Influence of Interannual Climate Conditions on the Composition of Olive Oil from Centenarian Olive Trees
, Susana Casal 4, Rebeca Cruz 4, António M. Peres 1,2, Paula Baptista 1,2 and Nuno Rodrigues 1,2,*Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Bortulozzi et al., in this paper evaluated the effects of two years characterized by different climatic trends on the qualitative characteristics of the oil obtained from 25 centenary olive trees grown in the Vale do Côa region. While the subject of this manuscript not highly novel, it is scientifically sound and there is important information pertaining the impacts of climate change on the olive groves. However there are deficiencies in trial description and presentation of results that make it difficult to judge the importance and real value of the study and therefore should be substantially revised. I suggest major revision before its consideration for publication.
Introduction
The objective of the study must be explained clearly. Evaluate the effects of climatic conditions in the Vale do Côa region on the composition of olive oil or evaluate the resilience of centenary olive trees to the different climatic trends of the year. These objectives are very different from each other. The content of the introduction part,must be aimed at the true objective of the study.
Materials and methods
The materials and methods must necessarily be integrated. More information on the genotypes studied should be reported. It is not possible to stay on this extremely generic level on an aspect of great importance. The genotype strongly influences the olive oil characteristics. It must be indicated whether it is a variety or seedling. In the latter case, information must be provided that excludes the possibility that they are identical genotypes and information on their main bio-agronomic characteristics (fruit weight, flesh/pit ratio, oil yield, etc.). Furthermore, the cultivation methods must be indicated (type of soil, soil management, fertilization, pruning, etc.). Agronomic management affects the olive oil characteristics. The geographical location of each genotype must be indicated. The experimental design adopted (number of replications) must be indicated.
Regarding climate data, the considerable territorial extension of the Vale do Côa region and its particular orography determines that different microclimates are present within it. This inevitably conditions the vegetative-productive performance of the olive trees and the quality of the oil. Analyzing the results obtained by reference to the climate data of just one meteorological station could be a bit of a gamble. Climate data from meteorological stations as near as possible to the selected olive trees should be used. Furthermore, using only climate data directly detected by weather stations to explain the results obtained is not sufficient. It is necessary to calculate specific thermal indices and correlate them with the results obtained. There is extensive literature on this topic.
Results
In the manuscript the results are presented as a comparison of the average of the twenty-five genotypes between the two years. In order to understand the level of resilience of the twenty-five genotypes studied regarding the climatic variations between the two years of observation (this is an extremely important aspect for evaluating their level of resilience towards climate change) it is appropriate to present the data as a comparison of the individual genotypes (and not as an average) between the two years.
Do the Authors have certain evidence that the differences in the olive oil composition between the two years are only due to a different climatic trend and not to a possible load effect? In old olive trees the phenomenon of alternate bearing is usually very pronounced. It would be advisable to also have data relating to yield per tree or yield efficiency over the two years to avoid the possibility of a load effect. If you have this information it would be appropriate to include it in the manuscript.
The differences in climatic trends should be evaluated based on the dynamics of growth and ripening of the olives. In this regard, there are lots of studies in the literature regarding the critical periods that most influence the composition of olive oil. The thermopluviometric data relating to these time frames should be correlated with the results obtained.
In the manuscript (lines 301-302) it is reported that “excessive water from storms was not infiltrated into the soil, depriving the crops of the benefits of precipitation during this period”. The Authors have any definitive evidence (e.g. monitoring of soil water content and tree water status) to make this statement, otherwise it is better to completely review the concept.
Comments on the Quality of English Language
The paper would benefit from moderate editing for grammar and spelling.
Author Response
Answers to Reviewer 1
We would like to thank the Reviewer for the effort and time spent in evaluating our manuscript. The points addressed by the Reviewer allowed us to improve considerably our work.
Comments to the Author
Bortulozzi et al., in this paper, evaluated the effects of two years characterized by different climatic trends on the qualitative characteristics of the oil obtained from 25 centenary olive trees grown in the Vale do Côa region. While the subject of this manuscript not highly novel, it is scientifically sound and there is important information pertaining the impacts of climate change on the olive groves. However, there are deficiencies in trial description and presentation of results that make it difficult to judge the importance and real value of the study and therefore should be substantially revised. I suggest major revision before its consideration for publication.
Answer: We sincerely appreciate your feedback and the tried to overcome the main concerns raised by thy Reviewer.
INTRODUCTION
The objective of the study must be explained clearly. Evaluate the effects of climatic conditions in the Vale do Côa region on the composition of olive oil or evaluate the resilience of centenary olive trees to the different climatic trends of the year. These objectives are very different from each other. The content of the introduction part must be aimed at the true objective of the study.
Answer: Thank you for the comment, with which we completely agree. In the revised version of the manuscript, we clarified the main objectives, and the introduction section was changed focusing now on the objectives. Our main objective was clearly to study the effect of annual climate variation on the composition of oils from different specimens of ancient olive trees. In this sense, the following changes were made.
MATERIALS AND METHODS
The materials and methods must necessarily be integrated. More information on the genotypes studied should be reported. It is not possible to stay on this extremely generic level on an aspect of great importance. The genotype strongly influences the olive oil characteristics. It must be indicated whether it is a variety or seedling. In the latter case, information must be provided that excludes the possibility that they are identical genotypes and information on their main bio-agronomic characteristics (fruit weight, flesh/pit ratio, oil yield, etc.). Furthermore, the cultivation methods must be indicated (type of soil, soil management, fertilization, pruning, etc.). Agronomic management affects the olive oil characteristics. The geographical location of each genotype must be indicated. The experimental design adopted (number of replications) must be indicated.
Answer: Once again, we agree with the Reviewer’s comment, and in the revised version of the manuscript, we tried to improve this section. Concerning the genotype, in the present work, very old specimens of olive trees were studied, and the varieties are not known neither easily identified. After consulting the growers, the technicians of the Ministry of Agriculture and also our colleagues from the sector, no conclusive answers were obtained. To overcome this gap, the olive oil was extracted from olives collected from each tree (so, each tree was used as an independent sample) and then analysed individually, and so all the changes were reported each specific tree (please check these data in the supplementary material). This procedure; allowed evaluating, independently, the changes that took place on the olive oil from each of the different specimens studied. Nevertheless, we consider that the general trend is the most important, which can provide insights regarding the inter-annual effect, and so, the data were analysed as a whole, using average values that reflect the global trend; reason why the average data are presented in the manuscript. In terms of cultural practices used, they are typical for the region under study, with traditional dryland olive groves, with a mark of plantation of 10x10 m, which corresponds to 100 plants per hectare. These orchards are located in an area characterized by skeletal soils, specifically Leptosols, acid soils (pH between 5 to 6) and very poor in organic matter (less than 1.5%). As we analyse the oil from each plant, no true replications are available once each tree is considered as one independent sample. As mentioned in the material and methods section, from each tree, 4 to 5 kg of olives were collected, divided into three lots, being extracted independently, and so, allowing obtaining three olive oil samples per tree, being reported for each tree the average value of the independent analysis of each oil. (See line 78-86)
Regarding climate data, the considerable territorial extension of the Vale do Côa region and its particular orography determines that different microclimates are present within it. This inevitably conditions the vegetative-productive performance of the olive trees and the quality of the oil. Analyzing the results obtained by reference to the climate data of just one meteorological station could be a bit of a gamble. Climate data from meteorological stations as near as possible to the selected olive trees should be used. Furthermore, using only climate data directly detected by weather stations to explain the results obtained is not sufficient. It is necessary to calculate specific thermal indices and correlate them with the results obtained. There is extensive literature on this topic.
Answer: We understand the Reviewer's point of view. However, taking into account the knowledge of the region and also considering some published studies developed in the Côa Valley, about the climate and future scenarios for the region (e.g. Rodrigues, D.; Fonseca, A.; Stolarski, O.; Freitas, T.R.; Guimarães, N.; Santos, J.A.; Fraga, H. (2023). Climate Change Impacts on the Côa Basin (Portugal) and Potential Impacts on Agricultural Irrigation. Water, 15, 2739) we assumed that the climate conditions on the different studied olive grove areas, within the Côa region, were similar. So, in our work, we used the data collected in one meteorological station, which, in our opinion, is representative of the region and is the one that best reflects the climatic conditions of the whole region under study.
RESULTS
In the manuscript the results are presented as a comparison of the average of the twenty-five genotypes between the two years. In order to understand the level of resilience of the twenty-five genotypes studied regarding the climatic variations between the two years of observation (this is an extremely important aspect for evaluating their level of resilience towards climate change) it is appropriate to present the data as a comparison of the individual genotypes (and not as an average) between the two years.
Answer: Thanks for the comment, which we understand. In general, we choose to use average values in this manuscript rather than values per specimen/tree, since, in our opinion, it is the best approach to detect and report the changing trends observed in the olive oil quality due to the inter-annual climate changes. However, a more detailed analysis of the results, genotype by genotype, can be found in the tables included in the supplementary material. These tables show that there are some significant inter-annual differences on the oils’ compositions, being the observed changes depending on the genotype, i.e., increasing/decreasing trends were observed and differ from genotype to genotype. However, for the main compounds, similar trends (increasing/decreasing) for the majority of the studied genotypes were observed. For example, regarding the fatty acids relative abundance, for SFA, MUFA and PUFA, the year had a significant effect in the related contents found in 20%, 48% and 76% of the oils/genotypes. Concerning tocopherols, the year showed a significant effect on the vitamin E content of the oils extracted in 68% of the studied genotypes, with an increasing content observed in 60% of those genotypes. Also, although the annual variation of the total content of tyrosol+hydroxytyrosol after acids hydrolysis was genotype-dependent, a decreasing trend was observed in 64% of the oils extracted from each single genotype. On the other hand, the oxidative stability and the total phenolic content, showed an increasing trend with the year in the oils extracted from 60% and 88% of the studied genotypes, but the DPPH and ABTS antioxidant capacities showed a decreasing trend for 88% and 68% of the oils extracted from the studied genotypes. So, besides the initial discussion based on the year’s average and to meet the reviewer's recommendation, information on the different parameters evaluated per plant was added to the article and the previous discussion was inserted, aiming a more refined overview of the data. Regarding the fatty acids, they are shown in Table S1 (see line 277), tocopherols in Table S2 (see line 358), hydroxytyrosol, tyrosol and total hydroxytyrosol + tyrosol in Table S3 (see line 408) and oxidative stability, antioxidant activity and total reducing capacity in Table S4 (see line 461).
Do the Authors have certain evidence that the differences in the olive oil composition between the two years are only due to a different climatic trend and not to a possible load effect? In old olive trees the phenomenon of alternate bearing is usually very pronounced. It would be advisable to also have data relating to yield per tree or yield efficiency over the two years to avoid the possibility of a load effect. If you have this information it would be appropriate to include it in the manuscript.
Answer: It is true that, in traditional olive groves, the low intensification of the crop together with the agricultural traditional practices can be responsible for the occurrence of inter-annual variations, being the phenomenon of alternate quite common in these traditional olive groves. These phenomena may be intensified by the management of the grove and cultural conditions. In general, pruning has a great influence, and the high production load in one year is normally followed by a low fruit load (alternate). In the present case, we did not observe the occurrence of this phenomenon. The plants were slightly pruned every year without major impacts on production, and due to cultural conditions (low intensification and input of production factors), the plants have low production, less than 1500 kg/ha, without major annual fluctuations. Thus, the sentence "The plants undergone a light pruning each year, not being observed a great impact on olive production, and so, for the two studied years, no major annual yield fluctuation was observed" was included in the revised manuscript.
The differences in climatic trends should be evaluated based on the dynamics of growth and ripening of the olives. In this regard, there are lots of studies in the literature regarding the critical periods that most influence the composition of olive oil. The thermopluviometric data relating to these time frames should be correlated with the results obtained. Answer: Thanks for your comment. We agree with you. In fact, there are two important periods that can influence the fruit and its composition. The period related to the hardening of the endocarp and that of the synthesis of oil in the fruit. However, we did not make this correlation, although in Figure 1 we can see some differences, mainly in precipitation. However, we know that the referred difference was due to a stormy day with more intense rainfall than usual. As explained in the manuscript, in our opinion, this water was not used by the plant since it resulted from a short but intense peak in rainfall, thus ruling out this factor in influencing the composition of the oils.
In the manuscript (lines 301-302) it is reported that “excessive water from storms was not infiltrated into the soil, depriving the crops of the benefits of precipitation during this period”. The Authors have any definitive evidence (e.g. monitoring of soil water content and tree water status) to make this statement, otherwise it is better to completely review the concept.
Answer: The Côa Valley region is a rugged region on the slopes of two rivers, the Côa River and the Douro River, with a high rate of sloping land. When a high quantity of rain occurs in this kind of soil, the water doesn`t have the capacity to infiltrate into the soil, and some erosion phenomena are observed. On the other hand, previous studies carried out in the Douro region reported that high rainfall on sloped and unprotected soils (like that of olive groves) generally results in runoff and some frequent erosion phenomena. Although the referred studies were focused on vineyards, which predominate in the Douro’s region, similar soils and erosion problems are described (e.g. Figueiredo T.; Poesen, J.; Fonseca, F.; Hernández, Z. (2021). Long-term erosion rates in Douro Vineyards, Portugal: Effects of rainfall characteristics and plant density. Soil Conservation: Strategies, Management and Challenges, 67 – 105).
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
Comments are included in the pdf document
Comments for author File: Comments.pdf
Author Response
Answers to Reviewer 2
We would like to thank the Reviewer for the effort and dedicated time to evaluate our manuscript. The points addressed by the Reviewer allowed us to improve considerably our work.
Comments to the Author
Line 80: Please indicate country Answer: As suggested, the country name was included in the new version of the manuscript.
Line 89: Include the meaning of theses abbreviations before they appear. Answer: As requested, in the revised manuscript, the meaning of each abbreviation was inserted at its first appearance in the text.
Line 106: Specify temperature Answer: In the revised manuscript the temperature was specified.
Line 109: Subsequently abbreviations are used, include them Answer: Thanks for your remark. In the revised manuscript, we used the abbreviations after they were described.
Table 1, 2 and 3: Explain what the letters mean Answer: We apologize for this. Lowercase letters in the different tables indicate the significance of the statistical analysis. The same letter means that no significant differences are observed (P≥0.05). Different letters mean there are significant differences (P< 0.05).
Table 4: - Explain what the letters mean Answer: Vitamin E is the name given to a group of eight fat-soluble compounds in their natural, vegetable state, with antioxidant activities. They are classified into two families, tocopherols and tocotrienols, which are divided into four subfamilies: α (alpha), β (beta), γ (gamma), and δ (delta). The other letters (a, b, c, …) are related to the statistical analysis, as explained in the previous question.
- If the units are put in the title of the table, it is not necessary to put them back in the table. Answer: We agree with the Reviewer and so, in the revised manuscript the units were deleted from the table.
Line 551: - The title of the figure is far from the figure. Answer: In the revised manuscript this format issue was taken into account.
- The axes are missing Answer: In the Figure, the axis are plotted (PC1, PC2 and PC3, corresponding to the first, second and thirs pricinpal components). In caption of Figure 2, the variance percentage explained by each PC, is included:. 3D PCA plot (PC1: 37%, PC2: 16.5%, and PC3: 1.2%).
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors
The manuscript is interesting and showed good goals to understand the quality and production of olive oil according different climate conditions. The authors made a good review and introduction about the climate variations and olive oil composition. The Introduction section showed a good size and format and explain the context for this manuscript.
Line 79-80: “A total of twenty-five specimens of centenarian olive trees were selected and georeferenced in eight dispersed plots in the Côa Valley region.”, in this sentence, please indicated because just this plants was selected also, the authors can provide a map with the localization of this plants, that can be submitted as supplementary material.
Line 288: Table 1, the values was a mean value obtained of each olive oil from each plant (n=25) or the olives from each plants were collected and combined before oil extraction? Please, the author can provide a better explanation about this experimental procedure.
Line 389: the tocopherols arte differences, but just for β-tocopherol, and the author described that this differences are “is influenced by various factors”. Please, the author can discuss more about the climate variations in tocopherol content, mainly in α-tocopherol.
Line 454-464: in this discussion about phenols and antioxidant activity increase and decrease the author considered the maturity index of olives, please clarify some discussion about this subject.
Line 518: review the place of figure legend in the text
Line 536: “was found to potentially alter”, please the author can describe in Conclusion section as the annual climate conditions influence of phenol composition of oils
Line 537-539: these conclusions were just for centenary plants? The author can provide some conclusion to another plants
Comments on the Quality of English Language
A review of English language are required.
Author Response
Answers to Reviewer 3
We would like to thank the Reviewer for the effort and dedicated time to evaluate our manuscript. The points addressed by the Reviewer allowed us to improve considerably our work.
Comments to the Author
The manuscript is interesting and showed good goals to understand the quality and production of olive oil according different climate conditions. The authors made a good review and introduction about the climate variations and olive oil composition. The Introduction section showed a good size and format and explain the context for this manuscript. Line 79-80: “A total of twenty-five specimens of centenarian olive trees were selected and georeferenced in eight dispersed plots in the Côa Valley region.”, in this sentence, please indicated because just these plants was selected also, the authors can provide a map with the localization of this plants, that can be submitted as supplementary material. Answer: Thank you for your comment. This work is part of a big project that intends to identify and characterize centenarian olive plants that grow in the region of Côa Valley. So, the plants included in this study were chosen considering that these centenarian plants belong to traditional productive olive groves, being as important asset for maintaining the quality and quantity of the olive production, according to the growers. To evaluate their relevance, in two consecutive seasons, olive fruits were collected from each tree, and the olive oil was extracted form the olives collected from each genotype. Also, considering that these plants have a “long history” of adaptation to the climate conditions, we consider that they are the ideal plants for this kind of study.
Line 288: Table 1, the values was a mean value obtained of each olive oil from each plant (n=25) or the olives from each plants were collected and combined before oil extraction? Please, the author can provide a better explanation about this experimental procedure.
Answer: Regarding the Reviewer's question, the values presented in Table 1 are the average of the results of the olive oils from the 25 plants under study. Each olive oil was extracted using only the olives collected from one tree/genotype, and no mixing of olives from different trees was performed. The supplementary material includes the individual values for the olive oil extracted from the olives collected from each plant and per year.
Line 389: the tocopherols arte differences, but just for β-tocopherol, and the author described that this difference is “is influenced by various factors”. Please, the author can discuss more about the climate variations in tocopherol content, mainly in α-tocopherol.
Answer: Although no significant differences in the average contents of alpha tocopherol were observed in the present study, there are several authors who report that there are certain factors that affect the amount of this vitamer. Therefore, in this manuscript more information was included about which factors affect the alpha tocopherol contents.
Line 454-464: in this discussion about phenols and antioxidant activity increase and decrease the author considered the maturity index of olives, please clarify some discussion about this subject.
Answer: The fruit maturation index on the different plants was very similar between samples and between years, varying only between the maturation index 2 (fruits with less than 50% violet spots on the epidermis) and 3 (fruits with more than 50% violet spots in the epidermis) following the Jaén index.
Line 518: review the place of figure legend in the text
Answer: Thank you very much for the remark. In the revised manuscript the legend is in the correct place.
Line 536: “was found to potentially alter”, please the author can describe in Conclusion section as the annual climate conditions influence of phenol composition of oils
Answer: Thanks for your suggestion. In the conclusion, the sentence "Water scarcity creates a state of stress that increases the production of phenolics in olive oils. Thus, the excess of water will lead to a decrease in phenolic content" was added.
Line 537-539: these conclusions were just for centenary plants? The author can provide some conclusion to another plants
Answer: The work in question only intended to study century-old olive trees of the Côa Valley region, since the aim in the near future is to select plants that are resistant to the influences of the year on the composition and quality of their oils. Therefore, younger plants were not studied, being not possible to extrapolate the conclusions to other scenarios.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
I recommend publication of this manuscript after a moderate editing for grammar and spelling.
Comments on the Quality of English Language
The paper would benefit from moderate editing for grammar and spelling.
