pathways, enhancing the defense system of the plant [59].

Comment 5. Comments on the Quality of English Language:  Minor editing of English language is required

Minor editing of the English of the manuscript have been carried out and changes have been included in red ink.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

 

The author has done some modifications or give the explanations in the revised version, I think in can be accepted and published in the Agriculture with some detailed modifications. For example, reference 2.

“Line 344 should leave a blank space. I am sorry but we do not understand what you mean with this comment, it would be great if you can clarify it.” About the above sentence, I mean there was a space between the data and unit. The same problem of line 119 in revised MS.

 

 

I hope that the authors can check the MS again carefully.

 

I hope that the authors can check the MS again carefully andthen accept it.

Author Response

Dear reviewer,

Thank you very much for the second round of valuable comments. All changes have been included and highlighted in blue ink in the manuscript. 

Comment 1. The author has done some modifications or give the explanations in the revised version, I think in can be accepted and published in the agriculture with some detailed modifications. For example, reference 2.

The reference two has been modified as follows:

Line 417- FAOSTAT. Production Statistics. 2021. Available online: https://www.fao.org/faostat/es/#data/QCL

Comment 2. “Line 344 should leave a blank space. I am sorry but we do not understand what you mean with this comment, it would be great if you can clarify it.” About the above sentence, I mean there was a space between the data and unit. The same problem of line 119 in revised MS.

The blank spaces have been included as suggested:

Line 120- All treatments were applied by spraying 5 L per tree in leaves and lemon fruit.

Line 365- While in the present study, this increase in firmness was only observed in ‘Fino’ lemon fruit treated in preharvest with 100 µl/L of carvacrol and not in the lemon fruit treated with a combination of carvacrol and thymol.

                         

Comment 3. Comments on the Quality of English Language: I hope that the authors can check the MS again carefully and then accept it.

The Quality of English Language has been reviewed in the Material and Methods section, changes are presented in red ink:

Line 109- For the experiment, three blocks of five lemon trees were selected for each of the treatments. In the harvest season 2021-22, lemon trees were treated with Carvacrol (CV), Eugenol (EG) and Thymol (TH) at three different concentrations (100, 500 and 1000 µL/L) according to previous work [18,29,40].

Line 112- Based on the results obtained in the first season, the concentrations that provided the best results were selected for the second harvest season (2022-23): 1)CV at 100 µL/L; 2) a combination of CV and EG, both at 100 µL/L (CV + EG); 3) a combination of CV and TH, at 100 and 500 µL/L, respectively (CV + TH); and 4) a combination of the three EOs, CV and EG at 100 µL/L and TH at 500 µL/L (CV + EG + TH).

Line 123- Then, lemons/lemon fruit were transferred to the lab within a 2-hour period. Afterward, lots of 15 fruits (three replicates of five lemons each) uniform in size, colour and without any physical damage were selected for each of the treatments and sampling days and stored for 35 days at 10 ^⁰C and 85% of relative humidity (RH).

Decay incidence methodology has been reviewed and some changes have been included:

 

Line 123- Decay incidence was determined weekly in a parallel experiment where lemon fruit were stored in a commercial facility. For this experiment, ten boxes of 100 lemon fruit from each of the different treatments were stored in the commercial storage at 10 ^⁰C and 85% RH. Decay incidence was evaluated after 7, 14, 21, 28 and 35 days by identifying and discarding lemon fruit with disease symptoms [41]. The fungal decay was expressed as decay accumulated; the number of lemon fruit decayed on every previous sampling day were summed up. The formula used was:

Decay (%) = (total decayed fruit/total evaluated fruit) X 100

 

Line 148- All parameters were measured in each of the treatments, and results were expressed as mean ± SE.

Line 161- Results (mean ± SE) were expressed as mg of gallic acid equivalent to 100 g^-1 of fresh weight (FW). 

Author Response File: Author Response.pdf