Effects of Altering Carbohydrate Supply to Fruit during Development on the Carpometric and Qualitative Characteristics of “Feminello Zagara Bianca” Lemon

Round 1

Reviewer 1 Report

The authors presented very interesting results of various analyzes of lemon fruit. However, the description of the results has a lot of similar numerical errors and generalizations that do not coincide with the obtained results. It is difficult to read the results from the two graphs. Scientifically very interesting discussion. However, the conclusion lacks specific recommendations for practice and possible further research directions

 

Comments for author File: Comments.pdf

Author Response

Thanks for handling our manuscript. We are grateful for the reviewer's diligent effort to help improve our manuscript. Provided below is the detailed answers to reviewer observations.

 

1. The authors presented very interesting results of various analyzes of lemon fruit. However, the description of the results has a lot of similar numerical errors and generalizations that do not coincide with the obtained results. It is difficult to read the results from the two graphs. Scientifically very interesting discussion. However, the conclusion lacks specific recommendations for practice and possible further research directions.
2. We appreciate very much the time that the reviewer has taken to provide all the interesting comments and suggestions with respect to our manuscript. We have carefully considered all the reviewer´s comments and we think that the manuscript has been improved to a large extend. As requested by other reviewers we have shortened the introduction and discussion.

Along text Reviewer 1 have inserted comments. For all of them we have applied the request and/or replied to them as reported below with the lines of the manuscript’s first version. We have correctly listed the number of references , unit of measures, check the  and some comments are not reported due to some text reductions requested by other reviewers. We changed the Figures 1 and 2 with tables 6 and 7.

R (lines 29-31). “the importance of growing this species in Italy why the word 'Citrus' with a capital letter when it comes to the name the family  should be described differently in the text”

1. We appreciate the comment and we changed "Citrus" with citrus.

R (line 35). “briefly list the most important”

1. We have reported the most important nutritional properties of lemon.

R (lines 62-63).  “specify what the individual studies were about, not all of them together”

1. We have specified the single studies and the relative reference.

R  (lines 86 and 88).  “and the dose of potassium,  please give doses per unit area, then it is better to compare with other studies” and “please give details, we don't know what the manufacturer's specification is, unless it's a trade secret, then at least some generalities”

1. We have modified and more details about the “Limone di Rocca Imperiale” PGI specifications were inserted.

R (line 105). “maybe a little clearer (additional information in brackets should be confusing (16 fruits X 6 trees)”

1. We have move this information and now we hope it is clearer.

R (lines 170-172). “whether the percentages have been transformed”

1. For the statistical analyses we have used the unrefined data to perform ANOVA and Tukey-test.

R (lines 191-192) “this statement contradicts the statement in the first line on this page”

1. We have removed the contradiction by mean of deleting the general statement at the beginning of the page.

R (lines 211-212) “not all treatments, again generalization not justified, why do statistics if the authors know better without it”

1. We have specified the samples collected at 70 DAFB.

R (lines 272-274, 301-302 and 311-312) “turn your attention to the discussion”

1. We have largely modified the discussion part and we hope the new version responds to the reviewers’ request.

R (line 483) “provide research directions for the future in this field”

1. We have added a statement in the ending of introduction part. It underlined the possibly of future studies on agronomic strategies to enhance production. We did not want to change the end of discussion part where we have already identified some future possibly lines of practical research.

 

Author Response File: Author Response.docx

Reviewer 2 Report

1. The introduction needs to be substantially rewritten.

1-1 Currently there is only one paragraph, please divide it into paragraphs.

1-2 There is a lack of discussion related to this study, and the content is unclear. It is difficult to understand why this research is needed. Another example is line 65, ""a species with a ripening mechanism quite different from other citrus fruits [27,28,29,30,31]"". A lot of literature is mentioned here, but it is completely impossible to understand what the author wants to express, and what is the relationship between this so-called difference and this study.

1-3. There are still many related discussions that need to be explained more clearly, in order to understand the purpose of this study and the real related with current understanding.

2.Provide relevant processing photos, so that readers can understand the real state of girdling treatment of this experiment.

3.Table 4 shows that Titratable acidity is lower in early treatment, but Table 5 shows that most of the acids are higher in early treatment. Please explain the possible reasons.

4.Lines 326-333 mentioned a lot of assumptions about fruit growth, such as the first growth phase and the second growth phase, the state of cell division, etc., to illustrate the results of this study. However, lacking the real corresponding data in this study, it is difficult for us to know whether there is such a correspondence. More information is needed to make such a statement.

5."Usually, the period following flowering and fruit set 336 represents a time of scarcity of carbohydrates for the citrus trees." needed reference.

6.Later stages of girding should normally accumulate less carbohydrates than earlier stages, but this study found the opposite. Authors need to address this point. Although many hypotheses about acid conversion have been put forward, it is difficult for these previous research results to correspond to the results of this study. At the same time, there is no relevant direct data in this study, proving that the related compounds have indeed undergone conversion.

7.The Discussion section is the same as the Introduction, and it's hard to read with few paragraphs. At the same time, too much information is irrelevant to this research, and it needs to be discussed concisely and accurately in order to make the article have a higher impact.

8.If the author believes that the results of this study are more related to acid conversion, it is suggested to adjust the content direction and title. Let the research pay more attention to the discussion on the impact of girding on the conversion of acidity-related substances

Author Response

R 1. The introduction needs to be substantially rewritten.

1. As requested also by other reviewers we have shortened the introduction.

R 1-1 Currently there is only one paragraph, please divide it into paragraphs.

1. See our previous reply.

R 1-2 There is a lack of discussion related to this study, and the content is unclear. It is difficult to understand why this research is needed. Another example is line 65, ""a species with a ripening mechanism quite different from other citrus fruits [27,28,29,30,31]"". A lot of literature is mentioned here, but it is completely impossible to understand what the author wants to express, and what is the relationship between this so-called difference and this study.

1. We have changed the Introduction part and in current version we hope it should be clearer.

R 1-3. There are still many related discussions that need to be explained more clearly, in order to understand the purpose of this study and the real related with current understanding.

1. We have changed the Introduction part

R 2. Provide relevant processing photos, so that readers can understand the real state of girdling treatment of this experiment.

1. We have provided photos to better understand the girdling treatment.

R 3. Table 4 shows that Titratable acidity is lower in early treatment, but Table 5 shows that most of the acids are higher in early treatment. Please explain the possible reasons.

1. Reviewer has correctly observed that titratable acidity trend was opposite to some organic acids ones. The most represented organic acid was citric which showed a similar trend of the titratable acidy. We think the great amount of this acid (from 46 to 49 g/L) influenced the whole acidity as the statistical differences proved.

R 4.Lines 326-333 mentioned a lot of assumptions about fruit growth, such as the first growth phase and the second growth phase, the state of cell division, etc., to illustrate the results of this study. However, lacking the real corresponding data in this study, it is difficult for us to know whether there is such a correspondence. More information is needed to make such a statement.

1. The days after full bloom were reported as time reference, it is difficult to report a precise day. As requested by the reviewer we have implemented the discussion of this part.

R 5. "Usually, the period following flowering and fruit set 336 represents a time of scarcity of carbohydrates for the citrus trees." needed reference.

1. The reference previously numbered as [49] (Goldschmidt, E.E. Carbohydrate supply as a critical factor for citrus fruit development and productivity. HortScience 1999, 34, 1020-1024) actually [37] supported our argument.

R 6. Later stages of girding should normally accumulate less carbohydrates than earlier stages, but this study found the opposite. Authors need to address this point. Although many hypotheses about acid conversion have been put forward, it is difficult for these previous research results to correspond to the results of this study. At the same time, there is no relevant direct data in this study, proving that the related compounds have indeed undergone conversion.

1. Thanks to the reviewer for the observation. As the reviewer underlined, the interruption of phloematic flow prevented the carbohydrates produced by the foliage from migrating to the roots. So, the fruits of the annulate plants had a greater amount of carbohydrates than they would normally have without girdling. The results obtained in this study have shown that the increase in the availability of carbohydrates has increased the size and many qualitative characteristics of the fruits. This aspect confirms, also for lemon, that the period after fruit set represents a critical moment in which the growth of the fruit can be influenced by a carbohydrate deficit. The differences between the different treatments indicate that for an optimal growth of the fruit it is essential to have an adequate amount of carbohydrates, especially in the first part of the growth phase due to cellular distension. With regard to the decrease in the content of Soluble Solids of fruits from early girdled plants, it is connected to the advance of the ripening process determined by the increase in the availability of carbohydrates. In fact, for the typical composition of lemon juice (the main soluble constituents are organic acids), it is inevitable that the decrease in organic acids connected to the evolution of maturation determines a consequent decrease in the content of the total soluble solids of the juice.

R 7. The Discussion section is the same as the Introduction, and it's hard to read with few paragraphs. At the same time, too much information is irrelevant to this research, and it needs to be discussed concisely and accurately in order to make the article have a higher impact.

1. We thank reviewer for the comment. We have largely reduced the Discussion part and better organized it. We hope in actual version the manuscript could be better read.

R 8. If the author believes that the results of this study are more related to acid conversion, it is suggested to adjust the content direction and title. Let the research pay more attention to the discussion on the impact of girding on the conversion of acidity-related substances

1. The reviewer’s observation could be correct but as we have reported in the manuscript there is a relation between carbohydrate and organic acids.

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

Introduction

This is a scientific work, and the language should correspond to it, the introduction describes the plant species but does not mention the Latin (scientific) name of lemon….

Please complete the full scientific name and author (Citrus limon L.)

 

Methodology

There should be a brief description of the variety, its origin - that it is probably a “Femminello” bud mutation - mutations can have different properties than standard varieties... and also, of course, the grower's requirements and why this lemon variety was chosen for the study... briefly.

 

The cuts after girdling were somehow treated (meaning fungicide, disinfectant balm, etc.) ?

 

At what stage of maturity was the fruit harvested? what determined the harvest date?

Lane 139 digital retractometer  – correct is digital refractometer…

 

The results and discussion are clearly described, nevertheless are the authors able to state what effect girdling had on the subsequent differentiation of flowers and the intensity of growth in trees treated in this way in next year/season ?

 

Author Response

R 1. Introduction.  This is a scientific work, and the language should correspond to it, the introduction describes the plant species but does not mention the Latin (scientific) name of lemon….Please complete the full scientific name and author (Citrus limon L.)

1. Thanks to reviewer for the observation. We have inserted, when needed, the scientific name Citrus limon (L.) Burm.

R 2. Methodology. There should be a brief description of the variety, its origin - that it is probably a “Femminello” bud mutation - mutations can have different properties than standard varieties... and also, of course, the grower's requirements and why this lemon variety was chosen for the study... briefly.

1. We have described the variety and other requirements in the experimental part.

R 3. The cuts after girdling were somehow treated (meaning fungicide, disinfectant balm, etc.) ?

1. Girdling was done using a graft knife. Before each girdling, the knife blade was disinfected by immersing it in a glass containing a solution of 10-percent bleach (preparated with one part bleach and nine parts water). No specific disinfectant treatment was done on the cut. The fear of possible toxic effects caused on the wound by the substances usually used to disinfect the pruning cuts was the reason for this choice. The cut was, however, covered with a specific air-permeable but water-resistant grafting tape. At the end of the study all the wounds healed perfectly and no visible damage or disease occurred in the following years in all the girdled branches.

R 4. At what stage of maturity was the fruit harvested? what determined the harvest date?

1. We have better described the sampling date

R 5. Lane 139 digital retractometer  – correct is digital refractometer…

1. We have corrected the word.

R 6. The results and discussion are clearly described, nevertheless are the authors able to state what effect girdling had on the subsequent differentiation of flowers and the intensity of growth in trees treated in this way in next year/season ?

1. Although the floral density, the flower quality and the nutritional status of the plants specifically has not been studied, but only visual observations were carried out aimed at evaluating the general state of health of the plant and the development of any diseases, no substantial differences were highlighted in the productive vegetative behaviour between plants with girdled branches and those of the control. In the following season all the plants produced regularly.

 

 

Author Response File: Author Response.docx

Reviewer 4 Report

The authors should rewrite the manuscript 1) for clarity, 2) shorten it, 3) make right conclusions.

 

The English language needs serious improvements. In the title and abstract I highlighted various points needing improvement in English. I am not marking these points to the rest of the manuscript. Actually, the rest of the manuscript is better written than the abstract.

 

L33-34 useless for the scope of the manuscript, remove

Introduction could be shortened substantially. Actually, after an initial comment about significance, the Introduction could start from Line 52 and again could be further shortened.

L65-66 what is the different mechanism? This is something you should discuss to separate your work from others working with orange and mandarins.

L86 and how much K?

L101-102 Unclear. At each tree-replicate there were four branches girdled?

L115 and Table 1 and L187, and paragraph L188-201 and L360, you name ‘equatorial and polar’ and then present ‘width and height’ and then ‘transverse …’

Table 1 you have fruit gravity, and you have not mentioned how you measured it

L211 at: ‘fruits of the girdled trees the a*…’ add: at 70 DAFB

L212-213 remove the sentence, it is wrong

L226-227 rewrite the sentence

All results could be shortened by removing excessive wording or useless sentences.

L336-345 you discuss about carbohydrate availability and girdling increased it, but the girdled branches did not offer any carbs to roots for their growth and function. Thus, the rest of the tree supplies carbs to roots to increase fruit mass in the girdled branches. I am not sure you will gain in the whole tree yield!

L373 what is ‘via growth of arms’

L404-405 how do you connect the lightness of the pulp with the turgor of the sacs?

L415-416 and 410-420 are two same sentences

L445 ascorbate and ascorbic acid are the same

L473-475 OK, you did not increase tree yield! You also harvested the lemons in February. If you are talking about early ripening, I would expect the lemons to ripen in October. Nevertheless you made lemons of inferior quality in some aspects. Why do you forget that? What usefulness to farmers you talk about? How much the repeated girdling in multiple branches per tree will cost?

In the references various improvements are required

Comments for author File: Comments.pdf

Author Response

R 1. The authors should rewrite the manuscript 1) for clarity, 2) shorten it, 3) make right conclusions.

1. We thank reviewer for suggestion. We have changed many parts of the manuscript to make it more appreciate.

R 2. The English language needs serious improvements. In the title and abstract I highlighted various points needing improvement in English. I am not marking these points to the rest of the manuscript. Actually, the rest of the manuscript is better written than the abstract.

1.  We thank reviewer for suggestion. We have revised English form, but from our point of view and we think title runs well in actual version so we think the title could remain. The other manuscript’s parts were modified as request.

R 3. L33-34 useless for the scope of the manuscript, remove

1. We have completely modified the Introduction part. This period was removed.

R 3. Introduction could be shortened substantially. Actually, after an initial comment about significance, the Introduction could start from Line 52 and again could be further shortened.

1. We have applied the suggestion of reviewer.

R 4. L65-66 what is the different mechanism? This is something you should discuss to separate your work from others working with orange and mandarins.

1. We have changed this part of the Introduction. It has been implemented with more information. We hope actual version is clear.

R 5. L86 and how much K?

1. We have changed this part of material and methods, more details were described.

R 6. L101-102 Unclear. At each tree-replicate there were four branches girdled?

1. Yes, four branches were girdled. We have slightly modified text to make more understandable the method applied.

R 7. L115 and Table 1 and L187, and paragraph L188-201 and L360, you name ‘equatorial and polar’ and then present ‘width and height’ and then ‘transverse …’

1. We thanks reviewer. We have revised the entire text and we have adapted as width and height the terms.

R 8. Table 1 you have fruit gravity, and you have not mentioned how you measured it

1. We have described the method into experimental part.

R 9. L211 at: ‘fruits of the girdled trees the a*…’ add: at 70 DAFB

1. We have ad “at 70 DAFB”

R 10. L212-213 remove the sentence, it is wrong

1. We have removed the sentence.

R 11. L226-227 rewrite the sentence

1. The sentence was rewritten.

R 11. All results could be shortened by removing excessive wording or useless sentences.

1. Many parts of the Results section have been removed.

R 12. L336-345 you discuss about carbohydrate availability and girdling increased it, but the girdled branches did not offer any carbs to roots for their growth and function. Thus, the rest of the tree supplies carbs to roots to increase fruit mass in the girdled branches. I am not sure you will gain in the whole tree yield!

1. As already replied to another reviewer, we give some more explanation about the observation. The interruption of the phloem flow by girdling certainly negatively influenced the carbohydrate supply to roots. However, it was a temporary interruption that did not affect the growth and functionality of the roots too much. In fact, no state of suffering of the tree was visible both during the study and in the following year. Although the floral density, the flower quality and the nutritional status of the plants specifically has not been studied, but only visual observations were carried out aimed at evaluating the general state of health of the plant and the development of any diseases, no substantial differences were highlighted in the productive vegetative behaviour between plants with girdled branches and those of the control. In the following season all the plants produced regularly. Furthermore, the data obtained, taken as a whole, highlighted that in order to anticipate ripening and improve production performance, it is important to guarantee an adequate carbohydrate supply to the fruit, especially in the first part of the cell enlargement phase. This result indicates that the interruption of the phloem flow can occur for a very short time. However, the increase in carbohydrate supply to fruits does not necessarily have to be achieved through girdling alone. The same objective can also be achieved by increasing the leaf/fruit ratio with other agronomic strategies (e.g. through load of fruit regulation).

R 13. L373 what is ‘via growth of arms’

20. This is a terminology used by Coggins [51] Coggins, C.W. Fruit development and senescence. Citrus flowering, fruit set and development, March 26-27, 1986, Lake Alfred, FL, USA, pp.15-20. It is an anatomical part of citrus fruits’ albedo.

R 14. L404-405 how do you connect the lightness of the pulp with the turgor of the sacs?

1. The lightness of the pulp is explained by the more turgor of sacs and a consequently dilution of solutes, as Brix° revealed.

R 15. L415-416 and 410-420 are two same sentences

1. We have changed the sentences.

R 16. L445 ascorbate and ascorbic acid are the same

1. We have changed the sentences.

R 17 L473-475 OK, you did not increase tree yield! You also harvested the lemons in February. If you are talking about early ripening, I would expect the lemons to ripen in October. Nevertheless you made lemons of inferior quality in some aspects. Why do you forget that? What usefulness to farmers you talk about? How much the repeated girdling in multiple branches per tree will cost?

1. The experimental protocol did not contemplate for evaluation yield, but we can state the plants with the branches annulated at 70 and 100 DAFB recorded a yield greater than the control, due to a substantially higher weight of the fruits. In fact, as specified in the section of materials and methods, the trees used for the study were homogeneous in terms homogenous in terms of trunk and canopy size, number of main branches, vegetative vigor, leaf density and productive load. We would underline the beginning of the treatments took place after the June’s drop, when it was possible to estimate the load of the fruits on each plant. Lemon rarely shows pre-harvest fruit drop. Therefore, the number of lemons present on each plant could be quite similar.

The Femminello Zagara Bianca cv is characterized by having an intermediate harvest period, which in the study’s environment falls in February. However, if the increase in the availability of carbohydrates towards the fruits leads to anticipate ripening, the same effect will probably also occur in lemon cultivars with earlier maturation. The possibility of further anticipating ripening in an early cultivar (such as Femminello Siracusano clone 2KR which shows a harvest period in November) could allow lemons to be harvested at a time when there is a shortage on the market (September-October).

With regard to the cost, the objective of the study was to evaluate how the variation of carbohydrate intake at different stages of fruit development could influence the growth and ripening of lemons. In fact, although girdling is a technique used to improve plant productivity and fruit quality, in this specific case it has been used as a means of altering carbohydrate translocation. Ascertained that the increase in the availability of carbohydrates to the fruits can allow to anticipate the ripening of the lemons, this condition can be obtained using other agronomic practices (eg through the regulation of the load of the fruits).

R 18. In the references various improvements are required

1. From our knowledge, the scientific literature about the lemon’s topic discussed in this study is limited. We have reported and discussed citations referred to other Citrus species carbohydrates supply on fruit growth and their quality.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 4 Report

About the references: you present them in different ways, homogeneity lacks.

Again, about the practical use of girdling: you must not recemmend it without economic analysis. and why did not you try fruit thinning? Forget practical use in the way you present it.

Author Response

The Authors thank the Reviewer for the careful reading and evaluation of our work.

R: About the references: you present them in different ways, homogeneity lacks.

A: We took care to check and fix the references.

R: Again, about the practical use of girdling: you must not recemmend it without economic analysis. and why did not you try fruit thinning? Forget practical use in the way you present it.

A: The objective of this study was to evaluate as the change in the carbohydrates supply in the different stages of fruit development, could influence the growth and ripening of lemons and the girdling was used as a means to alter the translocation of carbohydrates. In fact, girdling is the most widely used technique to achieve this.

The girdling carried out in different periods (at 70, 100, 130 and 160 DAFB) have also allowed to evaluate the effects of the change in the carbohydrates supply in the different stages of fruit growth. This objective would be too complicated to achieve with fruits thinning, even if this would have been done in a cadenced way.

Only later the results showed that the increase in the carbohydrates supply to fruits (especially when this occurs in the first part of the growth phase due to cell enlargement) can anticipate ripening and improve the productive performance of trees and qualitative characteristics of the fruits.

The increase in carbohydrate supply to fruits does not necessarily have to be achieved through girdling alone but also by increasing the leaf/fruit ratio with other agronomic strategies (e.g. through load of fruit regulation with pruning).

However, if growers want to use girdling to increase in carbohydrate supply to fruits, the cost of this agronomic operation would be extremely low. In practice, the girdling is not done with the grafting knife but with specific girdling knifes or specific pincers with rotary cutting discs. The simple rotation of these tools around the branch causes the interruption of the phloem flow. This is an operation that growers know very well and perform quite easily. It takes no more than five minutes per tree to make it.

Therefore, the costs of the operation are much lower compared to earnings that are obtained from the increase productive performance of trees and qualitative characteristics of the fruits and from the anticipate ripening.

 

Author Response File: Author Response.docx