Inadequate Pollination Is a Key Factor Determining Low Fruit-to-Flower Ratios in Avocado

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments and Suggestions for Authors

Manuscript ID: horticulturae-2808032

The paper entitled “Inadequate pollination is a key factor determining low fruit to flower ratios in avocado” was carefully reviewed. The main aim of this study is to assess the role of honey bees in avocado production, and to identify factors affecting pollination efficiency and fruit set in avocado cultivars.

The authors conducted several experiments to measure and compare pollen deposition, germination and paternity of avocado flowers and fruits under different conditions, such as the number of beehives/ha, the presence and compatibility of pollen donors, and the temperature and timing of the flowering season. The authors hoped to provide useful information and recommendations for avocado growers and researchers to improve the yield and quality of this important crop.

Detailed comments:

Please check a few notes you can use to improve this manuscript:

-          The methodology is not well introduced in the abstract.  The abstract only mentions that the effect of different beehive densities on the percentage of flowers receiving pollen was evaluated, but does not explain how this was done. For example, it does not specify the number and location of experimental sites, the duration and timing of the experiment, the sampling and measurement methods, or the statistical analysis used. 

-          The main results are not well presented in the abstract. Add relevant quantitative data to the abstract (e.g. number of flowers analyzed, average percentage of flowers with pollen for each treatment group, fruit set, etc.).

-          Correctly write the common noun as honey bee (two words) and not honeybee, and stick to honey bee throughout the text. Refer to the following reference for more details:

o   William D. J. Kirk (2022) Is It Honey Bee or Honeybee? Bumble Bee or Bumblebee? Who Decides the Common Names of Bees?, Bee World, 99:2, 38-39, DOI: 10.1080/0005772X.2021.1982315).

-          Page 1, lines 31-36: Rewrite the entire paragraph to avoid plagiarism.

-          Page 1, lines 38-39: Add a reference for this sentence: “Most of the avocado international market worldwide relies on a single variety, ‘Hass’, which originated in California (USA) by a chance seedling almost 100 years ago”. Also, rewrite this sentence to avoid plagiarism.

-          Page 2, lines 41-49: Rewrite the entire paragraph to avoid plagiarism.

-          Page 2, lines 49-50: Add a reference for this statement: “This floral behavior is highly dependent on temperature and, consequently, the cycle can be different under warmer or colder conditions”.

-          Page 2, lines 57-58: Add a reference for this statement: “The duration of this overlap between floral stages is highly dependent on environmental conditions, mainly temperature”.

-          Materials & Methods: The experimental design of the subsection “2.1. Pollen deposition in a ‘Hass’ avocado orchard with a different number of beehives” is unclear, as it does not explain how trees were selected for sampling, and how sampling was randomized and repeated. This subsection could be improved by providing more details on methodology, such as beehive location, tree selection and identification, and sampling randomization and replication. Questions to be answered include: hives were placed in each row or block of the orchard; trees were numbered or labeled; sampling was stratified or balanced between treatments and orchard; hives were placed in each row or block of the orchard.

-          Materials & Methods (Subsection: 2.2. Pollen deposition in ‘Reed’, a late flowering avocado cultivar). The authors compared pollen deposition and fruit set of the 'Reed' and 'Hass' cultivars under different temperature conditions, but did not take into account other factors likely to affect pollination, such as the presence and diversity of pollinators, the availability and compatibility of pollen sources, and environmental conditions such as humidity and wind. These factors could vary between the two orchards and influence the results of the comparison. The authors should provide more details on how they controlled or measured these factors or acknowledge the limitations of their study.

-          Hive colony strength, pollinator diversity in the orchard, the number of bees per hive and the number of honeybee visits to each flower are all important factors that could affect pollination efficiency and fruit set in avocado. The authors should have measured or controlled these factors, or at least acknowledged their limitations and discussed their implications for the results and conclusions of the study. Perhaps you could suggest ways to improve the experimental design or data analysis to account for these factors.

-          Table 1: Indicate the number of hives introduced in each orchard and give the coordinates of each of the five orchards.

-          Page 5, line 202: The total number of fruits collected for paternity analysis was 728. However, the authors did not indicate the total number of fruits collected per orchard. This is important because the results could be biased or inaccurate.

-          The authors did not explain why they chose 10 and 24 beehives/ha as treatments for the pollen deposition experiment in the "Hass" orchard. But to evaluate pollen deposition in commercial avocado orchards (2.4. Pollen limitation in flowers of cv. Hass in commercial avocado orchards), they selected five 'Hass' orchards in which 6 beehives/ha were introduced. We know that the optimum hive density for avocado pollination varies according to region, cultivar, flowering period and availability of other pollinators. Therefore, the authors are requested to provide more details on the reasoning and justification for their choice of treatments.

-          The authors did not measure the number of bee visits per flower, but assumed that more hives would increase pollination efficiency and avocado fruiting. However, this assumption may not be valid, as bee visitation depends on many factors, such as flower attractiveness, availability of other pollen sources, weather conditions and hive colony strength. Therefore, measuring the number of bee visits per flower could provide more accurate and reliable information on the pollination process and fruit production. Different methods can be used to measure the number of bee visits, such as direct observation, video recording or radio frequency identification. I recommend that the authors measure the number of bee visits per flower, as this data will provide proof and precision for the current study.

-          Figures 6 and 7: Add standard deviations and letters to indicate significant differences between treatments.

-          I disagree with the authors regarding their conclusions, in particular this statement: "The number of adhered pollen grains to the stigma and the percentage of flowers with pollen at the female stage varies not only among years but also over the flowering season due to the fact that both floral behavior and insect activity are affected by environmental conditions, mainly temperature”. Temperature is not the only environmental factor influencing floral behavior and the activity of pollinating insects. Other factors, such as humidity, wind, light and precipitation, can also affect flower opening and closing, pollen viability and release, as well as pollinator foraging and movement. As well, orchard management practices can also vary and affect pollen deposition and pollination of avocado flowers. In addition, honey bees are affected by many factors other than temperature, such as flower availability and attractiveness, competition and cooperation with other pollinators, and colony health and management.

Author Response

We would like to thank the reviewer for the careful review of the manuscript. Below we describe the main changes made in response to the reviewers comments.

1- The methodology is not well introduced in the abstract.  The abstract only mentions that the effect of different beehive densities on the percentage of flowers receiving pollen was evaluated, but does not explain how this was done. For example, it does not specify the number and location of experimental sites, the duration and timing of the experiment, the sampling and measurement methods, or the statistical analysis used. 

The main results are not well presented in the abstract. Add relevant quantitative data to the abstract (e.g. number of flowers analyzed, average percentage of flowers with pollen for each treatment group, fruit set, etc.).

 

Following the recommendation of the reviewer, we have included additional result information in the abstract section.

 

2- Correctly write the common noun as honey bee (two words) and not honeybee, and stick to honey bee throughout the text. Refer to the following reference for more details:

William D. J. Kirk (2022) Is It Honey Bee or Honeybee? Bumble Bee or Bumblebee? Who Decides the Common Names of Bees?, Bee World, 99:2, 38-39, DOI: 10.1080/0005772X.2021.1982315).

 

We thank the reviewer for the reference and, following the recommendation, the manuscript has been carefully revised using honey bee along the new version of the manuscript.

 

3- Page 1, lines 31-36: Rewrite the entire paragraph to avoid plagiarism.

 

Done

 

4- Page 1, lines 38-39: Add a reference for this sentence: “Most of the avocado international market worldwide relies on a single variety, ‘Hass’, which originated in California (USA) by a chance seedling almost 100 years ago”. Also, rewrite this sentence to avoid plagiarism.

 

Done

 

5- Page 2, lines 41-49: Rewrite the entire paragraph to avoid plagiarism.

 

Done

 

6- Page 2, lines 49-50: Add a reference for this statement: “This floral behavior is highly dependent on temperature and, consequently, the cycle can be different under warmer or colder conditions”.

 

Done

 

7- Materials & Methods: The experimental design of the subsection “2.1. Pollen deposition in a ‘Hass’ avocado orchard with a different number of beehives” is unclear, as it does not explain how trees were selected for sampling, and how sampling was randomized and repeated. This subsection could be improved by providing more details on methodology, such as beehive location, tree selection and identification, and sampling randomization and replication. Questions to be answered include: hives were placed in each row or block of the orchard; trees were numbered or labeled; sampling was stratified or balanced between treatments and orchard; hives were placed in each row or block of the orchard.

 

Following the recommendation of the reviewer, we have included in this section more information about the methodology used.

 

8- Materials & Methods (Subsection: 2.2. Pollen deposition in ‘Reed’, a late flowering avocado cultivar). The authors compared pollen deposition and fruit set of the 'Reed' and 'Hass' cultivars under different temperature conditions, but did not take into account other factors likely to affect pollination, such as the presence and diversity of pollinators, the availability and compatibility of pollen sources, and environmental conditions such as humidity and wind. These factors could vary between the two orchards and influence the results of the comparison. The authors should provide more details on how they controlled or measured these factors or acknowledge the limitations of their study.

 

We have included more information indicating that both experimental orchards are in proximity one to another (less than 100m) and, therefore, no substantial differences on relative humidity and temperature conditions are expected. Moreover, no pollen donors are planted in proximity to any of the two orchards.

 

 

9- Hive colony strength, pollinator diversity in the orchard, the number of bees per hive and the number of honeybee visits to each flower are all important factors that could affect pollination efficiency and fruit set in avocado. The authors should have measured or controlled these factors, or at least acknowledged their limitations and discussed their implications for the results and conclusions of the study. Perhaps you could suggest ways to improve the experimental design or data analysis to account for these factors.

 

We fully agree with the reviewer in the fact that honey bee activity has not been analyzed in this work. Previous observations carried in the experimental orchards used in this work by our research group showed that honey bees are the main visitor of avocado flowers. In this work we decided to analyze the effect of beehive density on pollen deposition. Although differences could exist among beehives and additional factors could be playing a role, we still think that the results obtained highlight the importance of low pollen load on the low fruit to flower ratio observed in avocado.

In the new version of the manuscript we provide additional information on the approximate number of bees per beehive and that the health of the beehives was checked once a week. A sentence has also been included in the conclusion section suggesting that further work is necessary to evaluate the honey bee activity and its effect on yield.

 

 

10-  Table 1: Indicate the number of hives introduced in each orchard and give the coordinates of each of the five orchards.

 

Since the number of beehives per hectare was the same in those commercial orchards with beehives, we have included this information in the figure caption. However, due to data protection issues we are not allowed to publish the exact coordinates of the private orchards.

 

11- Page 5, line 202: The total number of fruits collected for paternity analysis was 728. However, the authors did not indicate the total number of fruits collected per orchard. This is important because the results could be biased or inaccurate.

 

We agree with the reviewer that having the production data would be of interest. However, we do not have access to those data from the private orchards.

 

12- The authors did not explain why they chose 10 and 24 beehives/ha as treatments for the pollen deposition experiment in the "Hass" orchard. But to evaluate pollen deposition in commercial avocado orchards (2.4. Pollen limitation in flowers of cv. Hass in commercial avocado orchards), they selected five 'Hass' orchards in which 6 beehives/ha were introduced. We know that the optimum hive density for avocado pollination varies according to region, cultivar, flowering period and availability of other pollinators. Therefore, the authors are requested to provide more details on the reasoning and justification for their choice of treatments.

 

In the new version of the manuscript, a sentence has been included in material and methods explaining this topic.

About 6 beehives per hectare is the usual recommendation in our avocado growing region and that is the reason why in the commercial orchards that number is the most frequent. However, preliminary results obtained by our research group showed a low percentage of flowers with pollen with that number of beehives and, consequently we decided to increase the number to 10 and 24 beehives per hectare. The results show that even with such a high number of beehives still most of the flowers do not receive pollen in the female stage.

 

 

13- The authors did not measure the number of bee visits per flower, but assumed that more hives would increase pollination efficiency and avocado fruiting. However, this assumption may not be valid, as bee visitation depends on many factors, such as flower attractiveness, availability of other pollen sources, weather conditions and hive colony strength. Therefore, measuring the number of bee visits per flower could provide more accurate and reliable information on the pollination process and fruit production. Different methods can be used to measure the number of bee visits, such as direct observation, video recording or radio frequency identification. I recommend that the authors measure the number of bee visits per flower, as this data will provide proof and precision for the current study.

 

Certainly, we also consider that the evaluation of  honey bee activity could have been of interest in the context of this manuscript. Unfortunately honey bee activity was not evaluated in this work since the main objective was not analyzing honey bee efficiency but testing the hypothesis that avocado productivity in our growing area is hampered by the low percentage of flowers that receive pollen at the female stage under different management conditions.

In our experimental orchards, based on preliminary results, flowers are mainly visited by honey bees. Also, the growers receive similar recommendations about the number of beehives to place in the orchard, regardless of the different pollen sources, pollinator diversity, beehive health, etc. For this reason, we decided to pay more attention on how the percentage of flowers that receives pollen changes when we increase the number of beehives placed in the orchard. The results obtained can open a way to maximize the percentage of flowers receiving pollen grains by increasing the number of beehives and the diversity or pollinating insects. Without a doubt, additional works must be carried out to evaluate insect activity, insect diversity visiting avocado flowers and how this activity could be affected by different environmental conditions.

 

14-   Figures 6 and 7: Add standard deviations and letters to indicate significant differences between treatments.

In figure 6, no changes have been made in the new version since in figure 6a, the results were already analyzed statistically and, in figure 6b, that shows the percentage of avocado flowers into each range of pollen load size on the stigma for each group, we consider that this is a complementary information to figure a, and statistical analyses are not necessary.

Figure 7 has been modified following the reviewer recommendation.

 

15- I disagree with the authors regarding their conclusions, in particular this statement: "The number of adhered pollen grains to the stigma and the percentage of flowers with pollen at the female stage varies not only among years but also over the flowering season due to the fact that both floral behavior and insect activity are affected by environmental conditions, mainly temperature”. Temperature is not the only environmental factor influencing floral behavior and the activity of pollinating insects. Other factors, such as humidity, wind, light and precipitation, can also affect flower opening and closing, pollen viability and release, as well as pollinator foraging and movement. As well, orchard management practices can also vary and affect pollen deposition and pollination of avocado flowers. In addition, honey bees are affected by many factors other than temperature, such as flower availability and attractiveness, competition and cooperation with other pollinators, and colony health and management.

 

We have removed temperature from this sentence because we agree that there are many more factors influencing insect activity and avocado floral cycle. We initially considered temperature as one of the most important factors since temperature was the most variable parameter through the flowering season under our environmental conditions.

With the objective to reduce the effect of different flower densities on bee attractiveness, homogeneous trees were selected. Moreover, to avoid external factors, that we could not control, we have compared the different treatments by collecting samples from trees with identical physiological conditions and under the same management practices. This information has been added in the new version of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The pollination deficiency in avocados is significant and needs to be pointed out with evidence like in this paper. but this paper has some missing points:

1. there is a lack of evidence on bee activity (the number of beehives is not sufficient).

2. there is a lack of overall flower counting or bloom index (high bloom low). The number of flowers would affect bee activity!

3. The yield data in the specific orchard over the years of research is missing. it should be correlated to the fruit set.

4. there is no correlation (and no explanation) between pollen count on the stigma and fruit set.

5. as any year has different conditions it is not obvious to have different bee treatments across years, especially in alternate species like avocados. 

6. the comparison between 'Reed' and 'Hass' in pollen count and its relation to temperature is weak as differences can relate to bee activity and different bee attractiveness to different cultivars. 

Author Response

We would like to thank the reviewer for the careful review of our manuscript. Below we describe in detail the changes made in response to the reviewers comments.

1-There is a lack of evidence on bee activity (the number of beehives is not sufficient).

We agree with the reviewer in the fact that the honey bee activity has not been directly evaluated in this work and we think that this would merit further more detailed analyses.

This comment was also made by Reviewer 1. As we stated above, previous observations carried in the experimental orchard by our research group showed that honey bees are the main visitor of avocado flowers. In this work, we decided to analyze the effect of beehive density on pollen deposition. Although differences could exist among beehives and additional factors could be playing a role, we still think that the results obtained highlight the importance of low pollen load on the low fruit to flower ratio observed in avocado.

In the new version, we include a sentence in the conclusion section suggesting that further work is necessary to evaluate the honey bee activity and its effect on yield.

2-There is a lack of overall flower counting or bloom index (high bloom low). The number of flowers would affect bee activity!

To avoid the effect of bloom index on pollen deposition due to differences in attractiveness to bees, we have included a sentence in the material and methods section explaining that trees showing similar flower density were selected for the study.

3-The yield data in the specific orchard over the years of research is missing. it should be correlated to the fruit set.

This is an interesting point to consider in future works. Our main objective was to evaluate the effect of beehive density on pollen deposition and percentage of fruit set retained mainly two months after the end of the flowering season. That is because in avocado, a high abscission of non-fertilized flowers occurs during the two months following the end of the flowering season. Also, in this period a high percentage of fruitlets drop mainly due to competition between developing fruits and the new vegetative growth; this is more conspicuous in indeterminate inflorescences that show new leaf growth at the top. The information of the initial fruit set together with that of the percentage of flowers that receive polled during the female stage indicates that a high number of flowers dropped due to inadequate pollination (low percentage of flowers with pollen and low pollen density on the stigma). For this reason, we decided to consider the percentage of flowers that are retained on the trees two months after the end of flowering season as a good indicator of pollination. Moreover, the yield could be affected by additional fruit drop due to environmental conditions (extremely high temperatures or strong winds), not related to pollination.

4-There is no correlation (and no explanation) between pollen count on the stigma and fruit set.

In this work, no significant correlation was observed among percentage of flowers receiving pollen during the female stage and the percentage of fruit retained neither at the end of June nor at the harvest time. Although we found significant differences on the percentage of fruit retained among treatments, this is probably due to the very low fruit set in all the cases (only 2 from 1000 flowers became into fruits).

 

6- As any year has different conditions it is not obvious to have different bee treatments across years, especially in alternate species like avocados. 

In this work, we have compared the effect of three different beehive density on the percentage of flowers receiving pollen and on fruit set. The treatments were made in different years because it is not possible to manipulate in the same orchard the number of honey beehives introduced. We have preferred to compare the treatments in different years in the same orchard, collecting samples from trees with identical physiological conditions and, moreover, avoiding external factors (such as proximity to nearby orchard with different management conditions) that we could not control. For that, we selected a monovarietal Hass orchard in which we have controlled production for the last 15 years with low alternation in productivity in which almost 100% of the fruits derived from close pollination from flowers of the same tree of different trees of the same cultivar. Certainly, differences on environmental conditions could exist among years, and for that, temperature and relative humidity was monitored during the flowering season for all the years. Moreover, the experiment was carried out during a minimum of two years to reduce the possible effect of environmental conditions in those parameters evaluated.

7-The comparison between 'Reed' and 'Hass' in pollen count and its relation to temperature is weak as differences can relate to bee activity and different bee attractiveness to different cultivars.

We agree with the reviewer that additional parameters could affect the attractiveness of Reed flowers for the bees such as nectar production and we mention this topic in the discussion. However, we consider that, in addition to other factors, the increase of the percentage of flowers with pollen observed in Reed, that flowered at the end of April when no other avocado pollen is available, may be explained by the optimal temperatures for avocado reproductive success. Moreover, this increase on pollen deposition under natural conditions was also observed in the cultivar Hass when we compared pollen deposition among different weeks along the flowering season when minimum, maximum and mean temperatures increase.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have carefully reviewed the revised version of the paper and the responses to my previous comments. The authors have endeavored to improve the manuscript on the basis of the comments provided during the initial review. However, it is essential to note that the manuscript still requires significant improvements, particularly in terms of methodology.

Major comments:

-          Although the authors provided additional details on the experimental set-up, including orchard location, tree characteristics and the introduction of hives in different years and densities, they did not explicitly address the concerns I raised:

o   Beehive location: More explicit details on the specific locations where hives were placed for each treatment and each year are needed. This information is crucial to understanding potential spatial effects on pollination.

o   Tree selection and randomization: My initial concerns about the clarity of the tree selection and randomization process remain. Additional information on how trees were selected, numbered or labeled, and how sampling was randomized and replicated would be appreciated.

o   Number of replicates: It would be useful to know the number of trees considered as replicates for each treatment and each year, as this information is crucial for assessing the statistical robustness of the results.

Taking these three points into account will considerably improve the clarity and transparency of the experimental design.

-          Materials & Methods (Subsection: 2.2): The authors have stated that the 2 experimental orchards are close to each other (100 m) and that no substantial differences in relative humidity and temperature conditions are expected. However, the authors have not provided any data or references to support this hypothesis. Furthermore, they did not explain how they measured or controlled other factors that could affect pollination efficiency and fruit set in avocado, such as colony strength, pollinator diversity in the orchard, number of bees per hive, number of bee visits to each flower and pollen source compatibility. Nor did they explain how they ensured that the rate of self-pollination was the same for both cultivars. These factors could also influence pollen deposition and fruiting in the 'Reed' and 'Hass' cultivars.

-          Line 218 (Total number of fruits collected per orchard):  It seems that the authors have not responded satisfactorily to my comment. They acknowledge that production data would be interesting, but they don't have access to such data from private orchards. However, they do not explain how they ensured that the sample was representative of the population.

-        I reiterate my previous comment regarding the measurement of the number of bee visits per flower. The authors provided no evidence or references to support their claims that flowers are primarily visited by honey bees and that increasing the number of hives would increase the percentage of flowers receiving pollen. Nor did they address other factors such as flower attractiveness, availability of other pollen sources, weather conditions and hive colony strength that might affect bee visitation and pollination efficiency.

Minor comments:

-        Lines 109-129: The paragraph is too long and could be divided into two paragraphs. I suggest splitting it to separate sampling and fixation methods from staining and observation methods.

-          Line 118: Replace “FAA” by “formalin-aceto-alcohol (FAA)”.

-          The manuscript still needs to be improved, especially in terms of grammar and formatting. For this reason, I suggest using an English-language editing service to proofread the manuscript, as some sentences are difficult to understand and contain numerous errors and typos.

-          Finally, I would like to remind the authors that in future revisions, the modified sections must be clearly marked for easier comparison by the reviewers.

Comments on the Quality of English Language

The manuscript still needs to be improved, especially in terms of grammar and formatting. For this reason, I suggest using an English-language editing service to proofread the manuscript, as some sentences are difficult to understand and contain numerous errors and typos.

Author Response

We would like to thank the reviewer for the comprehensive review that has helped to significantly improve our manuscript over our original submission. Here, we describe in detail the changes made in response to the reviewer’s comments.

 

- Beehive location: More explicit details on the specific locations where hives were placed for each treatment and each year are needed. This information is crucial to understanding potential spatial effects on pollination.

Following the recommendation of the reviewer, we have included additional information about beehive location in the new version of the manuscript.

 

-Tree selection and randomization: My initial concerns about the clarity of the tree selection and randomization process remain. Additional information on how trees were selected, numbered or labeled, and how sampling was randomized and replicated would be appreciated.

In the new version of the manuscript, we have included more information about the selection of trees, identification and also about sampling of the flowers.

Number of replicates: It would be useful to know the number of trees considered as replicates for each treatment and each year, as this information is crucial for assessing the statistical robustness of the results.

We have included more information about the replicates for each treatment in each experiment. 

- Materials & Methods (Subsection: 2.2): The authors have stated that the 2 experimental orchards are close to each other (100 m) and that no substantial differences in relative humidity and temperature conditions are expected. However, the authors have not provided any data or references to support this hypothesis. Furthermore, they did not explain how they measured or controlled other factors that could affect pollination efficiency and fruit set in avocado, such as colony strength, pollinator diversity in the orchard, number of bees per hive, number of bee visits to each flower and pollen source compatibility. Nor did they explain how they ensured that the rate of self-pollination was the same for both cultivars. These factors could also influence pollen deposition and fruiting in the 'Reed' and 'Hass' cultivars.

Since we expected no differences in temperature or humidity conditions between the two orchards due to their proximity, we did not record temperature or humidity in the Reed orchard during the flowering period. We agree with the reviewer that other potential factors could affect pollination efficiency and fruit set in avocado. Initially, we considered that the observed increase in the percentage of flowers with pollen in Reed, flowering at the end of April when no other avocado pollen is available, could be attributed to the optimal temperatures for avocado reproductive success. However, we are well aware of the limitations of this preliminary study in Reed to draw clear conclusions and, thus, in response to the reviewer’s concern, we have decided to remove this section in the new version of the manuscript.

 

-Line 218 (Total number of fruits collected per orchard):  It seems that the authors have not responded satisfactorily to my comment. They acknowledge that production data would be interesting, but they don't have access to such data from private orchards. However, they do not explain how they ensured that the sample was representative of the population.

Unfortunately, we do not have information about the number of fruits collected from each of these trees to determine whether the number of fruits analyzed represents the entire population. However, it is important to note that the number of fruits analyzed were the same in each tree of each orchard, and the results clearly show differences in the progeny genotypes among orchards with different management practices, highlighting the important role of the pollen source. Thus, an important pollen movement from neighboring orchards has been observed in monovarietal orchards.

 

-I reiterate my previous comment regarding the measurement of the number of bee visits per flower. The authors provided no evidence or references to support their claims that flowers are primarily visited by honey bees and that increasing the number of hives would increase the percentage of flowers receiving pollen. Nor did they address other factors such as flower attractiveness, availability of other pollen sources, weather conditions and hive colony strength that might affect bee visitation and pollination efficiency.

We agree with the reviewer in the fact that the honey bee activity has not been directly evaluated in this work and we mention that further work is necessary to evaluate the honey bee activity and its effect on yield. We emphasize that our primary focus was to examine the impact of increasing the number of beehives placed in the orchard on the percentage of flowers receiving pollen during the female stage. All the growers receive similar recommendations about the number of beehives in the orchard, regardless of the conditions of each one, and we think that the information obtained in this work is valuable to improve avocado management and fruit set.

The experiments were carried out using trees of the same variety and under the same management practices, thereby minimizing variations in flower attractiveness. Specifically, we chose a monovarietal Hass orchard in which we have controlled production over the past 15 years with low alternation in productivity in which molecular marker paternity analyses show that nearly 100% of the fruits derive from close pollination, occurring either from flowers of the same tree or of different trees of the same cultivar. Certainly, differences on environmental conditions could exist among years, and for that, temperature and relative humidity was monitored during the flowering season for all the years. Moreover, the experiment was carried out during several years to reduce the possible effect of environmental conditions in the parameters evaluated.

The main conclusion of this work is that, under our environmental conditions, the percentage of avocado flowers receiving pollen during the female stage is very low, even when a high number of beehives are placed in the orchard. We agree with the reviewer, and we mention that in the conclusion, that further work is necessary to explore not only the effectiveness of honey bees in avocado pollination but also the effect of increasing the presence of other pollinators on avocado pollination.

- Lines 109-129: The paragraph is too long and could be divided into two paragraphs. I suggest splitting it to separate sampling and fixation methods from staining and observation methods.

Done

-  Line 118: Replace “FAA” by “formalin-aceto-alcohol (FAA)”.

Done

-  The manuscript still needs to be improved, especially in terms of grammar and formatting. For this reason, I suggest using an English-language editing service to proofread the manuscript, as some sentences are difficult to understand and contain numerous errors and typos.

A thorough editing has been performed following the reviewers recommendation

 

-  Finally, I would like to remind the authors that in future revisions, the modified sections must be clearly marked for easier comparison by the reviewers.

We apologize for overlooking this and we have maintained the changes made in the new version of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

the authors are satisfactory in answering most of my comments.

 

Author Response

We would like to thank the reviewer for the thorough review that helped to significantly increase the quality of our first version of the manuscript.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the time and effort the authors have put into considering my comments and improving their manuscript. I'm pleased with the changes they've made and think they've added clarity and rigor to their work.